184

Biochimica et Biophysica Acta, 418 (1976) 184--194

© Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands

BBA 98485 I S O L A T I O N AND C H A R A C T E R I Z A T I O N OF TWO A L K A L I N E RIBONUCLEASES FROM C A L F SERUM

RICHARD G. VON TIGERSTROM and JANET M. MANCHAK Department of Microbiology, University of Alberta, Edmonton, Alberta, T6G 2E9 (Canada)

(Received August 5th, 1975)

S u m mar y T r e a t m e n t o f calf serum at 60°C and pH 3.5 followed by c h r o m a t o g r a p h y on c a r b o x y m e t h y l (CM) cellulose resulted in the separation of t w o major peaks o f alkaline RNAase activity. One was eluted from CM-cellulose at 0.075 M KC1 with an overall purification of 5400-fold and the ot her was eluted at 0.25 M KC1 with a 6700-fold purification. The RNAase eluted from CM-cellulose at 0.075 M KC1 was almost com pl e t e l y inhibited by anti-RNAase A serum and by the endogenous RNAase inhibitor and a 33% inhibition was observed in the presence o f 5 mM MgC12. This e n z y m e seems to be similar or identical to RNAase A. The o t h e r RNAase, eluted f r om CM-cellulose at 0.25 M KC1 was n o t inhibited by anti-RNAase A or 5 mM MgC12 and was m uch less sensitive to the endogenous inhibitor. Both enzymes degraded RNA endonucleolytically and the nucleoside m o n o p h o s p h a t e s obtained after partial hydrolysis of RNA by the two serum RNAases were primarily 2'- or 3'-CMP and 2'- or 3'-UMP. Poly(A), native DNA and d e n a t u r e d DNA were degraded slowly or n o t at all. The RNAase A-like e n z y m e degraded poly(C) at a significantly faster rate, and p o l y (U) at a slower rate, than RNA. However, the o t h e r serum RNAase was m o r e active with poly(U) than with RNA and almost inactive w i t h p o l y ( C ) a s the substrate.

Introduction Mammalian tissues and b o d y fluids contain a n u m b e r o f di fferent RNAdegrading enzymes. Their properties, intracellular distributions and possible functions have been reviewed in a n u m b e r of articles [ 1 - - 4 ] . Som e progress has been made recently in elucidating the specific roles of several of these enzymes in RNA metabolism [ 5 - - 1 0 ] . However, the physiological functions of many o t h e r RNA-degrading enzymes, including alkaline RNAase II, are still uncertain.

185 Alkaline RNAase II is an enzyme(s) with physical and catalytic properties similar to those of pancreatic RNAase [3,4]. It is widely distributed in animal cells and in most cells it is associated with an endogenous inhibitor [11--14] which seems to be specific for alkaline RNAase II [15]. Both the endogenous inhibitor activity and alkaline RNAase II activity may vary greatly depending on the tissue or fluid investigated [16] and the activities may change in one particular tissue after administration of cytostatic agents [17--19] or after starvation of the animal [20]. It was proposed that the ratio between the endogenous inhibitor and the alkaline RNAase II activity might reflect the physiological state of a particular cell type by determining the rate of RNA catabolism and, in turn, the rate of protein synthesis [21--24]. However, the growth rates of several hepatomas [25] and the rate of protein synthesis in Ehrlich ascites cells [19], did not seem to be affected by a change in the inhibitor/RNAase ratios. Animal sera contain an RNAase specific for double-stranded RNA [26] and other, relatively non-specific RNAases [27,28]. Our primary investigations showed that an alkaline RNAase II-type activity also was present in calf serum which we were using in a cell culture medium for growth of Ehrlich ascites cells. In contrast to the cells grown intraperitoneally, these cultured Ehrlich ascites cells had very low alkaline RNAase II activity (von Tigerstrom, R.G. and Manchak, J.M., unpublished). We were interested in determining whether this low alkaline RNAase H activity of cultured cells might be due to contamination of the cells with calf serum RNAase from the medium. Therefore, we have partially purified and characterized the alkaline RNAases of calf serum to compare them with the alkaline RNAase of Ehrlich ascites cells. This report describes the purification and some of the properties of two major acid- and heat-stable alkaline RNAases. A comparison of the calf serum enzymes with the alkaline RNAases of Ehrlich ascites cells will be the subject of a later report. Materials RNAase A and crude snake venom (Russell's Viper) were purchased from Worthington Biochemical Corp. and Calbiochem, respectively. Calf serum was obtained from Morse's Biological Supplies. Microgranular carboxymethyl cellulose (CM-52) was purchased from Whatman, DEAE-cellulose from the Sigma Chemical Company and Sephadex G-75 superfine from Pharmacia. Highly polymerized RNA from yeast was the product of Calbiochem; poly(C), poly(U) and poly(A) were from Miles Laboratories and calf thymus DNA was from Worthington Biochemical Corp. Methods

