Properties of a ribonuclease inhibitor from bovine lens

Exp. Eye Res. (1971) 12, 120-127

Properties of a Ribonuclease Inhibitor from Bovine Lens* B. J.

ORTWERTH

AND

R. J. BYRNES

Departments of Ophddm~log~ and Biochemistry, University of Missouri Medical Center. Columbia, Missouri 65201, U.S.A. (Received 7 October 1970, Boston) Aribonuoleaae inhibitor has been purified 409fold from bovine lene. It exhibits the properties of a very negatively charged protein, which suggests that it may bind to ribonuolease in place of the RNA substrate. The inhibitor requires a free sulfhydryl group for activity and remains fairly stable in the presence of sulfydryl reagents. The addition of sufficient inhibitor will completely inhibit ribonuclease, however, this inhibition can be reversed by the addition of p-mercuribenzoate with complete recovery of the original ribonuolease activity. When rats were fed a diet containing galactose, a sharp decrease was seen in the activity of the lens ribonuolease inhibitor. This occurred after about 21 days on the diet and correlated with the appearance of a nuclear cataract. This inhibitor, however, was localized almost completely in the cortex of the lens. This indicates that the inhibitor wa8 inactivated during the transition from a reversible to an irreversible cataract in the cortex. Samples of senile cataracts also were assayed for inhibitor activity and similar results obtained. Every senile cataract had lost some inhibitor activity, and mature cataracts showed 9995% inactivation in every case.

1. Introduction of alkaline ribonuclease (RNase) have been reported from the liver (Roth, 1958; Shortman, 1961, 1962), adrenals (Giriza and Sreenivasan, 1966) and parotid gland (Rabinovitch, Sreebny and Smuckler, 1968) of the rat. Other reports show these inhibitors to be present in cerebral cortex (Takahashi, Mase and Suzuki, 1967) red blood cells (Ambellian and Hollander, 1968) and lymphosarcoma cells (Gupta Inhibitors

and Herriott,

1963).

An RNase

inhibitor

from

lens tissue has not been previously

reported, but its presence has been suggested by the work of Zigman, Burton, Fontaine and Lerman (1963) and Maione, Maraini and Carta (1968). These workers were unable to detect significant RNase activity in normal lenses,but RNase activity did appear in both senile and experimental cataracts. It was therefore suggestedby Maione et al. (1968) that the increase in RNase activity was more probably due to the inactivation of an RNase inhibitor. The data presented here show that there are high levels of an RNase inhibitor in normal lens tissue that decreasesharply in both galactose and senile cataracts. This inactivation, therefore, allows for the apparent induction of RNase in cataractous lenses.

2. Materials

and Methods

Assays of the RNase inhibitor were carried out by a modification of the method described by Shortman (1961). Purified E. CO& transfer RNA (Schwarz Bioresearch Corp., Orangeburg, N.Y., U.S.A.) was used as substrate. This substrate contained neither acid-soluble nucleotides nor residual RNase activity and permitted the use of higher levels of RNase in the assay system. The acid-insoluble precipitates were chilled and separated by filtration through nitrocellulose filters (0.45~ pore size, Millipore Filter Corp., Boston, Mass., U.S.A.) instead of by centrifugation. Crystalline pancreatic RNase (Sigma Chemical Co., St. Louis, MO., U.S.A.) was dissolved at a concentration of 50 pg/ml in O*1O/o gelatin and in

* This investigation was supported part by the Lions Eye Tissue Bank

in part by a General Research Support Grant (FR 6387-07 182) and of the University of Missouri. 120

