Upload
tellest
View
237
Download
0
Embed Size (px)
Citation preview
8/3/2019 ivanov 1997
1/18
Vadim T. Ivanov,
Andrei A. Karelin,Hemoglobin as a Source
Marina M. Philippova,
of Endogenous BioactiveIgor V. Nazimov,Vladimir Z. Pletnev Peptides: The Concept ofShemyakin-Ovchinnikov
Institute of Bioorganic Tissue-Specific Peptide PoolChemistry
Russian Academy of
Sciences,
Ul. Miklukho-Maklaya 16/10,
117871 Moscow, Russia
Abstract: Scattered literature data on biologically active hemoglobin-derived peptides are
collected in the form of tables. Respective structurefunctional correlations are analyzed and
the general conclusion is reached that hemoglobin fragments must have a profound physiologi-
cal function. Evidence is presented that generation of hemoglobin fragments starts inside the
erythrocytes. At that stage a- andb-globin chains of hemoglobin predominantly give rise to
relatively long peptides containing ca. 30 amino acid residues. The primary proteolysis is
followed by the next degradation step coupled with excretion of newly formed shorter peptides
form red blood cells. Both the primary and the secondary proteolysis products are subjected
to further stepwise C- and N-terminal chain shortening, giving rise to families of closely
related peptides that are actually found in animal tissue extracts. The possible sites of primary
proteolysis are compared with the positions of the exposed secondary structure elements within
the monomeric a- andb-globins as well as the tetrameric hemoglobin. Two tentative schemes
are proposed for hemoglobin degradation, one of which starts at the globin loops exposed on
the surface of the tetramer and the other, at monomeric globins where more sites are available
for the action of proteases.
The concept of a tissue-specific peptide pool is formulated, describing a novel system of
peptidergic regulation, complementary to the conventional hormonal and neuromodulatory
systems. According to that description, hemoglobin is only a single example, although an
important one, of a vast number of functional proteins providing their proteolytically derived
fragments for maintaining the tissue homeostasis. 1997 John Wiley & Sons, Inc. Biopoly
43: 171188, 1997
Keywords: hemoglobin-derived peptides; hemoglobin proteolysis in vivo; peptide excretion
from erythrocytes; tissue homeostasis; tissue specific peptide pool
INTRODUCTION tide faded as soon as it turned out to be a 110
fragment of the b-chain of hemoglobin. Moreover,other 416 membered peptides isolated later fromAs early as 1971, Schally et al. in their pursuit of
novel hypothalamic factors discovered in pig hypo- the same source were also considered artefacts
solely because they reproduced the amino acid se-thalamus a decapeptide with growth hormone re-
leasing activity.1 However, the interest in that pep- quences ofa- or b-globin chains.2,3 The apparent
Correspondence to: Vadim T. Ivanov
1997 John Wiley & Sons, Inc. CCC 0006-3525/97/020171-18
171
8/3/2019 ivanov 1997
2/18
172 Ivanov et al.
Table I Hemoglobin Fragments Isolated by Schally et al. (19711980) from Pig Hypothalamus
Sequence Fragment Activity
FLGFPTTKTYFPHFNL2 a 3348
FLGFPTTKTYFPHF2 a 3346 Corticotropin release in vitro2
VHLSAEEKEA1 b 110 Growth hormone release in vivo and in vitro1
GKVN3 b 1619 Prolactin release in vitro3
LVVYPWTQRF3
b 3241 VVYPWTQRF3 b 3341
although never explicitly formulated reason for such laboratories (Table IV) , including that of Nyberg
et al., as described in his contribution to this issue.36conclusion was the belief that since the function
of hemoglobin as an oxygen carrier is firmly estab- The list of endogenous peptides with hemoglobin
amino acid sequences was extended by a group oflished, one should not expect other significant func-
tions from the same protein. The subsequent dis- coronaro-constrictory peptides from bovine hypo-
thalamus (Table V) . Five of them, positionedcovery in bovine brain in 1979 and in 1982 of
kyotorphin 4 and neokyotorphin5 reproducing corre- within the b-chain (3241) segment, apparently
belong to the hemorphin group, although their opi-spondingly the 140141 and 137141 C-terminal
sequences ofa-globin, also were not interpreted as oid properties have not been studied.The family of myelopeptides, i.e., peptides se-an indication of the second function of hemoglobin.
In part that was due to the short length of the above- creted by pig red bone marrow cell primary culture
contains b-chain (3237) peptide anda-chain (33mentioned peptides, which therefore could originate
from another protein. 38) peptide fragments.38 Both these peptides were
studied in a variety of immunological test systems.In 1986 Brantl et al. found that proteolytic treat-
ment of hemoglobin gives rise to a series of pep- The results reviewed in this issue add another di-
mension to the spectrum of bioactivities of hemo-tides, called hemorphins, with opioid-like activity.6
That pioneering work was followed by a series of globin peptide fragments.39 Although the above-
mentioned myelopeptides induce naloxon-supressedcommunications dealing with proteolytic degrada-
tion of hemoglobin as well as with bioactive prod- analgesia in vivo, the a-chain (3338) peptide only
weakly inhibited binding ofm- and d-opiate ligandsucts resulting from such treatment.710 Since these
experiments were done in vitro, it remained uncer- to their receptors, and the b-chain (3237) pep-
tide was completely inactive in that test (IC50tain whether similar processes occur in the livingorganism. 1004M) .40 Therefore one should also not expect
any significant opioid activity from the above-men-Only in 1990s did it become clear that hemoglo-
bin serves in vivo as a powerful source of bioactive tioned coronaro-constrictoryb-chain (3337) pep-
tide shown in Table V. We believe the minimalpeptides that must have a profound role in homeo-
stasis. The present paper reviews the available data hemoglobin-derived unit displaying genuine opioid
activity is the tetrapeptide Tyr-Pro-Trp-Thr, i.e.,b-on the structure and properties of these peptides,
and describes new results related to the mechanism chain (3538) fragment or hemorphin-4. In other
words, not all biological properties of peptides fromof their biosynthesis.
the strategic b-chain (3241) zone should be
ascribed opioid receptor binding.
Systematic study of peptide composition of vari-STRUCTURE AND BIOLOGICALACTIVITY OF HEMOGLOBIN-DERIVED ous tissue extracts attempted several years ago in
our laboratory resulted in an avalanche of new pep-PEPTIDES FROM TISSUE EXTRACTStide structures.16,30 As seen from Tables VIVIII,
many of these peptides originate from a- or b-glo-Data on peptides mentioned in the introduction are
summarized in Tables IIII. The search for opioid- bin chains.