Enzyme assays. Alkaline RNAase activity was routinely determined in 0.08 ml of 16 mM potassium phosphate, 18 mM Tris/acetate, 1 mM Na2 EDTA, 1 mg/ml RNA (pH 7.8), using 0.02 ml of enzyme appropriately diluted with 0.14 M NaC1, 10 mM Tris/HC1, 4 mM sodium phosphate, 0.1% gelatin (pH 7.4). Samples were taken at four different time intervals for each assay except when RNAase activities were determined in fractions eluted from columns in

186 which case only one time interval was used. The reaction were terminated by the addition of 0.1 ml 8% perchloric acid and the acid-soluble nucleotides were determined spectrophotometrically after chilling the reaction mixtures in ice for 20 min. The same procedure was carried out when poly(C), poly(A}, native DNA or heat, denatured DNA was the substrate. The activity of RNAase on poly(U) was determined in a similar way except the reactions were terminated with 0.1 ml 8?0 perchloric acid containing 0.0625% uranyl acetate. This activity was compared to the activity obtained with RNA as the substrate, when the reactions were also terminated with perchloric acid containing uranyl acetate. The RNAase activity at acid pH was determined in identical reaction mixtures except the pH was adjusted to 5.5 with acetic acid. With RNA as the substrate, one unit of enzyme is defined as the a m o u n t which will produce 1 pmol of acid-soluble nucleotides per min. The extinction coefficient of the acid-soluble products from RNA was assumed to be 11 000 [29]. Purification o f alkaline RNAase from calf serum. 50 ml of calf serum were diluted with an equal a m o u n t of distilled water, adjusted to pH 3.5 with M H2 SO4 and kept at 60°C for 20 min. After cooling, a further 100 ml of water were added and the diluted, heat-treated serum was centrifuged at 16 300 X g for 20 min at 0--4 ° C. All subsequent manipulations were also carried out at 0--4°C. The supernatant was adjusted to pH 6.6 with M KOH. After a second centrifugation at 16 300 X g to remove a small a m o u n t of precipitate the supernatant (165 ml) was diluted with an equal volume of water to reduce the conductivity from 3.2 mMHO to 1.6 mMHO. It was then applied to a 2.5 X 19 cm CM-52 cellulose column equilibrated with 50 mM potassium phosphate, I mM EDTA (pH 6.6). The column was washed with 100 ml of the same buffer and the enzymes were eluted with a linear salt gradient prepared from 250 ml 50 mM potassium phosphate, 1 mM EDTA (pH 6.6) and 250 ml 50 mM potassium phosphate, 1 mM EDTA, 0.6 M KC1 (pH 6.6). Fractions (5.15 ml) were collected at a flow rate of approx. 0.5 ml/min. Gelatin was added to each fraction (to 0.01%} before RNAase assays were carried out or dilutions were made into buffer containing gelatin. The fractions containing RNAase activity were pooled, dialysed against 1 mM potassium phosphate, 0.1 mM EDTA (pH 7.4), freeze-dried and dissolved in one-tenth the original volume using 0.14 M NaC1, 10 mM Tris/HC1, 4 mM sodium phosphate, 0.1% gelatin (pH 7.4}. Gel filtration. Each enzyme solution, approx. 2 ml, was applied to Sephadex G-75 superfine (2.5 X 39.3 cm) equilibrated with 50 mM potassium phosphate, 0.1 M KC1 (pH 7.8), and eluted with the same buffer at a hydrostatic pressure of 37 cm. Blue dextran and (NH4)2 SO4 were used as marker compounds and 1.98-ml fractions were collected. BNAase activities were determined and the partition coefficients (Kav) for the enzymes were calculated as suggested in the brochure supplied by Pharmacia. Preparation o f anti-RNAase A. Anti-BNAase A serum from rabbits was prepared by Dr. G. Stemke, Department of Microbiology, University of Alberta. Each animal received 1 mg of RNAase A (bovine pancreatic RNAase) in complete Freund's adjuvant initially and one week after the first injection, and th.en i mg of RNAase A in incomplete Freund's adjuvant 3 and 5 m o n t h s after the first injection. The animals were bled 11 days after receiving the last injec-

