Biochem. J. (1984) 218, 601-608 601Printed in Great Britain

Conformation, structure and activation of bovine cathepsin DUnfolding and refolding studies

Tamara LAH,*t Marinka DROBNI1t-KO9OROK,* Vito TURK* and Roger H. PAINt*Department of Biochemistry, Joief Stefan Institute and Faculty of Chemistry, E. Kardelj University,

Ljubljana, Yugoslavia, and tDepartment of Biochemistry, University of Newcastle upon Tyne,Newcastle upon Tyne NEJ 7RU, U.K.

(Received 22 August 1983/Accepted 17 November 1983)

Cathepsin D is found in the cell in two forms, one a single polypeptide chain (M,44000) and the other a non-covalent complex of two peptides ofMr 14000 and 30000.These correspond to the N-terminal and C-terminal regions of the single chain fromwhich they originate. It has been shown that the two forms of the enzyme are closelysimilar in secondary-structure content, in aromatic amino acid environment and indenaturation behaviour. The two-chain enzyme has half the specific activity of thesingle-chain form. The denaturation and renaturation of the single-chain cathepsin Dhas now been studied by c.d., fluorescence and enzyme activity. Activity is lost irre-versibly on unfolding, but the loss of backbone ellipticity and of folded aromaticenvironment is 75% reversible. The enzyme unfolds in two main stages, and thekinetics of these transitions indicate the existence of at least two intermediate formsbetween the native and the fully unfolded states. A further form of the enzyme existsin 0.5M-guanidinium chloride. It is characterized by having an activity 40% greaterthan that of the native state. This increase is not reversed on removing the denaturant.The similarities between cathepsin D and pepsin are discussed.

Cathepsin D (EC 3.4.23.5), purified from bovinespleen by Press et al. (1960), is a lysosomal asparticproteinase that plays a major role in intracellularprotein degradation. On extraction from bovinespleen, cathepsin D is obtained as two species eachof M, 44000, as determined by gel filtration. One isthe single-polypeptide-chain cathepsin D and theother a non-covalently associated complex consist-ing of two chains of Mr 28000-30000 and M,14000-15000 (Turk et al., 1981). Polypeptideshaving these three molecular masses are obtainedin preparations of cathepsin D isolated from avariety of tissues and species (Ogunro et al., 1980;Whitaker, 1981; Takahashi & Tang, 1981). Ratspleen cathepsin D, however, is reported to be asingle chain of M, 44000 (Yamamoto et al., 1979),whereas cathepsin D isolated from human liver isobtained only in the two-chain form (Barrett,1979).Abbreviation used: GdmCl, guanidinium chloride.$ To whom requests for reprints should be addressed

at: Department of Biochemistry, Jozef Stefan Institute,Jamova 39, Ljubljana 61 000, Yugoslavia.

Cathepsin D is formed in vivo by proteolyticcleavage of a precursor after glycosylation andphosphorylation (Erickson & Blobel, 1979; Erick-son et al., 1981). The two chain species may arise aspart of the processing of cathepsin D in the cell(Erickson et al., 1981), possibly by autolysis (Lah &Turk, 1982). Its widespread occurrence suggeststhat a region of cathepsin D that is particularlysensitive to proteolysis is a feature basic to theenzyme conformation.We have studied the conformation and

denaturation of cathepsin D in order to comparethe intact and the cleaved forms, to examine thereversibility of unfolding and to look for possibleintermediate conformers.During the course of the denaturation studies,

apparent activation of the enzyme was observedsimilar to that reported earlier (Wojtowicz &Odense, 1970). A re-investigation of this phenom-enon showed, contrary to previous suggestions,that the proteolytic activity of cathepsin D is sig-nificantly increased by the action of low concentra-tions of GdmCl or urea.

