ANALYTICAL BIOCHEMISTRY 128, 213-216 (1983)

Isolation of Bovine Seminal Ribonuclease by Affinity Chromatography

WOLFGANG K.G.KRIETSCH,FRANZC.SIMM,BARBARA HERTENBERGER, GUNTER W.K. KUNTZ, AND ELMARWACHTER

Institut ftir Physiologische Chemie, Physikalische Biochemie und Zellbiologie der Universitiit Miinchen, Goethestrasse 33, 8000 Munich 2, West Germany

Received July 8, 1982

Pyrimidine base-specific RNase was isolated from bull semen by ammonium sulfate precip nation followed by affinity chromatography with ATP- ribosyl-adipoyldihydrazo-Sepharose. The enzyme is also bound to the AMP- or CMP-Sepharose gel. The binding capacity is 7 mg RNase/ ml ATP-Sepharose. Using this procedure, a homogeneous protein with 74% yield could be isolated. The enzyme is a dimer with a molecular weight of 26,000.

Pyrimidine base-specific ribonuclease (RNase)’ from bull semen was first isolated by D’Alessio and Leone (I). The molecular weight of this dimeric enzyme was deter- mined to be 29,000 compared to 13,700 for bovine pancreatic RNase A. Probably the same enzyme with a molecular weight of 25,500 was purified and characterized from the seminal vesicles by Hosokawa and Irie (2). The carbohydrate content of the seminal plasma enzyme was evaluated to be two ga- lactosamines per mole of protein (3). The seminal vesicle enzyme was found to contain 0.2 mol amino sugar and 0.5-2.0 mol neutral sugar per mole of enzyme (2). A second py- rimidine-specific RNase in bovine seminal plasma with different properties has been described by Reddy et al. (4). However, D’Alessio et al. (5) could not confirm these findings. The most interesting aspect of the seminal plasma RNase is its tumor-specific cytostatic effect both in vitro and in vivo in animal cells (6-S). In order to study this ef- fect in detail in human cells, a pure, native enzyme must be available in adequate amounts.

’ Abbreviations used: RNase, ribonuclease; AS, am- monium sulfate; HPLC. high-performance liquid chro- matography; SDS, sodium dodecyl sulfate.

We have isolated a dimeric RNase (A4, 26,000) from seminal plasma by affinity chromatography with ATP- (or AMP- or CMP-) ribosyl-adipoyldihydrazo-Sepharose. This method is a quick and gentle procedure allowing a high RNase yield. The sequencing showed that our enzyme is definitely identical to D’Alessio’s, although small inconsistencies in amino acid sequence (the entire sequence is under investigation) and carbohydrate con- tent exist (9).

MATERIALS AND METHODS

RNase activity. The enzyme was assayed at 25°C in 50 mM sodium acetate buffer, pH 5.0, containing 0.5 mg yeast RNA/ml, ac- cording to the method of Kunitz ( 10) with one modification: the decrease in absorbance was measured at 300 nm against 320 nm in a double-wavelength spectrophotometer to allow recording in turbid solutions.

Gel electrophoresis. The purity of the RNase was checked in sodium dodecyl sul- fate (SDS)-polyacrylamide gel ( 10% acryl- amide, 0.27% bisacrylamide, or 15% acryl- amide, 0.4% bisacrylamide) with and without mercaptoethanol according to Laemmli (11). Gels were stained overnight using 0.025% Coomassie brilliant blue dissolved in meth-

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214 KRIETSCH ET AL.

anal/acetic acid/water ( 10:2: 10) and de- stained using the same solvent.

Protein determination. The protein con- centration in the seminal plasma was assayed by a modified Biuret method (12) with one- tenth of the given volume and a color-con- verting factor of 37. The protein concentra- tion of the purified native enzyme was de- termined spectrophotometrically at 278 nm with an EiTm = 4.65 (3).

AJinity gels. The periodate-oxidized li- gands ATP, AMP, adenosine, CMP, and cyti- dine were coupled to adipoyldihydrazo-Se- pharose Cl-4B, as described previously for ATP (13). For further use the gel must be washed to remove any carbohydrates, lipids, peptids, and proteins (both native and dena- turated), which are still bound to the gel. AMP and ATP gels were used a minimum of three times after regeneration. The regen- eration procedure was performed at room temperature by stirring the gel in 6 M urea containing 2% (w/v) SDS, pH 7.0, for 15 min followed by exhaustive washing with water and buffer. After suction on a Buchner fun- nel, the gels were stored as moist, crumbly cakes at 4°C with merthiolate added to pre- vent microbial growth. Before use, the gels were suspended in an appropriate buffer and were then degased.

All chemicals were of the highest purity available. They were purchased from Boeh- ringer (Mannheim, FRG), Deutsche Phar- macia (Freiburg, FRG), and Merck (Darm- stadt, FRG).

All procedures were carried out at 4°C unless otherwise stated. Buffer pH adjust- ments were made at 22°C.