Neokyotorphin and its fragment (1 4 ) werelike hemoglobin fragments (hemorphin-containing
peptides) obtained by in vitro proteolysis was con- found in the brain of ground squirrels as well as in
the brain, lung, and heart of rat (Tables II and VI).tinued in publications of Piot et al. reviewed in this
issue of Peptide Science.8 Moreover, hemorphin- Neokyotorphin was shown to terminate the hiberna-
tion state in ground squirrels and to enhance thecontaining peptides were found in vivo in several
8/3/2019 ivanov 1997
3/18
Hemoglobin as a Source of Peptides 173
Table II Neokyotorphin Family of Hemoglobin Fragments
Peptide Fragment Source Activity
YR Kyotorphin a 140 141 Bovine brain4 Analgesic4
Rat spinal cord11
Rat brain11
TSKYR Neokyotorphin a 137 141 Bovine brain5 Analgesic5
Hibernating ground squirrel12
Antihibernatic12
Rat brain13 Ca2//K/ current regulation12
Rat lung13
Rat heart13
Human lung carcinoma14
Human erythrocytes15
TSKY a 137 140 Hibernating ground squirrel16 Ca2//K/ current regulation16
Rat brain13 Cytotoxicity13
Rat lung13
Rat heart13
Human erythrocytes15
SKYR a 138 141 Hibernating ground squirrel16
inward potential dependent Ca2/ current in cardiac ERYTHROCYTES AS A PRIMARYSOURCE OF HEMOGLOBIN-DERIVEDmyocytes of rat42 ; it was not cytotoxic in human
erythroid leukemia (K562) and murine-transformed PEPTIDESfibroblast (L929) cell lines.13 In contrast to neokyo-
torphin, the shortened by one C-terminal amino acid The abundance of hemoglobin fragments in variousfragment inhibited the Ca2/ current42 and showed tissues raises a question of the mechanism of theirpronounced cytotoxic effects in the above-men- formation and delivery. In principle, the globintioned transformed cell lines.13 genes can be expressed not only in red blood cells
Peptides belonging to the hemorphin family [i.e., and therefore respective peptides might form di-with sequences spanning the b-globin (3241) se- rectly within any cell.46 However, the huge amountquence] were amply represented in the bovine brain of hemoglobin circulating inside the erythrocytes
extracts ( Table VII) . suggest itself as the most probable precursor of pep-Several of the peptides from bovine red bone tides listed in Tables I, II, and IVVIII.
marrow were able to restore the hemopoietic func- Chromatographic analysis of the peptide contenttion of mice subjected to radiation or treated with 5- in human erythrocytes (Figures 2A and 3A) demon-fluorouracil (Table VIII) .30,41 It is worth mentioning strated that intensive proteolytic degradation of he-that one of these hemoregulatory peptides, a-globin moglobin takes place under normal conditions, giv-(116), spans three quarters of the sequence of the ing rise to peptides shown in Table X. In additionpeptide a-globin (121) from Table V. to the earlier published sequences, the latter also
A number of hemoglobin-derived peptides were shows the relative concentrations of isolated pep-isolated from brain extracts by Sleemon et al. (Table tides. The overall amount of these peptides com-IX). Although the structural approach applied in prised ca. 0.5% of the hemoglobin content. With thethat work did not allow the determination of the exception of neokyotorphin and its des-C-terminuscomplete amino acid sequences, it was shown that derivative found in very minor amounts, the intraer-
some of these peptides can serve as markers of such ythrocytic peptides have notably longer sequencespathologies as Alzheimers disease and brain isch- than most of the above-discussed endogenous he-emia.4345 moglobin fragments. Of them, the longest a-chain
In summary, hemoglobin fragments isolated (133) peptide is by far the most abundant frag-from various sources cover almost the entire se- ment present inside the erythrocytes.quences ofa- and b-globins. In turn, a considerable The reason for the above-mentioned discrepancypart of these sequences display distinct bioactivity. was found in our recent study of peptides released
The current state of the above-mentioned relation- by erythrocyte primary culture. The supernatant of
erythrocytes incubated in the phosphate buffer (pHship is illustrated in Figure 1.
8/3/2019 ivanov 1997
4/18
174 Ivanov et al.
Table III Biologically Active Hemoglobin Fragments Obtained by In Vitro Proteolysisa
Peptide Fragment Proteolitic Treatment Activity
ASHLPSDFTPAVHASL a 110 125 Bovine hemoglobin treated Bradykinin potentiation7
with pepsin7
LANVST a 129 134 Bovine hemoglobin treated Bradykinin potentiation8
with pepsin8
LVVYPWTQRF b 3241 Bovine hemoglobin treated Specific opiate receptor binding17
with pepsin9 Inhibition of electrically induced
contractions of GPI9
Inhibition of ACE18
Coronaro-constrictory19
VVYPWTQRF b 3341 Bovine hemoglobin treated Specific opiate receptor binding17
with pepsin9 Inhibition of electrically induced
contractions of GPI9
Bitter peptide20
Inhibition of ACE18
YPWT Hemorphin-4 b 3538 Bovine hemoglobin treated Naloxon-depending analgesia21
with pepsin/cathepsin6 Specific opiate receptor binding22
Inhibition of electrically induced
contractions of GPI6
YPWTQ Hemorphin-5 b 3539 Bovine hemoglobin treated Naloxon-depending analgesia21
with pepsin/cathepsin6 Inhibition of electrically induced
contractions of GPI6
GKKVLQ b 6469 Pig hemoglobin treated Inhibition of ACE10
with trypsin10
FQKVVA b 130 135 Pig hemoglobin treated Inhibition of ACE10
with trypsin10
FQKVVAG b 130 136 Pig hemoglobin treated Inhibition of ACE10
with trypsin10
a In Table III and the following tables the numbering of bovine b-globin chain starts with No. 2 in order to allow correct alignment
with sequences from other animal species.
7.2) was fractionated on size-exclusion (Figure 2B) chains as peptides found earlier in bovine extracts:a-(112), b-(211), b-( 2 9) (Table VIII) andand reverse phase high performance liquid chroma-
tography ( RP-HPLC; Figure 3B) columns. As with b-(133146) (Table V). As a result, these pep-
tides, as well as most of the other peptides fromthe erythrocytes lysates, the chromatographic pro-
files obtained with the supernatant did not show Table XI [with the exception ofa-chain (8488)
and b-chain (8791) fragments], show a very con-any significant individual differences, i.e., peptide
content of both, the erythrolysate and the superna- siderable overlap with respective peptides in Tables
I, IV, VII, and VIII. Accordingly, the b-chain (34tant did not depend upon the blood group, sex, or
age of the donor. 39) peptide is expected to display opioid properties
(Table IV) ; b-chain (1 11 ) peptidegrowth hor-The obtained fractions were rechromatographed
and substances corresponding to main peaks were mone release activity1 ; b-chain (34 37) peptide
coronaro-constrictory28 and/or immunomodula-analyzed by Edman technique. As a result, 15 amino
acid sequences of isolated peptides were estab- tory38 activities; a-chain (117), a-chain (112),
a-chain ( 1 11), b-chain (1 10) , b-chain (1 6 ),lished. Thirteen peptides were identified as hemo-globin fragments, (Table XI) the remaining two andb-chain (7284) peptideshemopoietic activ-
ity 41 (Table VIII).(NDKVQPLE and KEATQE, not shown in Fig. 3B
and Table XI) are derived from other proteins. The A striking feature of the obtained result is the
fact that not a single peptide found inside erythro-overall content of peptides in the supernatant after
4.5 h incubation was ca. 25% of the peptide con- cytes was present in the supernatant. From that ob-
servation we conclude that release of peptides fromtent inside erythrocytes.