187 tion of antigen. A 1/7500 dilution of the crude antiserum inhibited RNAase (5 • 10 -3/~g/ml) approx. 50%. The antiserum was subjected to gel filtration using a column of Sephadex G-75 superfine (2.5 X 39.3 cm) to remove any RNAase activity. 4 ml of antiserum were applied to the column equilibrated with 50 mM potassium phosphate, 0.1 M KC1 (pH 7.8) and then eluted with the same buffer. Fractions of approx. 2.16 ml were collected and the anti-RNAases A activity was obtained in fractions 38--56 and coincided with the early part of the major protein peak. Alkaline RNAase activity was eluted as a broad peak in fractions 64--90• Fractions 40--55 were pooled, adjusted to pH 7.0 and stored a t - - 2 0 ° C . They contained 4.2 mg protein/ml. The partially purified anti-RNAase A at final dilutions of 1/100 inhibited a 5 • 10 -3 pg/ml solution of RNAase A 98%. Preparation of endogenous RNAase inhibitor. Ehrlich ascites cells grown in suspension in Eagles' minimal essential medium + 10% (v/v) calf serum have low alkaline RNAase II and high RNAase inhibitor activity (yon Tigerstrom, R°G. and Manchak, J.M., unpublished). They were used as a source of the inhibitor and it was prepared as previously reported [19]. 4 pl of the inhibitor preparations inhibited 5 • 10 -3 pg RNAase A in 1 ml approx. 60%. Partial degradation of RNA and isolation of products. To obtain R N A degradation products the reaction mixture for the assay of alkaline RNAase was used with 2 mg/ml instead of 1 mg/ml RNA. An 8-ml reaction mixture was incubated at 37°C for 18 h with an amount of RNAase from peak I or peak II which would have activity equivalent to 0.25 • 10 -1 pg RNAase A. The reactions were terminated by acidification to pH 1 with 6 M HC1. The mixtures were then incubated at 37°C for 60 min to open any cyclic phosphates produced during the enzymatic hydrolysis. After adjusting the pH to 8, the products were isolated by DEAE-cellulose chromatography using an NH4 HCO3 gradient [30]. The initial, tentative identification of low molecular weight products was made according to the points in the salt gradient at which they were eluted from DEAE cellulose and their ultraviolet spectra at acid and alkaline pH. Further identification of nucleoside monophosphates was carried out by descending paper chromatography of the products before and after treatment with crude snake venom [3] using isobutyric acid/M NH4 OH (50 : 30) as the solvent system• Analytical. Protein determinations were carried o u t according to the method of Lowry et al. [32] using bovine serum albumin as the standard and by determining the absorbance at 280 nm. Ammonia was determined with Nessler's reagent {Fisher Scientific) and by measuring the absorbance at 490 nm. Results