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LENS

121

diluted for each day’s assays. Under these conditions t’he enzyme remained stable for several months. Each assay mixture contained 1-O mg of transfer RNA and 0.05 pg of RNase in O-10 M Tris buffer (pH 7.5). The reaction was routinely run at room temperature for 8 min. Under these conditions only 50-60% of the RNA was hydrolyzed. If hydrolysis was allowed to continue longer, the inhibitor gave a nonlinear response since the substrate began limiting the reaction. Aliquots of the inhibitor solution were added to several R,Nase assay mixtures and the inhibition was measured. One unit of inhibitor activity is defined as the amount of inhibitor required to inhibit 0.005 pg of RNase 50%. Bovine lenses, both fresh and frozen, were homogenized with 3 volumes of demineralized water in a Waring blender for 1 min at top speed. After centrifuging at 30,000 g for 20 min. the supernatant was adjusted to 0.14 M NaCl, 0.02 M phosphate buffer (pH 6+3), O*OOl M EDTA and O@Ol M P-mercaptoethanol (MSH) with concentrated solutions of each eomponent. A slight precipitate that formed was removed by centrifugation. A 2 x 10 cm diethylaminoethyl (DEAE) cellulose column was prepared and lo-50 ml of lens extract was applied. The column was washed with 0.14 M NaCl, 0.02 1v1phosphate buffer (pH 69, 0901 M EDTA and 0.001 M MSH until. the optical density at 280 mp (O.D.& of th e effl uent was below O-05. The RNase inhibitor activity was removed by an elution with 1.4. M NaCl, O-2 M phosphate buffer (pH 6-S), 0.001 31 EDTA and O*OOl 51 MSH. Fractions of 5 ml were collected. The peak tubes were pooled, concentrated by vacuum filtration with $ in diam. dialysis tubing and dialvzed against 0.02 M phosphate buffer (pH 6*8), 0.001 M EDTA and 0.001 M MSH. _ This preparation was again placed on a DEAE cellulose column at 0.15 M NaCl in O-02 M phosphate buffer (pH 6*5), and 0.001 1\1MSH. The column was eluted with a linear salt gradient from 0.15 M to 1.5 M NaCl in the same buffer. The active fractions were pooled and the prot’eirl was precipitated with the addition of 2 vol. of saturated ammonium sulfat’e. Sprague-Dawley male rats weighing about 50 g ea.& were placed on a diet containing 50th galactose and 50% lab chow. At various intervals several rats were decapitated and the lenses were removed and placed in ice. Each pair of lenses was weighed and homogenized in demineralized water (12.5 mg lens tissue/ml) with a glass homogenizer. Each homogenate was centrifuged at 12,000 g for 10 min and the supernatant was assayed for RNase inhibitor activity. A similar procedure was followed for the preparation and assay of human lens extracts. The lenses containing senile cataracts were obtained after surgery and placed on ice. The normal human lenses were obtained from eyes sent to the Lions Eye Tissue Bank of the University of Missouri. These eyes were kept from 1 to 3 daya before the lenses were removed, and therefore, the values determined may not be the maximum activity obtainable from normal human lenses. All localization studies were carried out using bovine lenses. The capsules were removed from the lens and the nuclei were punched out with a No. 9 cork borer. The remainder \Y:IS called the cortex. Each lens fraction was homogenizrd alld assayed separately. 3. Results The presence of an RNase inhibitor in bovine lens was demonstrated easily by adding increasing amounts of a crude lens extract to an RNase assay. The RNase activity decreased in a linear fashion as shown in Fig. 1. These data have been corrected for the absorbance of any acid-soluble material in the lens extract. It should be noted that in most cases a small RNase activity remains even at high inhibitor concentrations. This has also been reported by Shortman (1961) for the rat liver inhibitor and may reflect a reversible RNase-inhibitor interaction. Since this assay involves the addition of several milligrams of lens extract to a solution containing only 0.05 pg of RNase, it was felt that a purification was necessary

122

B. J.

ORTWERTH

AND

R.

J.

BYRNES

to demonstrate that the inhibition was due to the action of a discrete molecule. Studies on the whole lens extract revealed that the inhibitor was nondialyzable and heat-labile at 60°C for 2 min. These results suggested that the inhibitor was a protein, therefore a fractionation of lens proteins was carried out on DEAE cellulose.

pl of inhibltor FIG.

1. The effect of the addition

of increasing

amounts

solution

of lens extract

on the activity

of pancreatic

RNase.