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Materials and methodsMaterialsGdmCl (AristaR grade) was from BDH Chemi-

cals, Poole, Dorset, U.K. Urea (ultra pure andcyanide-free), dansyl (5-dimethylaminonaph-thalene-1-sulphonyl) chloride, sodium dodecylsulphate, 2-mercaptoethanol, acrylamide (4 x crys-tallized), NNN'N'-tetramethylethylenediamineand ammonium persulphate were from Serva,Heidelberg, Germany, and Folin-Ciocalteaureagent was from Merck, Darmstadt, Germany.All other chemicals were of analytical grade.Sepharose CL-6B and Sephadex G-100, G-25and G-10 were from Pharmacia, Uppsala,Sweden. Pepstatin was from the Peptide Institute,Osaka, Japan. Standard marker proteins for gelfiltration were from Serva, and those for sodiumdodecyl sulphate/polyacrylamide-gel electro-phoresis from Pharmacia, Uppsala, Sweden.

Isolation of cathepsin DCathepsin D was isolated from bovine spleen(Smith & Turk, 1974; Kregar et al., 1977). Thisprocedure results in three active fractions, DI con-sisting of single-chain enzyme, and D2 and D3,each containing a mixture of single-chain and two-chain species. The two forms of cathepsin Dstudied in the present work are the fractions Dland D2. They were dialysed against 0.1 M-NaCl/lOmM-Mes (4-morpholine-ethanesulphonicacid)/NaOH buffer, pH6.6, and concentrated byultrafiltration. Cathepsin D concentration wasdetermined by using the value of A0 1 == 1.05 at280nm.

Separation of the light chain (Mr 14000) fromcathepsin fraction D2

Cathepsin D2 was denatured in 5mM-Mesbuffer, pH6.6, containing 2.5M-GdmCl and frac-tionated on a column (63cm x 1.5 cm) of SepharoseCL-6B, equilibrated with the same buffer/GdmClsolution.

C.d.Ellipticity values are reported as mean-residue

ellipticities [O]m.r.w. calculated by using a mean resi-due Mr value of 110. Cells were 1 cm for the nearu.v. and 0.05cm or 0.01 cm for the far u.v. region.The spectral bandwidth was set at 1 nm (near u.v.)and 2nm (far u.v.). Measurements were made atroom temperature on a Jobin-Yvon dichrographIII standardized with epiandrosterone (Jobin-Yvon, Longjumeau, France).

Fluorescence

Fluorescence was measured at 25°C with aHitachi Perkin-Elmer MPF3 spectrofluorimeter.

The excitation wavelengths were 278nm and285nm and the excitation bandwidths 4nm and7nm for measurements of spectra and unfoldingrespectively. First-order rate constants and ampli-tudes of the fast and the slow reactions from bi-phasic processes were calculated as described byFrost & Pearson (1961).

Sodium dodecyl sulphate/polyacrylamide-gel electro-phoresis

This was performed in 12.5% acrylamide intubes and on slabs as described by Laemmli (1970).

Assay of cathepsin D activityProteolytic activity was determined with haemo-

globin as substrate in a modification of the methodof Anson (1939). Enzyme samples (5-50ul) wereincubated with 2ml of 2% (w/v) substrate solutionfor 5min at 37°C. Preincubation of substrate withdenaturant caused no change in the extent ofproteolysis on subsequent incubation with protein-ase. Refolding of partially or fully denaturedenzyme on dilution into substrate solution wasshown not to occur by measuring the time course ofproteolysis. A linear increase of trichloroaceticacid-soluble products over the first 5min was ob-served with both native and partially denaturedenzymes. In a third control enzyme was incubatedwith substrate for 5min at 37°C and denaturant orbuffer was added immediately before the additionof trichloroacetic acid. Under these conditionsdenaturant was found to have no effect on the solu-bility of the products of haemoglobin digestion.

Determination of N-terminal amino acidsThis was done by using the dansyl chloride tech-

nique of Hartley (1970) (Narita, 1970), with t.l.c.on F-1700 micro polyamide-coated t.l.c.-readyplastic sheets (Schleicher und Schiill, Dassel,Germany).

Denaturation experimentsSamples of filtered stock enzyme solution (3-

4mg of cathepsin D/ml) were diluted into solutionsof denaturant. Optical properties and enzymeactivity were measured after the mixture had stoodat 25°C for from 60min to 22 h. In kinetic experi-ments the changes in optical properties werefollowed within 30s of the addition of protein tothe denaturant solution. The decay of enzymeactivity was measured by the withdrawal ofsamples (5-50u1) from the denaturing solution. Inrenaturation experiments the denaturant was re-moved by dilution (1 :10) into buffer, by dialysis orby gel filtration on Sephadex G-10.