Four of the peptides present in the supernatant erythrocytes is accompanied by proteolytic degrada-
tion of initially formed long peptide sequences, pos-occupy the same positions in the a- and b-globin
8/3/2019 ivanov 1997
5/18
Hemoglobin as a Source of Peptides 175
Table IV Hemorphins and Related Peptide Fragments ofb-Globin
Sequence Position Source Activity
LVVYPWTQRF 32 41 Pig hypothalamus3 Specific opiate receptor binding17
Bovine hypothalamus19 Inhibition of electrically induced
contractions of GPI9
Bovine brain23 Coronaro-constrictory activity19
Human liquor24
Inhibition of ACE18
Human gingival crevicular fluid25
LVVYPWTQR 32 40 Human pituitary gland26 Specific opiate receptor binding26
Inhibition of electrically induced
contractions of GPI26
Inhibition of ACE27
LVVYPWTQ 32 39 Bovine brain23
LVVYPWT 32 38 Bovine brain23 Coronaro-constrictory activity28
Bovine hypothalamus28 Inhibition of enkephalin-degrading enzyme29
Bovine spinal cord29 Convulsion in vivo30
VVYPWTQRF 33 41 Pig hypothalamus3 Specific opiate receptor binding17
Bovine brain23 Inhibition of electrically induced
contractions of GPI9
Mice peritoneal macrophages31 Inhibition of ACE18
Bitter peptide20
VVYPWTQR 33 40 Bovine hypothalamus32 Coronaro-constrictory activity32
VVYPWTQ 33 39 Bovine hypothalamus33 Specific opiate receptor binding33
Bovine brain23 Inhibition of electrically induced
contractions of GPI33
Cytotoxicity34
VVYPWT 33 38 Bovine hypothalamus28 Coronaro-constrictory activity28
YPWTQRF 35 41 Human plasma35 Specific opiate receptor binding9
Inhibition of electrically induced
contractions of GPI17
sibly by membrane-associated proteases. The partic- There are all grounds to assume that release of
short peptides from erythrocytes makes an im-ular type of protease as well as the mechanism oftransmembrane passage (direct transport or secre- portant contribution to the overall peptide content
of various tissues. At the same time the array oftion involving vesicle formation, energy, and carrier
requirement, etc.) require further investigation. concrete peptides found in three thus far studied
tissues (bovine bone marrow, bovine brain, and hu-
man cerebellum, see Tables VII, VIII, and IX ) dis-Table V Hemoglobin Fragments Isolated from plays considerable differences. For instance, hemor-Bovine Hypothalamus by Galoyan et al.
Sequence Fragment Table VI Hemoglobin Fragments Isolated from
Brain of Hibernating Yakut Ground SquirrelsVLSAADKGNVKAAWGKVGGHA37 a 121 Citellus undulatus16
ASHLPSDFTAPAVAS37 a 110124
SHLPSDFTPAV32 a 111121 Sequence FragmentHLPSDFTPAVHASLD32 a 112126LVVYPWTQRF19,a b 3241 VLSPA a 15LVVYPWT28,a b 3238 TSKYR a 137141VVYPWTQR32,a b 3340 TSKY a 137140VVYPWT28,a b 3338 SKYR a 138141VVYPW28,a b 3337 VHLSDGEKNAISTAWG b 116FQKVVAGVANALAHRYH32 b 130146 VHLSDGEKNAISTA b 114VVAGVANALAHRYH37 b 133146 IVIVMA b 110115
VVAGVANA b 133140a These peptides display coronaro-constrictory properties.
8/3/2019 ivanov 1997
6/18
176 Ivanov et al.
Table VII Hemoglobin Fragments Isolated from Bovine Brain30
Content
Sequence Fragment (nmol/g Tissue)
VLSAADKGNVKAAWGKVGGHAAEYGAEALERM a 132 0.11.0
VLSAADKGNVKAAWGKVGGHAAEYGAEALE a 130 0.11.0
VLSAADKGNVKAAWGKVGGHAAEY a 124 0.11.0
DKGNV a 610 0.11.0LSHSL a 101105 0.11.0
ASHLPSDFTPAVHASLDKFLANV a 110132 1.03.0
ASHLPSDFTPAVHASLDK a 110127 0.11.0
FLANVSTVL a 128136 0.11.0
MLTAEEKAAVTAFWGKVKVDEVGGEALGRL b 231 0.11.0
MLTAEEKAAVTAFWGKVKVDEVGGEALG b 229 0.11.0
MLTAEEKAAVTAF b 214 0.11.0
MLTAEEKA b 29 0.11.0
MLT b 24 0.11.0
WGKVKVDEVGGEA b 1527 0.11.0
WGKVKVDEVG b 1524 0.11.0
EVGGEALG b 2229 0.11.0
EVGGEAL b 2228 0.1
GGE b 2426 0.11.0LVVYPWTQRF b 3241 1.03.0
LVVYPWTQ b 3239 1.03.0
LVVYPWT b 3238 0.11.0
LVVYP b 3236 0.11.0
VVYPWTQRF b 3341 1.03.0
VVYPWTQ b 3339 0.11.0
VVVLARNFGKFFTPVLQADFQKVVAGVAN-? b 111139? 0.11.0
VVVLARNFGKFFTPVLQ b 111127 0.11.0
VVVL b 111114 0.11.0
ARNFGKFFTPVLQ b 115127 0.11.0
VLQ b 125127 0.11.0
FQKVVAGVANALAHRYH b 130146 0.11.0
phins were not found in bovine bone marrow ex- taining ca. 30 amino acid residues and found in
tissue extracts come from the intraerythrocyte pooltracts and are well represented in bovine brain (1
nmol/ g of tissue) . In other words, in spite of argu- of the peptides where they are represented in high
amounts (Table X) . The shorter peptides are genu-ments favoring the role of erythrocytes as a common
primary source of hemoglobin fragments, each tis- ine tissue specific peptides and are either absorbed
from the blood stream or result from proteolyticsue has its own, specific set of hemoglobin-derived
peptide components. degradation of the absorbed peptides.