Purification of alkaline RNAase To determine total alkaline RNAase II activity in extracts of Ehrlich ascites cells, liver and other tissues, we normally carry out an acid treatment and a heat treatment [3,19]. These treatments inactivate the endogenous inhibitor and other RNAases that might interfere with the assay of alkaline RNAase II. They also allow for the specific determinations of acid RNAase II

188

activity which is due to acid-stable, but heat-labile, enzymes [ 3 , 3 3 ] . Our initial work with calf serum indicated that it contained little or no endogenous inhibitor and acid RNAase II activity and that the usual acid treatment did not result in a significant purification of the alkaline RNAase activity. Therefore, the acid treatment was omitted and the serum was just heat treated at pH 3.5 as described in the Methods. The heat treatment resulted in a 23-fold purification of alkaline RNAase II activity. The heat-treated serum was then chromatographed on a carboxymethyl cellulose column. The elution profile is shown in Fig. 1. Two major peaks of alkaline RNAase activity, peak I (fractions 46--52) and peak II (fractions 68--74) were obtained. It was found that bovine pancreatic RNAase (RNAase A) was eluted from the same column at a position identical to that of peak I. The overall purifications obtained for the peak I and peak II enzymes were 5400 and 6 7 0 0 fold, respectively. A summary of the purification is given in Table I. On some occasions we first applied the heat-treated calf serum to a column of Bio Rex 70 (2.5 × 18 cm) and eluted the RNAase activities in batches with M KCI, 50 mM potassium phosphate, 1 mM EDTA (pH 7.2). This resulted in a significant purification and the active fractions were then subjected to ion-exchange chromatography using CM-52 cellulose, as described above. Gradient elutions from Bio Rex 70 was also tried but resulted in poorer resolution of the RNAases than that obtained with carboxymethyl cellulose. Adsorption of the RNAase to Bio Rex 70 was not routinely done since it did not increase the overall purifications significantly. However, a preliminary adsorption of the RNAases to Bio Rex 70 may have an advantage in a large-scale purification

2.0



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Fig. I. C a r b o x y m e t h y l cellulose cl~omatography of calf serum R N A a s e . Calf serum (50 ml) was heat treated at p H 3.5 and diluted as outlined under M e t h o d s and applied to a 2.5 × 19 c m C M - 5 2 cellulose column. 3 1 2 m l of effluent with an absorbance of 0.3/ml at 2 8 0 n m were collected before fraction n u m b e r 1. This effluent contained negligible R N A a s e activity. T h e c o l u m n w a s then eluted with 100 m l 50 m M potassium phosphate. 1 m M EDTA ( p H 6.6) and with a linear gradient of 2 5 0 m l 50 m M potassium phosphate. 1 m M EDTA ( p H 6.6) and 2 5 0 m l 50 m M potassium phosphate, 1 m M E D T A , 0.6 M K C I ( p H 6.6). Fractions containing 5.15 m l were collected and the R N A a s e activity, the absorbance at 2 8 0 n m and the K C I concentration were determined.

189 TABLE I

PURIFICATION O F C A L F S E R U M RNAases Step

Total Activity

Protein (mg)

Spec. Act. (units/mg)

Purification

Yield

(fold)

(%)

1 23

100 72

(units) 1. S e r u m 2. H e a t - t r e a t m e n t 3. CM-52 cellulose peak I p e a k II

77 55.3 26.5 14.1

4700 148.5 0.46 0.37

0.016 0.37 57.6 38.1

5400 * 6700 *

34 18

* C a l c u l a t e d b y assuming that 65% of t h e a c t i v i t y in s e r u m w a s d u e to p e a k I a n d 35% due to peak II enzyme.

because of the higher exchange capacity of Bio Rex 70 compared to CM-52 cellulose.