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ResultsComparison of cathepsins DI and D2The results of sodium dodecyl sulphate/poly-

acrylamide-gel electrophoresis on the single-chainspecies Dl (Mr 44000) and the two-chain speciesD2 confirm that the complex (M, 44000) of heavychains (Mr 29000) and light chains (Mr 14000) isheld together by non-covalent interactions only.The absence of any detectable change in the gelpattern of banding in the presence of mercapto-ethanol shows that there is little, if any, otherproteolytic nicking in either cathepsin DI orcathepsin D2. N-Terminal analysis showed thatglycine is at the N-terminus of cathepsin DI andalso of the light chain separated from species D2,confirming the position of the light chain at the N-terminal end of the cathepsin D molecule (Taka-hashi & Tang, 1981; Huang et al., 1980). In theother fraction from cathepsin D2, which contains44000-Mr and 29000-M, chains, phenylalanineand smaller amounts of valine, lysine and alaninewere found in addition to glycine, supporting thesuggestion (Lah & Turk, 1982) that the cleavage isautolytic.The specific activities of cathepsin Dl and D2

against haemoglobin at pH3.5 are 125+5 and

1.4

.- 1.21

-t1.0,E

0

E 0.8-4

Cd 0.6

X. 0.4

01-

° 0.2:>b._--

C<

1.0

0

.aE

r:

U._

0

4-o

0.8

0.6

0.4

0.2

7s

I-~0 0

00.0

i. I p,!

00

0 1 2 3 4 5 6 7[GdmClI (M)

Fig. 2. Unfolding transitions of cathepsin Dl in GdmCl:c.d. and activity

Cathepsin D was diluted into GdmCl solutions atthe stated concentrations and time-dependent reac-tions were allowed to go to completion. Values ofellipticity were normalized to the value in zero[GdmCI]. Enzyme activities were calculated relativeto the activity of native enzyme stored under thesame conditions. No loss of activity was detected fornative enzyme over similar periods of time. A,Activity; 0, ellipticity at 291 nm; 0, mean-residueellipticity at 220nm., Activity and ellipticities weremeasured as described in the Materials and methodssection.

Wavelength (nm)Fig. 1. Near-u.v. c.d. spectra of cathepsins Dl and D2Spectra were measured with a 1 cm-pathlength cellat protein concentration 1 mg/ml and 1 nm band-width. The spectra shown are those of cathepsin Dlin lOmM-Mes buffer, pH 6.6, containing no GdmCl( ), containing 2.9M-GdmCl (. ) and con-taining 6.2M-GdmCl (-----) and of cathepsin D2 inlOmM-Mes buffer, pH6.6 (- - -).

95 + 4 Anson units/mg respectively. Since cathep-sin D2 contains approx. 50% single-chain cathep-sin D, and assuming that the latter has the samespecific activity as the DI single-chain enzyme, thespecific activity of the two-chain complex isapprox. 65 units/mg or half that of the single-chainenzyme, DI.The c.d. and fluorescence spectra of cathepsins

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0

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T. Lah, M. Drobnic-Kosorok, V. Turk and R. H. Pain

Dl and D2 were compared. The far-u.v. c.d.spectra indicate a very close similarity of second-ary structure, with 4% a-helix, 50% fl-structure,18% reverse turn and 28% aperiodic structure(Provencher & Gl6ckner, 1981). Near-u.v. c.d.spectra show a general decrease in intensity forcathepsin D2 compared with species DI at theshorter wavelength end, though without loss orshift of the tyrosine and phenylalanine fine struc-ture (Fig. 1). This suggests a small change intryptophan environment or interaction, associatedwith the ILb transition (Strickland, 1974). There isno significant difference in fluorescence spectrabetween cathepsins DI and D2.