The results of the step-wise elution of the RNase inhibitor from DEAF cellulose are shown in Table I. Purifications of 40-fold have been routinely obtained with frozen lenses; however, higher activity was observed when fresh lenses were used. It was important that the pH of the purified extract was kept between 6.0 and 7.0 at all times since at higher pH the inhibitor became inactivated. At pH 9-O all activity was lost in 10 min. TABLE

Purification Fraction

Original extract 0.14

I

of the bovine lens ribmuclease inhibitor on DEAE Volume

(ml)

Total

0.D.p8,,

20

2200

80

2000

Total

activity

3660

cellulose

Activity/o.D.,,,

1.7

M NaCl

wash NaCl wash

0

_-

1.4 M

10

54

3680

68

It was known that this purified fraction also contained considerable quantities of RNA. Therefore, further chromatography on DEAE cellulose was carried out to remove this RNA. When the purified fraction obtained by step-wise elution from DEAE cellulose was placed on a second DEAE-cellulose column and eluted with a linear salt gradient, the results shown in Fig. 2 were obtained. As can be seen by the absorbance values at 280 and 260 rnp, the protein and RNA eluted in two distinct peaks. The RNase inhibitor activity, which is represented by the shaded area in Fig. 2, was eluted at the trailing edge of the protein peak. The specific activity of the pooled peak was 650 units/mg protein. This was true when the protein was estimated by either absorbance at 280 rnp or by the method described by Lowry, Rosebrough, Parr and Randall (1951).

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1%

LENS

Tube number

FIG. 2. Separation of a partially purified preparation of lens RNase inhibitor on diethylaminoethyl cellulose. Elution was carried out using a linear salt gradient from 0.15 to 1.5 M NaCl in 0.02 M phosphate buffer (pH 64) and 0401 M MSH. The shaded area represents inhibitor activity.

Confirmation of the protein nature of the RNase inhibitor was obtained by the use of the purified RNase of proteolytic enzymes. Table II shows the inactivation inhibitor by several proteolytic enzymes. All incubations were carried out at 37°C for 10 min, and in every case 100 pg of proteolytic enzyme were sufficient to inactivate 1 mg of partially purified inhibitor protein. It has been reported that a free sulfhydryl group is required for the activity of the purified RNase inhibitor from rat liver (Shortman, 1962). This is also true for the RNase inhibitor from bovine lens. The results in Table III show that when the TABLE

II

Effect of proteolytic enzymes on ribonuclease inhibitor Activity (units/ml)

Addition

SO

TABLE

Activity (units/ml)

J&O

92

~xIO-~MPMB 1 X lCFs M 1 A lo-” Y 1 x 1O-6 M 1 x lo+’ Y

0 0 0 32 67

PMB PMB PMB PMB

Los.9 (%I -___ 100 100 100 65 27

(l/u)

0 88 6 61 35 88

III

E$ect of su~hydy~ bkdcing age&s on kns Addition

Loss

6i 67 9 63 26 42 9

Papain 10 pg Papain 100 pg Pronase 10 pg Pronase 100 jkg Trypsin 10 pg Trypsin 100 pg

activity

ribonuclease

Addition ____-~-___ H,O 1 x 1O-3 a~ NEM 1 x IO-” N[ NEM 1 x 1O-5 M NEM

inhibitor activity Activity (units/ml)

Lose (%I

147 13: 140

97 6 5

124

B. J.

ORTWERTH

AND

R.

J.

BYRNES

partially purified inhibitor was incubated with either p-hydroxymercuribenzoate (PMB) or N-ethylmaleimide (NEM), it was completely inactivated. The presence of these sulfhydryl blocking agents in the assay mixture, however, had no effect on the RNase activity. Larger concentrations of PMB were required to completely inhibit the lens RNase inhibitor, than those reported for the rat liver inhibitor. However. many other proteins that also have free sulfhydryl groups and that also react with the PMB are present in our partially purified preparation. The experiment shown in Table IV was performed to determine the type of binding between the inhibitor and the RNase. These data show that the inhibitor used was TABLE

IV

Reversal of RNase inhibition First preincubation 1. 2. 3. 4. 5. 6. 7.

Second preincubation

Blank RNwe INH.+PMB RNasefINH. INH. +PMB RNase+INH. RNase+PMB

RNase activity (%)

“J’.se.o 0.18 0.57 O-18 0.18 0.57 0.60 0.57

Buffer RNase PMB INH.