The available quantitative, data although rather
fragmentary on the content of hemoglobin-derived
peptides in various samples, also speak of tissue PROTEOLYTIC DEGRADATIONOF HEMOGLOBINspecificity. For instance, the level of neokyotorphin
in rat lung is 45 times higher than in erythro-cytes.13 We believe that peptides released from the With more that 150 established amino acid se-
quences of endogenous hemoglobin fragmentserythrocytes either accumulate in the tissue sur-
rounding the blood vessel or are further degraded available for comparison, it is tempting to analyze
the pathways of hemoglobin proteolysis in moreby tissue-specific proteases. The observed level of
peptides in the tissue extract is a sum of the contri- detail. For that purpose we collected in Figures 4
and 5 all peptides from Tables IXI and alignedbutions from the erythrocytes always present in
blood vessels and from the rest of the tissue. them under the a- and b-globin sequences of re-
spective species.Accordingly, we assume that long peptides con-
8/3/2019 ivanov 1997
7/18
Hemoglobin as a Source of Peptides 177
Table VIII Hemoglobin Fragments Isolated from Bovine Red Bone Marrow30
Content
Sequence Fragment (nmol/g Tissue)
VLSAADKGNVKAAWGKa a 116 0.01
VLSAADKGNVKAAWG a 115 0.01
VLSAADKGNVKAAa a 113 0.010.1
VLSAADKGNVKA a 112 0.01LSAADKGNVKAA a 213 0.010.1
SAADKGNVa a 310 0.01
KVGGHAAEYGAEAa a 1628 0.010.1
KVGGHAAEYGAE a 1627 0.010.1
KVGGHAAEYGA a 1626 0.010.1
VGGHAAEYGAEALa a 1729 0.01
VGGHAAEYGAEAa a 1728 0.010.1
AEYGAELa a 2229 0.01
AEYGAE a 2228 0.10.3
GAEALERa a 2531 0.010.1
AEALERM a 2632 0.010.1
EALERM a 2732 0.010.1
EALEa a 2730 0.010.1
LSFPTTK a 3440 0.01DLSHGSAQV a 4755 0.11.0
ALTKA a 6569 0.11.0
LPGALSELS a 7684 0.11.0
LPGALSEa a 7682 0.11.0
LPGA a 7679 0.010.1
LASHLPSDFTPAV a 109121 0.010.1
ASHLPSDFTPAVHA a 110123 0.10.3
ASHLPSDFTPAVH a 110122 0.010.1
ASHLPSDFTPAV a 110121 0.010.1
ASHLPSDFTPA a 110120 0.010.1
ASHLPSDF a 110117 0.010.1
ASHLPS a 110115 0.10.3
ASHLP a 110114 0.010.1
LPSDFTPAVH a 113122 0.010.1LPSDF a 113117 0.10.3
LDKFLA a 125130 0.010.1
MLTAEEKAAVTa b 212 0.010.1
MLTAEEKAAVa b 211 0.010.1
MLTAEEKAAa b 210 0.010.1
MLTAEEKA b 29 0.010.1
MLTAE b 26 0.010.1
LTAEEKA b 39 0.010.1
AEEKAA b 510 0.010.1
GKVKVDEVGGEALGRLa b 1631 0.010.1
VKVDEVGGEALGRL b 1831 0.010.1
DEVGGEALGR b 2130 0.010.1
EVGGEALGRLa b 2231 0.10.3
EVGGEALGR b 2230 0.10.3EALG b 2629 0.010.1
ALG b 2729 0.10.3
SNGMKGLDDLK b 7282 0.010.1
KLHVDPEa b 95101 0.010.1
ARNFGKFFa b 115122 0.010.1
NFGKEFTPV b 117125 0.010.1
VLQA b 125128 0.010.1
a These peptides display hemopoietic activity.41
8/3/2019 ivanov 1997
8/18
178 Ivanov et al.
Table IX Hemoglobin Fragments as Peptide Markers of Brain Pathologies (Sleemon et al. 19921996)
Sequence Fragment Source Pathology
VLSPADKTNVKAAWGKVGAHAGEYGA-? a 126? Human cerebellum Alzheimers disease43
VLSPADK-? a 17? Human cerebellum Healthy donor44
?-MFAAFPTTK-? a (32 40)? Rat hippocampus Ischemia45
FLSFPTTKTYFPHFDLSHGSAQV-? a 3355? Human cerebellum Alzheimers disease43
FLSFPTTKTYFPHFDLSH-? a 3350? Human cerebellum Alzheimers disease43
FLSFPTTK-? a 3340? Human cerebellum Alzheimers disease43
?-TYFNHIDVSP-? a (41 50)? Rat hippocampus Ischemia45
LVTLAAHLP-? a 106114? Human cerebellum Alzheimers disease43
AAHLPAEFTP-? a 110119? Human cerebellum Alzheimers disease43
?-LASVSTVLT-? a (129 137)? Rat hippocampus Ischemia45
VHLTPEEKSAVTAL-? b 114? Human prefrontal cortex Alzheimers disease45
?-VVYPWTQRY-? b (33 41)? Rat hippocampus Ischemia45
FESFGDLSTPDAV-? b 4254? Human cerebellum Alzheimers disease43
(AV)MGNPK-? b 53 59? Human postcentral gyrus Alzheimers disease45
SELHCDKLHVEFTPPVQAAYQK-? b 89132? Human cerebellum Alzheimers disease43
VCVLAHHFGKEFTPPVQAAY-? b 111130? Human cerebellum Alzheimers disease43
The obtained overall picture clearly speaks of a several smaller pieces, e.g., at sites (13) (Ala/ Cys) -
Trp-(Gly/ Glu) -Lys-( Val/ Ile)( 17) , (127)Lys-Phe-nonrandom, stepwise manner of hemoglobin degra-
dation, regardless of the chosen species and tissue. Leu-(Ala/Ser) -(Asn/Ser)-Val-Ser( 133) , or (135)
Val-Leu-Thr-Ser(138) in the a-globin, and (13)As seen from the positions of the nonoverlapping
sequences, the primary splitting might occur at the (Ala/ Gly/ Thr)-(Phe/Leu/ Ala) -Trp-Gly-Lys-Val
(18), (30)Arg-Leu-Leu-Val-Val(34), (108)Asn-sites marked in Figures 4 and 5 by arrows, giving
rise to 2 4 fragments 3060 amino acid resi- (Val /Met) -( Leu/Ile )-Val-( Val/ Cys/Met) -Val-
(Leu/Met)- (Ala/Gly)( 115) , or (127)Gln-Ala-dues long. These positions for a-globin are
(33) Met-Phe-(Leu/Ala)(35), (55)Val-rrr-Ala(65), (Asp/ Ala) -( Phe/ Tyr) -Gln( 131) in the b-globin.
The products of both primary and secondary proteo-(69)Ala-rrr-( Leu/Met/ Val) (76) , (88) Ala-rrr-
Leu(101) and (105)Leu-Leu(106). For b-globin lytic attack are degraded by carboxy- and aminopep-
tidases, giving rise to ladders of stepwise C- andthe respective sites are (41)(Phe/Tyr)-Phe(42),
(54)(Val/Ile)-rrr-(Ser/Asn)( 72), (85)Phe-rrr N-terminally shortened peptides. The products ofcarboxypeptidase action are more frequently repre-(Ala/Thr/Lys/Ser/His)(87).
The obtained polypeptides are further cut into sented in Figures 4 and 5. However, part of that
FIGURE 1 Hemoglobin sequences covered by peptides isolated from animal tissues and
biological activities associated with respective sequences. Sequences covered by isolated pep-
tides are marked by dark shading. Types of biological activities: HPhemopoietic; HR
hormone releasing; CCcoronaro-constrictory; NKpeptides from neokyotorphin group;
IMimmunomodulatory; AGantigonadotropic; OPopioid.