Unfolding transitions of cathepsins Dl and D2Cathepsin D exhibits two consecutive unfolding

transitions in GdmCl on the basis of static andkinetic experiments. The first is steep and occursbetween 0.5M- and 2M-GdmCl. The loss ofenzymeactivity is paralleled by loss of ellipticity at 291 nm(Fig. 2) and by a decrease in fluorescence emissionat 314nm (Figs. 3 and 4). There is also a decrease inthe ellipticity at 220nm, corresponding to 60% ofthe total change in [01220 between OM- and 6M-GdmCl (Fig. 2). The superposition of these transi-tions measured by changes in enzyme activity, aro-matic amino acid asymmetry and backbone con-formation indicates co-operative unfolding of asubstantial element of the cathepsin Dl structurein 2M-GdmCl.The molecule is not, however, completely un-

folded under these conditions, as shown by thefurther changes in ellipticity at 220nm and in

ca

._r.

U)

-~ac)U

031

300 320 340 360 380Wavelength (nm)

Fig. 3. Fluorescence emission spectra of caiFluorescence was measured with a 1 cm ctein concentrations of 0.07mg/ml. Bandwii4nm for the exciting beam at 285nm andthe emitted beam. The fluorescence emisstra shown are those of cathepsin DI in Hbuffer, pH6.6, containing no GdmCl (-taining 3.OM-GdmCl (----) and containGdmCl (......). Fluorescence is given inunits.

c)0aa) 0.6-0

:3

0.4

0.2

0 a

* . 0

*00 1 2 3 4 5 6 7

[GdmClI (M)Fig. 4. Unfolding and refolding transitions ofcathepsin Dl

followed by fluorescenceFluorescence, calculated as the emission at 314nmrelative to the emission for native enzyme, is plottedagainst increasing concentrations of GdmCl (un-folding transition) (0) and against decreasing con-centrations of GdmCl, obtained by diluting enzymefrom 3.0M-GdmCl (0). Also, intensity of fluores-cence emission at 348nm is plotted as a fraction ofchange with increasing concentration of GdmCl(El). All solutions contained 0.07mg of protein/mlin lOmM-Mes buffer, pH6.6, and were measured at25°C. Aexc. is 278nm; bandwidths are 7mm and6mm for excitation and emission respectively.

fluorescence emission at 348nm that take placebetween 2M- and 6M-GdmCl. This second transi-tion is broad and much less co-operative than is thefirst transition. The kinetics of these changes (seebelow) show them to be associated with the unfold-

400 420 ing of molecular structure.The unfolding of cathepsin D2 is closely similar

thepsin Dl to that of cathepsin Dl (Table 1). The specificell at pro- cleavage in cathepsin D2, besides decreasing the

5Snm for asymmetry of one or more tryptophan residuession spec- (Fig. 1), also renders the remaining tryptophanOmM-Mes asymmetry more sensitive to low concentrations of

), con- GdmCl. The small 'pre-transition' detected byLing 5.6M- measurements of ellipticity at 291 nm accounts forarbitrary the increased breadth and lower average midpoint

of the first transition (Table 1). Similar changes in

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Structure of cathepsin D

Table 1. Comparison of the first unfolding transition in GdmCl for cathepsins Dl and D2The range of GdmCl concentration covering each transition, the concentration of GdmCl at which 50% of thetransition has taken place (Cm) and the fraction of the total change between native and fully unfolded enzyme arelisted. Transitions were measured at pH6.6.

Cm

(M)

1.11.31.11.10.61.40.95

Percentagechange10060.10010010066100

ParameterEllipticity at 291 nmEllipticity at 220nmFluorescence at 314nmEnzyme activityEllipticity at 291 nmEllipticity at 220nmEnzyme activity

backbone ellipticity occur on denaturation ofcathepsins DI and D2 in GdmCl. Since the heavychains and light chains separate in 2.5M-GdmCl(see the Materials and methods section), theremaining structure in 2M-GdmCl must exist inone or both of the isolated chains.