0 100 0 0 100 100 100

active enough to inhibit the RNase completely (line 4), that the PMB concentration (1O-4 M) was sticient to completely inactivate the inhibitor (line 5), that PMB can reverse the inhibitor and restore full RNase activity (line 6) and that PMB has no effect directly on RNase (line 7). We have not been able to determine, however, whether the PMB reacts directly with the RNase-inhibitor complex to cause release of the inhibitor, or whether the PMB continually reacts with free inhibitor, causing the equilibrium to shift, releasing more inhibitor from the complex. Rats were fed the galactose-containing diet and cataracts developed in accordance with previously reported descriptions (Sippel, 1966). Measurements of the RNase inhibitor activity at various times during the feeding are shown in Fig. 3. This figure shows that the inhibitor activity decreases after day 14, reaching a minimum at day 28, while lenses from control animals show no decrease. The values determined on day 21 are individual measurements on three lens pairs. These rats showed obvious

6.0 r’ .Ft

/p-

------y----”

5.0

2 4-o e 3.0 \ 1, 9 5 EO

,\

0

I.0

o-~----p

0, 5

FIO. 3. The effect of galactose A, control.

feeding

to 15 Doys on a 50%

20

25

gatactose

diet

on the RNase

inhibitor

30

activityiof

I 35

rat lens.

0, Galactoee.

fed;

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differences in the development of cataracts. One rat had a marked nuclear cataract in both lenses, and had the lowest level of inhibitor activity. Another had no nuclear opacities and gave an activity value comparable to control values. The third rat had a nuclear cataract in one lens only and showed an intermediate activity. These data suggest that the inactivation of the RNase inhibitor occurs at the particular time in galactose cataract development when a nuclear cataract appears. Localization studies on bovine lenses were carried out and are presented in Table V. It can be seen that almost all of the RNase inhibitor activity was located in the cortex. The small amount of activity seen in the nucleus could be accounted for by TABLE

Localization Fraction

Total

Capsule cortex Nucleus

V

of the lens ribmuckme

inhibitor

Total activity (units)

o.D.~~,, 2.5 400 400

Specific activity (units/o.D.,,,)

35 2ooo 20

14 5 0.05

contamination with some of the cortex. The capsular activity also was low, but on a specific activity basis was higher than the cortex and could be significant. It remains certain, however, that the loss of activity seen during gala&se feeding must have been due to an inactivation process taking place in the cortex. This event, therefore, must occur while the cortex is relatively clear and accompanies the formation of the irreversible opacity. To see if inhibitor inactivation was an effect of gala&se feeding, or a general property of cataract formation, measurements of RNase inhibitor activity were made on several lenses with senile cataracts and compared to normal human lenses. These measurements are shown in Table VI. These data show that cataracts which involve TABLE

Ribordease

inhibitor

VI

activity in normal

Lens description Normal Cataract A. Nuclear 1. 2. B. Mature 1. 2. 3. 4. 5.

lenses and senile cu&racts Activity

(unit/lens)

440&-110 and posterior

subcapsular 270 170 45 30 < 10 (10
only a portion of the lens still show considerable inhibitor activity. It can be seen, however, that essentially all activity was absent in lenses that contained mature cataracts. Therefore, the inactivation of the lens RNase inhibitor seemsto correlate directly with the extent of cataract development.

126

B. J.

ORTWERTH

AND

R.

J.