8/3/2019 ivanov 1997
9/18
Hemoglobin as a Source of Peptides 179
(Ala/Leu/Phe)-Trp, Trp-Gly, and Gly-Lys sites.
Such diffuse specificity might be also associated
with conformational features of the products of pri-
mary splitting. Certainly, more work should be done
to confirm the above speculation since possible par-
ticipation of more than one enzyme in degradation
as well as inclusion into Figures 4 and 5 of peptides
from various tissues and various species can serveas alternative explanations.
We tried to correlate the putative positions of
primary cutting with the secondary structure of glo-
bin chains around those positions as well as their
availability to intermolecular interactions with pro-
teases. Figure 6 presents the space filling model of
the tetrameric hemoglobin globule in which helix-
connecting loops of the polypeptide chains are
marked with different colors and labeled with num-
bers, as shown in the figure caption. It is clear from
a brief glance at the picture that all loops, although
FIGURE 2 Size-exclusion chromatography of peptide
material from the lysate of human erythrocytes (A) and
the supernatant of the primary culture of human erythro-
cytes (B). The collected fractions are underlined.
abundance might be due to increasing uncertainty
in identification of the last C-terminal residues in
the course of automatic sequence analysis by theEdman procedure.
Following the arguments given in previous sec-
tion, we assume that primary proteolysis of hemo-
globin takes place exclusively inside the erythro-
cytes while all the following steps predominantly
occur on crossing the erythrocyte membrane and
later within the tissue.
There are no obvious regularities in amino acid
sequences around the primary or secondary splitting
sites. It is only clear that trypsin-like proteases do
not participate in the processing of hemoglobin,
since there are no arginines and very few lysines in
those sequences. All splitting sites contain hy-drophobic residues.
As for the secondary splitting, it is worth men-FIGURE 3 RP-HPLC of the fractions obtained after
tioning that it takes place at several adjacent peptidesize-exclusion separation. (A) The lysate of human eryth-
bonds rather than at strictly defined single pairs of rocytes; (B) the supernatant of primary culture of humanamino acid residues. For instance, the a-chain (32 erythrocytes. Absorbance range in mV (1800 mV 2.5634) segment (Met-Phe-Leu) is cut at Met-Phe and AU) . The peaks containing peptides subjected to seque-Phe-Leu sites and the b-chain (1417) segment nation are marked with numbers. The obtained sequences
are given in Tables X and XI at respective numbers.[( Ala/Leu/Phe) -Trp-Gly-Lys] is apparently cut at
8/3/2019 ivanov 1997
10/18
180 Ivanov et al.
Table X Peptides Isolated from Human Erythrolysate15
Content
No. Sequence Fragment (nmol/mL cells)
6 VLSPADAKTNAVKAWGKVGAHAGEYGAEALERMF a 133 140160
5 VLSPADAKTNAVKAWGKVGAHAGEYGAEALERMa a 132 612
3 VLSPADAKTNAVKAWGKVGAHAGEYGAEALERa a 131 35
4 VLSPADAKTNAVKAWGKVGAHAGEYGAEALE a 130 487 VTLAAHLPAEGFTPAVHASLDKFLASVSTVL a 107136 2550
9 AAHLPAEGFTPAVHASLDKFLASVSTVLTSKYR a 110141 2040
1 TSKYR a 137141 24
2 TSKY a 137140 24
8 AHHFGKEFTPPVQAAYQKVVAGVANALAHRYH b 115146 2550
a The content of these peptides was 515 times higher in the blood samples of patients with Hodgkins disease than those of healthy
donors.47
to a varying degree, are represented at the surface of to loops 2 and 7, as well as the adjacent a-helical
amino acid residues [in particular, a-chain (3334)the globule. However, more detailed analysis proves
that the above-mentioned sites a-chain (3335), and b-chain (4142)] become a plausible targetfor the protease. Such splitting explains the forma-a-chain (88101), and b-chain (4142) are not
sterically accessible. Of the remaining sites, the tion of the peptides a-chain (133)15 and b-chain
(141).26symmetric a-chain (6976) and b-chain (5672)
ones, belonging respectively to loops 3 and 8, seem It is too early to give a preference to either of the
proposed degradation pathways. One should alsoto satisfy the steric requirements. The remaining a-
chain (59 65), a-chain (105107), and b-chain consider that conformational rearrangement might
accompany the nicking of both the tetramer and the(8587) sites fall into the middle of respective heli-
cal segments. dissociated forms, leading to formation of additional
degradation sites. In other words, if general princi-Besides the tetramer, monomeric a- and b-glo-
bins whose content in the equilibrium is ca. 0.001 ples of hemoglobin proteolysis leading to formation
of peptide families are reasonably established, the0.01% of the total protein 48 can also be a subject
of enzymatic degradation. Secondary structures of concrete sites of primary splitting require identifi-
cation of longer peptides containing ca. 60 aminothese chains are shown in Figure 7. In that case sitesa-chain (3652) and b-chain (35 57 ) , referring acid residues.
Table XI Hemoglobin Fragments Released from Human Erythrocytes in Primary Culture
Content
No. Sequence Fragment (nmol/mL Cells)
13 VLSPADKTNVKAAWGKV a 117 0.040.06
6 VLSPADKTNVKA a 112 0.030.05
5 VLSPADKTNV a 110 0.030.05
3 VLSPADKTN a 19 0.030.05
4 SDLHA a 8488 0.050.07
8 VHLTPEEKSAV b 111 0.100.12
2 VHLTPEEKSA b 110 0.060.08
1 VHLTPEEK b 18 0.030.05
10 VYPWTQ b 3439 0.030.05
9 VYPW b 3437 0.010.02
11 SDGLAHLDNLKGTF b 7284 0.060.08
7 TLSEL b 8791 0.030.05
12 VVAGVANALAHRYH b 133146 0.100.12
8/3/2019 ivanov 1997
11/18
Hemoglobin as a Source of Peptides 181
FIGURE
4
a-G
lobin
fragmentsisolatedfrom
differentsour
ces.Sequencesofa-g
lobins:(A)bovi
ne,
(B)human,
(C)pig,
(D)Yakutg
roundsquirrelsCitellus
undulatus,
and(E)rat.
Theprimarysplittingsitesaremarkedbyarrows.Sequencesofbiologicallyactivepeptidesareprintedinbolditalic.
8/3/2019 ivanov 1997
12/18
182 Ivanov et al.
FIGURE5
b-G
lobinfra
gmentsisolatedfrom
differentsources.S
equencesofb-g
lobins:(A)bovine,
(B)
human,
(C)pig,
(D)YakutgroundsquirrelsCitellusundulatus,
and(E)rat.Theprimarysplittingsitesaremarkedbyarrows.Sequencesofbiologicallyactivepeptidesa
reprintedinbolditalic.