Activation at low GdmCl concentrationsWhen cathepsin DI is incubated in concentra-

tions of GdmCl below the first main transition andsamples are diluted into haemoglobin substratesolution and assayed, the specific activity is foundto be significantly increased (Fig. 2). Enzyme incu-bated under the same conditions, but withoutGdmCl, maintains the native specific activity. At0.5M-GdmCl the activity was 140% of that ofnative enzyme, reaching this maximal value onlyafter 10min incubation. Controls, described in theMaterials and methods section, show that this in-crease in specific activity is not due to increasedsusceptibility ofhaemoglobin or decreased precipi-tation of substrate by trichloroacetic acid, as wassuggested for the apparent activation observed atmuch higher concentrations of denaturant(Schlamowitz et al., 1961; Wojtowicz & Odense,1970). This activation of cathepsin D is not revers-ible within the period of the assay (see theMaterials and methods section). Both near-u.v.c.d. (Fig. 2) and fluorescence (Fig. 4) are decreasedsignificantly in intensity at concentrations ofGdmCl less than 1 M. These observations suggestthat the activation of cathepsin D is associatedwith a separate conformational transition, sincethe main transition involves loss of activity (Fig.2).

Reversibility of denaturationActivity. Solutions of cathepsin D denatured in

GdmCl concentrations up to 6M were adjustedback to low or zero concentrations of GdmCl bydilution, by gel filtration and by dialysis. In no casewas any recovery of activity observed. The rate of

1.0

._

4-

0._

0

ro_- .-

en a

0 oUv. C

-,

0.5

U

0

0

0

U~~~~~~~~

I.0. -0

0 1 0 20 30 40 50 60 70Time (min)

Fig. 5. Comparison of the decay offluorescence and ofactivity for cathepsin Dl in 2.32M-GdmCI

Fluorescence emission at 314nm (M) and enzymeactivity values (0) were normalized and plottedagainst time after placing the enzyme in 2.32M-GdmCl. Activity was measured by diluting samples10-fold into haemoglobin substrate solution.Cathepsin D was 0.05mg/ml for fluorescence and0.25mg/ml for activity measurements, both at 25°C.

loss of activity on denaturation was measured bydiluting samples of enzyme unfolding in 2.32M-GdmCl into excess substrate solution. The result-ing decay curve coincides with the decrease influorescence measured in 2.32M-GdmCl over thesame time period (Fig. 5), demonstrating the lackof reversibility to native enzyme in the presence ofsubstrate. A similar experiment, but with themixture left to stand overnight before measure-ment of the activity, led to the same curve, con-

firming the complete inability of the denaturedenzyme to recover activity.

Conformation. In contrast with enzyme activity,the loss of fluorescence emission at 314nm ondenaturation was rapidly though only partiallyreversed when denaturant was removed by dilu-tion. Fig. 4 shows the values of fluorescence inten-

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Cathepsin DlCathepsin DlCathepsin DlCathepsin DlCathepsin D2Cathepsin D2Cathepsin D2

Range ofGdmCl concn.

(M)

0.8-2.00.8-2.20.5-2.30.8-2.00.2-2.11.0-2.00.6-2.1

605

T. Lah, M. Drobnic-Kosorok, V. Turk and R. H. Pain

4.)co

IC0U

*0P

06

Time (min)Fig. 6. Kinetics ofunfolding ofcathepsin Dl with GdmClat

314nmFluorescence emission at 314nm (F) was used tofollow the unfolding of cathepsin D in 1.1 M-GdmCl(M), 1.89M-GdmCl (M), 2.42M-GdmCl (0) and3.02M-GdmCl (A), each in 10mM-Mes buffer,pH 6.6. Protein concentration was 0.088 mg/ml.Results are plotted according to first-order kinetics.

sity measured between 30 and 60s after dilutionfrom 3M-GdmCl into the indicated concentrationof denaturant.

Ordered secondary structure as indicated byellipticity at 220nm is also recovered on renatura-tion, again with a half-time of less than min.Under certain conditions, the recovery of fluores-cence and far-u.v. ellipticity is followed by slowchanges with high-order kinetics, indicating aggre-gation of the partially refolded polypeptide. Extra-polation of these kinetics to zero time shows thatapprox. 75% of native fluorescence and ellipticityare recovered by rapid refolding.