Sephadex G-75 filtration Samples of RNAase A and the RNAases obtained in peak I and peak II of the CM-52 column were subjected to gel filtration as described under Methods. The partition coefficients (Kay) for RNAase A and the enzymes in peak I and II were 0.695, 0.718 and 0.832 respectively. Thus, the RNAase in peak I was eluted at a position very similar to that of RNAase A. The RNAase in peak II, however, was eluted from Sephadex G-75 significantly later. Whether this is due to a difference in the molecular weight of the two enzymes remains to be determined by other methods. Inhibition of calf serum RNAases by anti-RNAase A and the endogenous inhibitor o f alkaline RNAase H To investigate the relationship between the two major alkaline RNAases that we obtained from CM-52 cellulose chromatography of calf serum and between these enzymes and RNAase A, we studied the inhibition of these enzymes by anti-RNAase A and the endogenous inhibitor. The enzyme concentrations of the calf serum RNAases were adjusted by dilution into 0.14 M NaCI, 10 mM Tris/HC1, 4 mM sodium phosphate, 0.1% gelatin (pH 7.4), so t h a t each preparation had an activity approximately equivalent to that obtained with 5 • 10 -3 #g/ml RNAase A. Anti-RNAase A dilutions were prepared in the same buffer solution. Residual RNAase activity was determined after incubation of the enzyme preparations with appropriate dilutions of the antibodies for 15 min at 37°C. The effects of anti-RNAase A on the activities of RNAase A and the calf serum RNAases is shown in Table II. The endogenous inhibitor of alkaline RNAase II was prepared from Ehrlich ascites cells as described under Methods, and appropriate dilutions of inhibitor were added to the enzyme preparations. The remaining RNAase activity was determined after incubation of the RNAase-inhibitor mixtures for 10 min at 22 ° C. The effects of the inhibitor on RNAase A and the calf serum RNAase activities are shown in Table III. Calf serum RNAase activity in peak I is inhibited by anti-RNAase A

190

TABLE II INHIBITION

O F R N A a s e A A N D C A L F S E R U M R N A a s e BY A N T I - R N A a s e

A

Partially p u r i f i e d a n t i - R N A a s e A s e r u m w a s a d d e d t o t h e R N A a s e s o l u t i o n s , t h e a c t i v i t i e s w e r e d e t e r m i n e d a f t e r 1 5 rain at 3 7 ° C a n d t h e % i n h i b i t i o n s w e r e c a l c u l a t e d a n d t a b u l a t e d . M i n u s v a l u e s i n d i c a t e activities higher than control values. Anti-RNAase A (final dilution)

RNAase A

Calf s e r u m R N A a s e peak I p e a k II

1

1

1

1O0

SO~

70~

0

95 98

78 80

73 --

0 0

86 --3.6 4.0

----

63 --17 20

0 0 0

antibodies and by the endogenous RNAase inhibitor to approximately the same extent as RNAase A. Therefore, it is an enzyme similar, if not identical, to RNAase A. The calf serum RNAase activity eluted in peak II, however, was unaffected by anti-RNAase A and was less sensitive to the RNAase inhibitor. These results, in addition to those obtained from ion-exchange chromatography and gel filtration, show that calf serum contains two major acid- and heat-stable alkaline RNAases and one of these may be identical to RNAase A.

Effect o f p H and Mg 2÷ concentration on purified calf serum RNAases Our preliminary work had indicated that crude calf serum contained little, if any, acid RNAases and that the RNAase activity in crude and heat-treated serum was not dependent on the presence of divalent cations. It was of interest, however, to verify, these initial observations with the purified enzymes. The peak I and II RNAases and RNAases A (for comparison) were assayed under three different conditions: at pH 7.8 with 1 mM EDTA (routine assay as described under Methods); at pH 5.5 with 1 mM EDTA; and at pH 7.8 without EDTA but with 5 mM MgC12. Both calf serum RNAases were 11 times more active, and RNAase A 17 times more active, at pH 7.8 that at pH 5.5 in the TABLE III INHIBITION ITOR