8/3/2019 ivanov 1997
13/18
Hemoglobin as a Source of Peptides 183
FIGURE 6 Space-filling model of human deoxyhemoglobin tetrameric structure. (A) front
view; (B) and (C) views of the model rotated by 90 around X and Y axes, respectively.
Helices of two a- and b-chains are shown in white and magenta, respectively. Loops of two
a-chains are shown in light and dark blue [position numbers: (1 ) fragment 1819; (2) fragment
3652; (3) fragment 7475; (4) fragment 9294; (5) fragment 113118] and those of two
b-chainsin red and brown [position numbers: (6) fragment 1819; (7) fragment 3550;
(8) fragment 7780; (9) fragment 9699; (5) fragment 119123].
HEMOGLOBIN FRAGMENTS AS globin carries out an important in vivo regulatoryfunction. In other words, we believe participationCOMPONENTS OF TISSUE-SPECIFIC
PEPTIDE POOL of hemoglobin fragments in homeostasis providesan example of a novel peptide-mediated regulatory
pathway complementary to traditional hormonal orThe broad spectrum of biological effects exhibited
by endogenous hemoglobin-derived peptides forces neuropeptide mechanisms. That concept originally
formulated in 1992 was further supported by thea conclusion that proteolytic degradation of hemo-
8/3/2019 ivanov 1997
14/18
184 Ivanov et al.
FIGURE 7 A view of the overall structure fold of human deoxyhemoglobin. (A) a-chain;
(B) b-chain. The a-helices and loops are shown in magenta and yellow, respectively. The
numbers indicate the beginning and the end of the a-helical secondary structure elements.
above-described data on intensive formation in and It is generally accepted that the proteins normally
present in the tissue are digested by proteolytic en-release from the erythrocytes of active peptides sub-
sequently found in various tissues.30 zymes after fulfilling their function. We suggest that
8/3/2019 ivanov 1997
15/18
Hemoglobin as a Source of Peptides 185
Table XII Distinctive Features of Peptidergic Regulatory Systems
Peptidergic Regulatory System
Characteristic Nervous Endocrine and Paracrine Tissue-Specific Peptide Pool
Peptides Neurotransmitters Hormones Fragments of functional proteins
Precursor Specific protein precursor Functional protein
Type of processing Discrete site specific processing Action of tissue proteinasesLevel (pM/g tissue) 0.001 1.0 0.001 1.0 1010,000
Type of regulation Synaptic secretion Extracellular secretion Alteration of the level in the tissue
Mechanism of action Binding to receptors Binding to receptors in Binding to receptors of homologous
in postsynaptic cellular membranes hormones
membrane
Receptor binding 1 1000 0.110 10010,000
constants (Kd, nM)
Time range of action Secondsminutes Minuteshours Hoursdays
Biological role Transmission of nerve Regulation of physiological Maintenance of tissue homeostasis
impulse processes in the tissue or
the whole organism
protein elimination does not occur by random hy- of proteolytic processes depends on such a relatively
conservative parameter as the metabolic state of thedrolysis directly leading to amino acids, which are
further utilized in metabolic reactions. Instead, it organism, we suggest that the tissue-specific peptide
pool predominantly controls long-term processes,represents a specific process regulated by the level
of tissue-specific proteases and the availability of i.e., is responsible for tissue homeostasis. The latter
includes cell proliferation, differentiation, and celltheir substrates. As a result of that process, a large
number of peptides is formed that can be defined death. Prevention of cell transformation and lysis
of tumor cells also fall into that category. The recentas a peptide buffer, peptide background, or
more accurately as tissue-specific peptide pool. finding that hemoglobin fragments induce death of
transformed cells13,34 and regulate proliferation andThe properties of that pool depend upon the con-
crete set of peptide components as well on individ- differentiation of normal cells38,41 provides a con-
vincing argument favoring our view.ual levels of those components. Characteristic prop-
erties of peptides from the tissue-specific pool in Components of the tissue specific peptide poolshave several features in common with the so calledcomparison with the classical regulatory peptides
are summarized in Table XII. In contrast to peptides cytomedines, a term proposed in early 80-ies for
the components with low molecular mass fractionsderived from functional proteins, the signal peptide
molecules of the nervous tissue (neurotransmitters of the total tissue extracts. Similarly to the peptide
pool, cytomedines were ascribed a homeostatic roleand/or neurohormones) or endocrine system (hor-
mones and parahormones) are released from a nar- for the given tissue (see the review49 and the refer-
ences therein). However, most of the porperties ofrow circle of specific precursors for which no other
than having a precursor function is known. Compo- cytomedines were studied on complex, poorly char-
acterized mixtures containing all types of regulatorynents of the peptide pool bind to the same receptors
as neurotransmitters or hormones modulating peptides presented in Table XII.
There is a growing evidence of changes in tissuethereby the availability of the receptors to their
true ligands. The binding affinities of the pool composition of hemoglobin-derived peptides ac-
companying various pathologies, such as humanpeptides are by several orders of magnitude lowerthan for hormones or neurotransmitters. However, lung adenocarcinoma14 (Tables II ) , Alzheimers
disease,43,45 brain ischemia45 (Table IX), and Hodg-these peptides are found in higher amounts and typi-
cally occur as families of related molecules rather kins disease47 (Table X) . Notwithstanding the clin-
ical and biochemical differences, all the above-men-than single representatives. Hemorphins and closely
related peptides with their opioid receptor binding tioned pathologies have a common principle fea-
ture they involve changes in tissue homeostasisability consistently found in most of the investigated
sources ( see Tables I, IV, V, and VII ) provide a or metabolic state of the cells, whatever the origin of
the disease: cell transformation ( adenocarcinoma) ,good example of that tendency. Since the intensity
8/3/2019 ivanov 1997
16/18
186 Ivanov et al.
tissue atrophy ( Alzheimers disease and ischemia) , Destruction of Erythrocytesor impaired lymphoproliferation ( Hodgkins dis-
The washed clean cells were subjected to two cycles ofease). Although it is not known whether the above- freezing (070C) and unfreezing (5C), homogenized inmentioned changes in peptide composition are the Potter S (B. Braun, Germany) (900 rev 1 5 min) andcause or the consequence of the disease, these centrifuged (10000 rev. 1 15 min) at Biofuge B (Her-changes are in full accord with our views of the aeus, Germany). The amount of 0.25 mL of supernatant
was dissolved in 10 mL 0.1M acetic acid and injectedbiological role of tissue-specific peptide pool.into the size-exclusion column.