Examination of the denatured enzyme bysodium dodecyl sulphate/polyacrylamide-gelelectrophoresis showed that, although a smallamount of autolytic cleavage can occur during theunfolding process even at pH 6.6, more than 90% ofthe polypeptide chain retains its covalent integrity.The amount of autolysis decreases with increasedspeed of unfolding at higher GdmCl concentra-tions. The inability of the denatured enzyme torefold is therefore not due to cleavage of covalentbonds.

0.4

Fig. 7. Unfolding ofcathepsin Dl: dependence of the rateconstants and amplitudes on GdmCl concentration

Biphasic rate plots (Fig. 6) were decomposed (Frost& Pearson, 1961) to give amplitudes (a) and first-order rate constants (b) for the fast phase and theslow phase. Amplitudes ofthe fast phase are plottedas a percentage of the total amplitude of fluores-cence change at 314nm. Rate constants (s- 1) for un-folding were calculated from fluorescence at 314nm(O and *), ellipticity at 291 nm (A and A) andellipiticity at 220nm (O and *). Open and filledsymbols refer to the fast phase and the slow phaserespectively. The lines for the rate constants areleast-squares-regression slopes for the values ob-tained from fluorescence experiments. Kineticswere measured at 250C for fluorescence and approx.22°C for c.d.

Kinetics of denaturation of cathepsin DThe progress of denaturation of cathepsin Dl

was followed by fluorescence emission at 314nm,by c.d. and by enzyme activity. The first-order rateplots are biphasic within the region of the first un-folding transition, becoming monophasic at higherconcentrations of GdmCl (Fig. 6). The rate con-stants and relative amplitudes for the two phasesare plotted as a function of GdmCl concentration(Fig. 7). The relative amplitude of the faster phaseis strongly dependent on GdmCl concentration, asare the rate constants for each phase. The values of

1984

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Structure of cathepsin D

0.1

0

0

0

0.2

0

0.3 - \0

0 10 20 30 40Time (min)

Fig. 8. Kinetics of unfolding of cathepsin Dl with GdmClmonitored by fluorescence at 348nm

Fluorescence emission at 348 nm was monitored as afunction of time for cathepsin D (0.08mg/ml) inlOmM-Mes buffer, pH6.6, containing 3.17M-GdmCI (e) or containing 4.19M-GdmCl (El). Theexperiment in 3.17M-GdmCl was also monitored at314nm for comparison (0). Results are plottedaccording to first-order kinetics.

d(logk)/d(log[GdmCl]) obtained by least-squares-regression analysis of the fluorescence results are4.4 and 5.3 for the slow phase and fast phase res-pectively.At higher concentrations ofGdmCl the fluoresc-

ence emission at 348nm changes (Fig. 3). Thekinetics of unfolding, followed at this wavelengthfrom 3M-GdmCl upwards, show biphasic first-order kinetics, the main, slower, phase of whichhas a positive dependence on GdmCl concentra-tion {d(logk)/d(log[GdmCl])= 1.5}. The fastphase has rates similar to the monophasic ratesmeasured at 314nm at the same concentrations ofGdmCl (Fig. 8).

DiscussionThe two-chain form of cathepsin D froni bovine

spleen has been shown to be very similar to the

single-chain enzyme in its conformation and in itsdenaturation behaviour. It possesses a lower butsignificant proteolytic activity. The structuralperturbation resulting from limited cleavage at arelatively highly accessible point in the intact poly-peptide chain is thus slight, and this form of theenzyme is capable therefore of having a physio-logical role, as suggested by Erickson et al. (1981).

Cathepsin D loses activity concomitantly withthe unfolding of a major part of the molecule (Figs.2 and 5). All attempts to reverse the unfolding andto recover activity, paying special attention to fac-tors that may affect the ability of a polypeptidechain to refold, including renaturation time, pres-ence or absence ofsubstrate and protein concentra-tion, have failed. In no case has activity beenrecovered.