OF

RNAase

A AND

CALF

SERUM

RNAases BY THE ENDOGENOUS

RNAase INHIB-

T h e i n d i c a t e d v o l u m e s o f t h e R N A a s e i n h i b i t o r w e r e a d d e d t o 1 m l o f t h e R N A a s e s o l u t i o n s , t h e rem a i n i n g a c t i v i t i e s w e r e d e t e r m i n e d a f t e r 1 0 rain at 2 2 ° C a n d t h e % i n h i b i t i o n s w e r e c a l c u l a t e d a n d tabu l a t e d . T h e m i n u s v a l u e i n d i c a t e s a n a c t i v i t y h i g h e r t h a n t h e c o n t r o l values. R N A a s e i n h i b i t o r (/~1) 0 RNAase A Calf s e r u m R N A a s e peak I p e a k II

4

0

63

0 0

60 --10

8

16

40

81

92

--

83 1.3

91 20

-60

191

presence of EDTA. The addition of 5 mM MgC12 inhibited the peak I enzyme and RNAase A 33%. A slight, perhaps insignifi.cant, stimulation was observed with the peak II enzyme in the presence of MgC12. These results show that both calf serum RNAases have alkaline pH optima and they are further evidence that the peak I RNAase, but not the peak II RNAase, is similar or identical to RNAase A.

Type of hydrolytic cleavage and substrate specificity RNAase activity was routinely determined by measuring the release of nucleotides soluble in 4% perchloric acid from RNA. To determine whether the enzymes degraded RNA endo- or exonucleolytically, duplicate reactions were carried out for each enzyme. In one we measured the release of nucleotides soluble in 4% perchloric acid; in the other we measured the release of nucleotides soluble in 4% perchloric acid 0.5% uranyl acetate. The ratio of the two is equal to one if the enzyme is an exonuclease but greater than one if it is an endonuclease or a mixture of exo- and endonucleases [19,34]. The ratios obtained for the calf serum RNAases of peaks I and II were 4.3 and 7.0 respectively, clearly indicating that both are endonucleases. The difference in the ratios obtained for the two enzymes also indicated that they have different specificities: the peak I enzyme being less restricted that the peak II enzyme. Further evidence that the two enzymes differ significantly with respect to their specificities was obtained by measuring the hydrolytic activity of the enzymes with different substrates (Table IV). In each case the activity obtained with RNA was called 100. T h e peak I RNAase degrades poly(C) more readily than RNA and shows relatively low activity with poly(U). The peak II RNAase, on the other hand, hydrolyses poly(U) faster than RNA and has negligible activity with poly(C). Poly(A), native DNA and denatured DNA are poor substrates for both enzymes. We also wanted to study the degradation products released by the two calf serum enzymes and determine whether the nucleoside monophosphates contained phosphate at the 3' or at the 5' position. The partial degradation of RNA with the calf serum enzymes and with RNAase A was carried o u t and the products were isolated and identified as described under Methods. Products which might be expected to be nucleosides were n o t detected. The nucleoside monophosphates (elution from DEAE-cellulose up to 0.1 M NH4HCO3) obT A B L E IV A C T I V I T I E S OF C A L F SERUM RNAase WITH D I F F E R E N T S U B S T R A T E S

T h e activities o b t a i n e d w i t h R N A as the substrate were arbitrarily assigned a v a l u e o f 1 0 0 . Substrate