RESULTS AND DISCUSSION

In order to test the affinity of RNase to the various gels, the following procedure was em- ployed. Before applying the sample, the col- umn was washed with 10 vol of 50 mM im-

idazole-HCl buffer, pH 6.6. Sixty-five mil- liliters of seminal plasma was adjusted with 0.2 M imidazole to pH 6.6 and poured onto a column (2.0 X 7 cm) containing 20 ml of affinity gel. When the optical density was lower than 0.2 at 280 nm, a linear gradient (2 X 250 ml) ranging from 0 to 2.5 M NaCl dissolved in the same buffer was used. The protein elution profile was determined by measuring the optical density at 280 nm and the enzymatic activity of RNase. Compari- son of the various gels (see Table 1) showed that under these conditions, the AMP, CMP, and ATP gels were optimal for the isolation of RNase.

Since the RNase isolated using an NaCl gradient shows some minor impurities (less than 5%) on SDS-PAGE, the procedure must be modified in the following manner to obtain the pure enzyme with high yield. Saturated ammonium sulfate (AS) solution, pH 7.8, was continuously (75 ml/h) mixed with seminal plasma to reach a final AS so- lution of 60% saturation (2.34 M ). After 30 min of stirring, this solution was centrifuged

TABLE 1

AFFINITY AND CAPACITY OF DIFFERENT GELS FOR SEMINAL PLASMA RNASE

Ligand U/ml gel

Spacer” 2.6 Cytidine 8 Adenosine 9.7 AMP 340 CMP 310 ATP 300

Percentage of AMP binding capacity

0.7 2.4 2.9

100 91 88

NaCl (M) Concentration at 50% enzyme elution

0.3 0.3 0.4 1.7 1.2 1.3

’ Adipic acid dihydrazide.

ISOLATION OF SEMINAL RIBONUCLEASE 215

for 20 min at 42,000g. The sediment was suspended in 50 ml of 2.34 M AS, stirred 3 h, and centrifuged as above. Both superna- tants were pooled and dialyzed for at least 12 h against a tenfold volume of 50 mM pi- perazine-HCl buffer, pH 5.3. This dialyzed solution was poured onto an ATP-Sepharose column (2.6 X 6 cm) at a rate of 75 ml/h. More weakly bound protein was eluted with the fivefold gel volume of 0.5 M NaCl in the piperazine-HCl buffer. Piperazine-HCI, pH 5.3, was used according to Stewart and Stev- enson ( 14) and Dixon (15) to reduce the un- specific binding to the matrix. The RNase was eluted with 1.0 M NaCl in 50 mM Tris, pH 7.8 (Fig. 1). The pooled peak fractions were desalted using a Sephadex G-25 super- fine column (3 X 70 cm). The enzyme was eluted with 20 mM ammonium hydrogen- carbonate, and was then lyophilized. A typ- ical preparation is summarized in Table 2.

In SDS-polyacrylamide gel electrophore- sis, the RNase exhibits one band with an M,

00

*is?

2.0

1.0

of 26,000 which dissociates into two identical subunits in the presence of mercaptoethanol.

Until now, the ATP-ribosyl-adipoyldihy- drazo-Sepharose has been used for the pu- rification of citrate synthase from mitochon- dria ( 16), nuclear poly(A)polymerase ( 17) myosin from rabbit muscle (18), and phos- phoglycerate kinase from different sources ( 13). A further application of this affinity gel is now the isolation of seminal plasma RNase. Not only the bovine seminal plasma RNase but also the pancreatic RNase was bound to the ATP-Sepharose although somewhat more weakly (0.8 M NaCl).

The capacity of the ATP-ribosyl-adipoyl- dihydrazo-Sepharose was 7 mg (0.25 pmol) RNase/ml compared to 5 mg (0.11 pmol) RNase/ml gel for the biospecifically-bound phosphoglycerate kinase ( 13).

In the isolation procedure used until now (2,3), the enzyme was exposed to such harsh steps as acid precipitation at pH 3.5, heat denaturation by boiling at the same pH, and

RNase D/ml]

FIG. 1. Affinity chromatography of seminal RNase. The dialyzed supematant from the AS precipitation was applied to an ATP-Sepharose column. The arrows indicate the change from protein solution to 0.5 M NaCl and to 1.0 M NaCl. The fraction volume was 8 ml. ODzso (W), RNase activity (A).

216 KRIETSCH ET AL.

TABLE 2 ACKNOWLEDGMENTS

PURIFICATION OF RNASE FROM BULL SEMINAL PLASMA

Activity

Fraction Total Specific Yield W) (U/m4 (%I

Seminal plasma Dialyzed supematant

4900 2.0 100

from the 2.34 M ammonium sulfate precipitation

After ATP-Sepharose 3850 13 19 3630 40 14

We wish to gratefully thank the Besamungsstation in Neustadt and Dr. H. Richter from the Besamungsstation in Reitmehring-Rottmoos for the bovine seminal plasma. We also thank L. Everett and R. Pery for proofing and typing the manuscript. Portions of this work have been submitted by F. C. Simm and accepted in the form of a dissertation in partial fulfillment of the requirements of the Medical School of the University of Munich. This work was supported by the Deutsche Forschungsge- meinschaft (Wa 286/3- 1).

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