More data should be accumulated before the con-cept of tissue-specific peptide pool finds its final
shape. However, regardless of these developments Supernatant of the Primary Culture ofthere is little doubt that besides the oxygen transport Erythrocytes(and possibly the transport of nitrogen oxide50) he-
Ten milliliters of washed cells were incubated with 40moglobin serves as a rich source of bioactive pep-mL of buffer A in culture flacks for 4 h at 37C. Aftertides. In that case the function of erythrocytes mightincubation, the cells were pelleted by centrifugation inbe compared with the function of endocrine gland.conditions described above. The obtained supernatants
Endogenous fragmentation of the hemoglobinwere collected and lyophilized.
and the properties of respective peptides are studiedA portion of lyophilized supernatant obtained from 50
much better than analogous processes with other mL of blood cells was dissolved in 8 mL 0.1M aceticfunctional proteins. Still, identification of active acid and subjected to centrifugation at 11000 rpm for 5peptides derived in vivo from cytochrome c oxi- min. Supernatant was immediately injected into the size-
dase,51
immunoglobulins,30,5254
albumins,55
fi- exclusion column.brinogen,56 and other functional proteins30,57 allows
us to conclude that the regulatory role of hemoglo-Size-Exclusion Chromatography
bin-derived peptides should not be considered aThe samples were separated on Sephadex G-25 sf (Phar-unique, isolated phenomenon. On the contrary, it ismacia, Sweden) column (2.5 1 85 cm) equilibrated withonly one of the components (although an important0.1Macetic acid. Elution was carried out at 120 mL/h,one) of the general system of peptidergic regulationwith detection at wavelengths 206/280 nm. The fractionsof tissue homeostasis. One can even suggest thatcorresponding to the zone of elution of substances withthis system is phylogenetically more ancient than0.54.0 kDa molecular masses about (Figure 2) were
the endocrine and the nervous system since the lattercollected and lyophilized.
deal with higher level of regulation, involving sev-
eral types of tissues, several organs, and the wholeRP-HPLC Separationorganism.
The material obtained after size-exclusion chromatogra-
phy was dissolved in 250 mL of 0.1% solution of triflour-
oacetic acid (TFA) in water and subjected to separation
on Nucleosil 120/5m C8 cartridge (4.0 1 250 mm; Mash-MATERIALS AND METHODSerey-Nagel, Germany), equilibrated with buffer A (0.1%
TFA in water). The column was washed for 5 min by
buffer A, then the elution was performed for 60 min byPreparation of Human Erythrocyteslinear gradient of acetonitril from 0 to 60% of buffer B
(0.1% TFA, 80% acetonitril solution in water) at 0.75Peripheral venous blood was obtained from 10 healthy
mL/min. The detection was carried out at 226 nm.volunteers [7 men and 3 women with different blood
groups A(II), B(III), AB(IV); aged 2035 years] by
veinpuncture after confirmation of their health status by Isolation of the PeptidesResearch Haematological Centre, Russian Academy of
The peaks corresponding to homogeneous substancesMedical Sciences.were collected as shown in Figure 3, lyophilized andBlood samples (25 mL) were placed into the tubessubjected to separation on Nucleosil 120/5m C18 cartridgecontaining citrate buffer (to final citrate concentration(4.0 1 250 mm) in linear acetonitril gradient from 8of 0.25%). The cells were separated from plasma byto 40%.centrifugation at 1000 rpm for 15 min at 0C. The ob-
tained pellet (1012 billions of cells, 99% of erythro-
cytes) was washed four times by buffer A (0.025M Amino Acid SequencesNaH2PO4 containing 0.1M NaCl, pH 7.2). Both lysate
and supernatant were prepared from the same batch of Amino acid sequences of isolated peptides were deter-
mined in the gas-phase sequencer Applied Biosystemscells.
8/3/2019 ivanov 1997
17/18
Hemoglobin as a Source of Peptides 187
447A (Foster City, USA). Estimation of the peptide con- Mikhaleva, I. I., Ivanov, V. T., Kokoz, Yu. M., Alek-
seev, A. E., Korystova, A. F., Sukhova, G. S., Eme-tent was made from the sequencing data.
lyanova, T. G. & Usenko, A. B. (1994) Bioorgan.
Khim. 20, 677702 (in Russian).Molecular Graphic 17. Garreau, I., Zhao, Q., Pejoan, C., Cupo, A. & Piot,
J.-M. (1995) Neuropeptides 28, 243250.The x-ray coordinates of the human deoxyhemoglobin18. Zhao, Q., Sannier, F., Garreau, I., Guillochon, D. &(Brookhaven PDP code 4hhb) solved at 1.74 A resolu-
Piot, J.-M. (1994) Biochem. Biophys. Res. Commun.tion 58 have been used for the analysis using CHAIN pro-
204, 216223.gram.59 Figures 6 and 7 were prepared by SETOR pro-19. Barkhudaryan, N. A., Kellermann, J., Galoyan,gram.60
A. A. & Lottspeich, F. (1993) FEBS Lett. 329, 215
218.
20. Aubes-Dufau, I., Capdevielle, J., Seris, J. L. &REFERENCES Combes, D. (1995) FEBS Lett. 364, 115119.
21. Davis, T. P., Gillespie, T. J. & Porreca, F. (1989)
Peptides 10, 747751.1. Schally, A. V., Baba, Y. & Nair, R. M. G. (1971) J.
Biol. Chem. 246, 6647 6650. 22. Liebmann, C., Schrader, U. & Brantl, V. (1989) Eur.
J. Pharmacol. 166, 523526.2. Schally, A. V., Huang, W. Y., Redding, T. W., Coy,
D. H., Chihara, K., Chang, R. C. C., Raymond, V. & 23. Karelin, A. A., Philippova, M. M., Karelina, E. V. &
Ivanov, V. T. (1994) Biochem. Biophys. Res. Com-Labrie, F. (1978) Biochem. Biophys. Res. Commun.
82, 582588. mun. 202, 410415.
24. Glamsta, E.-L., Meyerson, B., Silberring, J., Tere-3. Chang, R. C. C., Huang, W.-Y., Redding, T. W., Ari-
mura, A., Coy, D. H. & Schally, A. V. (1980) Bio- nius, L. & Nyberg, F. (1992) Biochem. Biophys. Res.Commun. 184, 10601066.chem. Biophys. Acta 625, 266273.
4. Takagi, H., Shiomi, H., Ueda, H. & Amano, H. 25. Maekinen, K. K., Sewon, L. & Maekinen, P. L.
(1996) J. Periodont. Res. 31, 4346.(1979) Nature 5737, 410412.
5. Fukui, K., Shiomi, H., Takagi, H., Hayashi, K., Kiso, 26. Glamsta, E.-L., Marclund, A., Hellman, U., Wern-
stedt, C., Terenius, L. & Niberg, F. (1991) Regul.Y. & Kitagawa, K. (1983) Neuropharmacology 22,
191196. Peptides 34, 169179.
27. Lantz, I., Glamsta, E.-L., Talback, L. & Nyberg, F.6. Brantl, V., Gramsch, Ch., Lottspeich, F., Mertz, R.,
Jaeger, K.-H. & Herz, A. (1986 ) Eur. J. Pharmacol. (1991) FEBS Lett. 287, 3941.
28. Barkhudaryan, N., Oberthuer, W., Lottspeich, F. &125, 309310.
7. Piot, J.-M., Zhao, Q., Guillochon, D., Ricart, G. & Galoyan, A. (1992) Neurochem. Res. 17, 1217
1221.Thomas, D. (1992) FEBS Lett. 299, 7579.