RNA poly(C) poly(U) poly(A) Native D N A Denatured DNA

Rates of Hydrolyses Peak I

Peak II

100 143 16 2.4 ~0.4 ~0.8

100 2.8 140 1.4 ~1.0 ~I.0

192 tained from the reactions were identified as CMP and UMP. Degradation of RNA by the peak I enzyme yielded CMP and UMP in approximately equal amounts, whereas the peak II enzyme produced two times more UMP than CMP. One product, which was tentatively identified as a dinucleotide containing only cytidine, was isolated from the hydrolysate with the peak II enzyme but not when the peak I enzyme was used. The products obtained from the digests of RNA using RNAase A and the peak I enzyme appeared to be very similar. All reaction mixtures yielded only very small amounts of ultravioletabsorbing material which might be expected to be AMP and GMP. These results are in general agreement with the substrate specificities of the two calf serum enzymes determined with the synthetic polynucleotides as substrates. The cytidine and uridine monophosphates obtained from DEAE-cellulose chromatography were concentrated by lyophillization and further characterized by paper chromatography. They could be cochromatographed with 2'(3')CMP and 2'(3')-UMP respectively, and were not converted to nucleosides by t r e a t m e n t with snake venom. Therefore, both enzymes degrade RNA to produce products with 3'-phosphate groups, probably with 2',3'-cyclic phosphate intermediates. Discussion Calf serum is widely used as a c o m p o n e n t of cell and tissue culture media and its constituents may be adsorbed to cell surfaces or be ingested by the cells. In determining the a m o u n t of enzyme produced by cells grown in culture it is essential to know whether an enzyme is produced by the cells or whether it has been derived from the medium. This is especially true when studying RNAases since some are small and stable proteins. Our primary objective was to characterize acid- and heat-stable calf serum RNAases to be able to differentiate their activities from the activities of similar RNAases produced by cells growing in cell culture. RNAase activity in animal plasma was reported as early as 1945 [27]. A detailed characterization of the activities was not possible at that time. More recently (1970), an RNAase specific for double-stranded RNA was detected by Stern [26] in a number of animal sera. Upon chromatography of crude bovine serum on Sephadex G-200, Stern located two peaks of what he called RNAase A-like activity in addition to the double-strand-specific RNAases. It is most likely that these are the same two RNAases that we have purified by the heat treatment and ion-exchange chromatograph of calf serum. In addition to the two major activities (peak I and peak II) a small peak of activity was eluted from CM-52 cellulose between the two major peaks when some batches of serum were used. Although we have used calf serum for the isolation and characterization of these enzymes, similar activities seemed to be present in foetal calf serum and serum of mature animals. The a m o u n t of acid- and heat-stable RNAase activity and the proportion of the two major activities may vary between different batches of serum. The reasons for this have n o t been investigated. The two calf serum RNAases in peaks I and II resemble RNAase A in t h a t they are optimally active at alkaline pH, are acid- and heat-stable, degrade RNA

193 endonucleolytically by hydrolyzing the bond between the phosphate and the 5' position of the adjacent nucleoside and seem to be relatively small proteins as evidenced by their elution from Sephadex G-75. Our results clearly indicate that the RNAase peak I is very similar, if not identical, to bovine pancreatic RNAase. The RNAase peak II, however, differs from RNAase A in several respects. It is a much more basic protein, it is not inhibited by anti-RNAase A antibodies, it seems to have a low affinity for the endogenous RNAase inhibitor and it has a different substrate specificity, especially with respect to hydrolysis of poly(C). The fact that this enzyme is eluted from Sephadex a significant period after RNAase A and the peak I RNAase may indicate that it has a lower molecular weight than that of RNAase A. However, since it is an unusually basic protein a non-specific interaction of the enzyme with Sephadex, even at the relatively high pH and high ionic strength, cannot be ruled out. Further characterization of this enzyme, especially an investigation of its chemical composition seems warranted. The isolation and characterization of a ribonuclease from human plasma was reported by Schmukler et al. [28]. This enzyme also seems to be a very basic protein b u t has a molecular weight of 32 000 or greater, has a high specificity for cytidylic acid residues and is strongly activated by polyamines. Although we have not tested the effect of the polyamines on the calf serum RNAases, this human plasma RNAase is clearly different from the enzymes we have isolated. Acknowledgements This work was supported by the National Cancer Institute of Canada and the 'National Research Council of Canada. We thank Dr. G. Stemke for the anti-RNAase A serum. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

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Isolation and characterization of two alkaline ribonucleases from calf serum.

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