Although the possibility that refolding is inhibit-ed by post-translational modification (Erickson etal., 1981; Hasilik & von Figura, 1981; Hasilik etal., 1982) cannot be excluded, the results are inkeeping with the fact that cathepsin D originatesfrom a precursor by proteolytic cleavage with theloss of an N-terminal peptide (Hasilik & Neufeld,1980; Erickson et al., 1981; Puizdar & Turk, 1981;Rosenfeld et al., 1982).The denaturation studies show a highly co-

operative transition between OM- and 2M-GdmClin which enzyme activity, aromatic amino acidenvironment and backbone structure are affectedin a concerted manner. The kinetics of this transi-tion are biphasic, with the fast phase taking over athigher GdmCl concentrations. Extrapolation ofthese kinetics to zero time shows that the observedphases account for the complete transition. Thisbehaviour is not due to proline isomerization(Brandts et al., 1975), since the slow phase is firstlytwo orders of magnitude slower than the rate of iso-merization of proline residues and secondly isstrongly dependent on GdmCl concentration. Thebiphasic kinetics therefore reflect the presence ofan intermediate species either on or off the unfold-ing pathway between the native state and the par-tially unfolded state that exists in 2M-GdmCl. Thefact that both phases are positively dependent onGdmCl concentration indicates that the inter-mediate species has a conformation intermediatebetween those of the native state and the partiallyunfolded state (Ikai & Tanford, 1973).At concentrations ofGdmCl higher than 2M, the

further transition shown by ellipticity at 220nmand fluorescence emission at 348nm results in lossof the remaining backbone structure and interac-tions of tryptophan residues. The kinetics areagain biphasic. Since the fast phase at 348nm issimilar in rate to the monophasic rates measured at314nm, this phase can be assigned to the change offluorescence occurring between OM- and 3M-

Vol. 218

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608 T. Lah, M. Drobnic-Kosorok, V. Turk and R. H. Pain

GdmCl (Fig. 3), i.e. to the first transition. The slowphase relates then to the subsequent changebetween 3M- and 6M-GdmCl, which characterizesthe second transition.A simple model that can reconcile the partial

reversibility to a non-native state with the kineticbehaviour is:

N - D1.D2.Uwhere D1 is transient, D2 exists at 2M-GdmCl andU is the fully unfolded protein. The denaturationbehaviour is indicative of a molecular structurethat unfolds in stages and with only partial reversi-bility. In these general properties cathepsin D issimilar to pepsin (Ahmad & McPhie, 1978; Lah etal., 1980; Privalov et al., 1981), which possesses amarked domain structure (Andreeva & Gustchina,1979).When the unfolding ofcathepsinD was followed

by measurements of proteolytic activity againsthaemoglobin, a further transition was found at lowconcentrations of denaturant (Fig. 2). Earlierreports have suggested that the autolytic activity ofpepsin may be raised in the presence of low con-centrations of urea or GdmCl (Perlmann, 1956;Blumenfeld et al., 1960), and its activation againsthaemoglobin substrate by low concentrations ofurea has been characterized (Lah et al., 1980). Theactivation of cathepsin D by GdmCl reported inthe present paper closely parallels that found forpepsin. These activated states of pepsin andcathepsin D may correspond to one of the twostates of pepsin demonstrated by Kozlov et al.(1979) at temperatures below the main thermallyinduced transition.

We thank Mrs. N. Pelicon, Mrs. M. Pregelj, Mrs. S.Kosir and Mr. R. Nicholson for technical assistance,Mrs. J. Komar for drawing the Figures and Miss M.Hamilton for typing the manuscript. We are indebted toDr. R. M. Thomas for carrying out the secondary-struc-ture analysis. This work was supported by the ResearchCommunity of Slovenia, the British Council and theScience and Engineering Research Council of GreatBritain.

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12, 155-163Andreeva, N. A. & Gustchina, A. E. (1979) Biochem.

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Frost, A. A. & Pearson, R. H. (1961) Kinetics andMechanism, 2nd edn., pp. 162-164, John Wiley andSons, New York

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Huang, J. S., Huang, S. S. & Tang, J. (1980) in EnzymeRegulation and Mechanism of Action (Mildner, P. &Ries, B., eds.), vol. 60, pp. 289-306, Pergamon Press,Oxford

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