8. Zhao, Q., Garreau, I., Sannier, F. & Piot, J.-M., this 29. Nishimura, K. & Hasato, T. (1993) Biochem. Bio-
phys. Res. Comm. 194, 713719.issue.
9. Piot, J.-M., Zhao, Q., Guillochon, D., Ricart, G. & 30. Ivanov, V. T., Karelin, A. A., Mikhaleva, I. I.,Vaskovsky, B. V., Sviryaev, V. I. & Nazimov, I. V.Thomas, D. (1992) Biochem. Biophys. Res. Com-
mun. 189, 101110. (1993) Soviet J. Bioorg. Khim. 18, 677702.
31. Dagouassat, N., Garreau, I., Zhao, Q., Sannier, F. &10. Mito, K., Fujii, M., Kuwahara, M., Matsumura, N.,
Shimizu, T., Sugano, S. & Karaki, H. (1996) Eur. Piot, J.-M. ( 1996) Neuropeptides 30, 15.
32. Galoyan, A. A., in issue.J. Pharmacol. 304, 9398.
11. Ueda, H., Shiomi, H. & Takagi, H. (1980) Brain 33. Erchegyi, J., Kastin, A. J., Zadina, J. E. & Qiu,
X.-D. (1992) Int. J. Peptide Protein Res. 39, 477Res. 198, 460464.
12. Vaskovsky, B. V., Ivanov, V. T., Mikhaleva, I. I., 484.
34. Blishchenko, E. Yu., Mernenko, O. A., Mirkina, I. I.,Kolaeva, S. G., Kokoz, Yu. M., Svieryaev, V. I., Zi-
ganshin, R. H., Sukhova, G. S. & Ignatiev, D. A. Satpaev, D. K., Ivanov, V. S., Tchikin, L. D., Ostrov-
sky, A. G., Karelin, A. A. & Ivanov, V. T. ( 1997)(1990) in Peptides, Chemistry, Structure and Biol-
ogy, Rivier, J. E. & Marshall, G. R., Eds., Leiden, Peptides 18, in press.
35. Glamsta, E.-L., Marclund, A., Lantz, I. & Nyberg,ESCOM, pp. 302 304.
13. Blishchenko, E. Yu., Mernenko, O. A., Yatskin, F. (1993) Regul. Pept. 34, 918.36. Nyberg, F., Sanderson, K. & Glamsta, E.-L., thisO. N., Ziganshin, R. H., Philippova, M. M., Karelin,
A. A. & Ivanov, V. T. (1996) Biochem. Biophys. issue.
37. Barkhudaryan, N., Kellerman, J., Lottspeich, F. &Res. Commun. 224, 721727.
14. Zhu, Y. X., Hsi, K. L., Chen, Z. G., Zhang, H. L., Galoyan, A. A. (1991) Neirokhimiya 10, 146154
(in Russian).Wu, S. X., Zhang, S. Y., Fang, P. F., Guo, S. Y., Kao,
Y. S. & Tsou, K. (1986) FEBS Lett. 208, 253 257. 38. Fonina, L. A., Guryanov, S. A., Nazimov, I. V., Ya-
novsky, O. G., Zakharova, L. A., Mikhaylova,15. Karelin, A. A., Philippova, M. M. & Ivanov, V. T.
(1995) Peptides 16, 693 697. A. A. & Petrov, R. V. (1991 ) Dokl. A. N. SSSR 319,
755757 (in Russian).16. Ziganshin, R. H., Svieryaev, V. I., Vaskovsky, B. V.,
8/3/2019 ivanov 1997
18/18
188 Ivanov et al.
39. Petrov, R. V., Mikhailova, A. A. & Fonina, L. A., 49. Kuznik, R.I., Morozov, V.G. and Khavinson, V.Kh.
this issue. (1995), Uspekhi sovremennoi biologii 115, 353-36740. Malukova, I. V., Zakharova, L. A. & Metaxa, E. E. (in Russian).
(1996) Biokhimiya 61, 440444 (in Russian). 50. Stamler, J. S. (1996) Nature 380, 108111.41. Ivanov, V. T., Karelin, A. A., Karelina, E. V., Ulya- 51. Bennett, H. P. J., Chang, R. Y., Nelbach, L. & Adel-
shin, V. V., Vaskovsky, B. V., Mikhaleva, I. I., Nazi- son, J. W. ( 1990) Regul. Peptides 29, 241250.mov, I. V., Grishina, G. A., Khavinson, V. Kh., Mor-
52. Julliard, J. H., Shibasaki, T., Ling, N. & Guillemin,ozov, V. G. & Mikhaltsov, A. N. (1992 ) in Peptides.
R. (1980) Science 208, 183185.
Chemistry and Biology, Smith, J. A. & Rivier, J. E., 53. Najjar, V. A. (1983) Ann. NY Acad. Sci. 419, 111.Eds., Leiden, ESCOM, pp. 939941.54. Zavjalov, V. P., Zaitseva, O. R., Navolotskaya, E. V.,42. Ziganshin, R. H., Mikhaleva, I. I., Ivanov, V. T., Ko-
Abramov, V. M., Volodina, E. Yu. & Mitin, Yu. V.koz, Yu. M., Alekseev, A. E., Korystova, A. F., Mav-(1996) Immunol. Lett. 49, 2126.lyutova, D. A., Emelyanova, T. G. & Akhrimenko,
55. Cochrane, D. E., Carraway, R. E., Feildberg, R. S.,A. K. (1995) in Peptides. Chemistry, Structure and
Biology, Kaumaya, P. T. P., & Hodges, R. S., Eds., Boucher, W. & Gelfand, J. M. (1993) Peptides 14,
Mayflower Scientific Ltd., pp. 244245. 117123.43. Slemmon, J. R., Hughes, C. M., Cambell, G. A. & 56. Standker, L., Sillard, R., Bensch, K. W., Ruf, A.,
Flood, D. G. ( 1994) J. Neurosci. 14, 22252235. Raida, M., Schulz-Knappe, P., Schepky, A. G.,44. Slemmon, J. R. & Flood, D. G. (1992) Neurobiol. Patscheke, H. & Forssmann, W.-G. (1995) Biochem.
Aging 13, 649660. Biophys. Res. Commun. 215, 896902.45. Slemmon, J. R., this issue.
57. Ohmori, T., Nakagami, T., Tanaka, H. & Maruyama,46. Ohyagi, Ya., Yamada, T. & Goto, I. (1994) Brain
S. (1994) Biochem. Biophys. Res. Commun. 202,
Res.635,
323327. 809815.47. Pivnik, A. V., Rasstrigin, N. A., Philippova, M. M.,58. Fermi, G., Perutz, M. F., Shaanan, B. & Fourme,Karelin, A. A. & Ivanov, V. T. (1996) Leukemia
R. J. (1984) J. Mol. Biol. 175, 159174.Lymphoma 16, 179184.59. Jones, T. A. (1978) J. Appl. Cryst. 11, 268278.48. Brittain, T. & Greenwood, C. (1982) Biochem. J.60. Evans, S. V. (1993) J. Mol. Graphics 11, 134138.201, 153159.