BYRNES

4. Discussiou It now seems apparent that the lack of detectable levels of RNase in the lens tissue is the result of the presence of high levels of an RNase inhibitor protein. The chromatographic properties of the inhibitor suggest that the protein contains a high concentration of negative charges. In this way it could mimic the negatively charged phosphate backbone of RNA. This protein has been purified 400-fold from bovine lenses; however the preparation is still impure. Shortman (1961) has reported a purification of 5000-6500-fold from rat liver with the final preparation having a specific activity of 40,000-50,000 units/mg protein. We have attempted to determine an approximate molecular weight of the inhibitor by gel diffusion on a Sephadex G-100 column; however no activity was recovered. The specificity of this inhibitor to various RNases has not been tested; however, our purified preparation showed no activity when tested with pancreatic deoxyribonuclease. It is tempting to speculate that the presence of the inhibitor in the cortex serves to protect the stable mRNA templates (Stewart and Papaconstantinou, 1967) from RNase attack. This is essential for continued protein synthesis in the cortex because new mRNA templates cannot be synthesized due to nuclear degeneration. The finding that a free sulfhydryl group was required for activity of the lens inhibitor is consistent with the observation of Shortman (1962) with the rat liver inhibitor. The reason why a free sulfhydryl group is required for activity is not known at this time and can only be considered as necessary for proper protein conformation. While no studies have been carried out on the mechanism of this inhibition, it is clear that the inhibition was reversed when the inhibitor was inactivated with PMB. The inhibitor did not hydrolyze or denature the RNase, since full RNase activity was restored. The importance of the reversibility experiment is that it shows that RNase could exist in an inhibited state in the normal lens, and then be released as active enzyme if the inhibitor is inactivated. Endogenous RNase has been reported to be present in normal lens epithelium by Swanson, Jeter and Raley (1967). Whether or not it is also present in the cortex is uncertain. The data presented here suggest that the endogenous RNase activity, which has been reported in extracts of cataractous lenses, can be measured because the inhibitor was inactivated by the cataractogenic process. Inactivation of the inhibitor in senile cataracts was greatest only in mature cataracts where the entire cortex was affected. Maione et al. (1968) similarly reported that RNase activity was the highest when the cataract involved the cortex of the lens. Both of these observations agree with the finding that almost all of the inhibitor activity was found in the cortex of the lens. It is not clear whether the endogenous RNase and the inhibitor actually exist as a complex in vivo. If they do, however, this complex can be dissociated by blocking the free sulfhydryl group of the inhibitor. This will result in the release of active RNase. This may be the mechanism that inactivates the inhibitor during cataract formation. The free sulfhydryl group of the inhibitor may become bound in disulfide linkages to the structural proteins of the lens, causing the release of RNase. Support for this idea comes from the recent report of Weller and Green (1969). They have shown an inactivation of the methionyl-tRNA synthetase enzyme during the formation of galactose-induced cataracts. Since this protein, like the RNase inhibitor, also requires a free sulfhydryl group, the same mechanism could be involved in the inactivation of both proteins. These events occur late in cataract development and

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appear to correlate with the change from a reversible to an irreversible cataract. The idea that protein alterations, which involve sulfhydryl groups, occur at this stage has been suggested by many investigators and has been recently reviewed by Dische (1968). Th ese protein alterations appear to involve more than structural proteins and can lead to the complete inactivation of biologically active macromolecules. ACKNOWLEDGMENTS

We would like to gratefully acknowledge the technical assistance of Mr Robert Hyatt., Mr Vernon Sackman and MI- Stephen Godfrey. Part of this work was presented at the National Meeting of the Association for Research in Ophthalmology at Sarasota, Florida in May, 1970. REFERENCES Ambellian, E. and Hollander, B. P. (1968). Proc. Sot. E’xp. Biol. Med. 127,482. Dische, 2. (1968). In Biochemistry of the Eye: Proceedings of the XXth International Congress of OphthaZmoZogy, pp. 413428. (Ed. by Dardenne. M. V. and Nordmann, J.) S. Karger AG, Basel. Giriza, N. S., and Sreenivasan, A. (1966). Biochem. J. 98, 562. Gupta,, S. and Herriott, R. M. (1963). Arch. Biochem. Biophys. 101,88. Lowry, 0. H., Rosebrough, K. J., Farr, A. L. and Randall, R. J. (1951). J. Biol. Chem. 193,265. Maione, M., Maraini, G. and Carta, F. (1968). Exp. Eye Res. 7,546. Rabinovitch, M. R., Sreebny, L. M., and Smuckler, E. A. (1968). J. Biol. Chem. 243,3441. Rot’h. .J. S. (1958). J. Biol. Chem. 231, 1985. Shortman, K. (1961). B&him. Biophys. Acta 51, 37. Shortman, K. (1962). Biochim. Biophys. Actu 55,88. Sippel. T. 0. (1966). Invest. Ophthulwwl. 5, 576. Stewart,. J. A. and Papaconstantinou, J. (1967). Proc. Nrct. Acad. Sci. U.S.A. 58, 95. Swanson, A. A., Jeter, J. and Raley, L. W. (1967). Exp. Eye Res. 6, 351. Takahashi, Y., Mase, K. and Suzuki, Y. (1967). Experientia 23,525. Weller. C. A. and Green, M. (1969). Exp. Eye Res. 8, 84. Zigman, S., Burton, M., Fontaine, J. and Lerman, S. (1963). Invest. Ophthalmol. 2, 621.