Age changes in bovine lens endopeptidase activity

Mechanisms of Ageing and Developmen tl 31 (1985) 37 -47

37

Elsevier Scientific Publishers Ireland Ltd.

AGE CHANGES IN BOVINE LENS ENDOPEPTIDASE ACTIVITY*

KAREN R. FLESHMAN, JOYCE W. MARGOLIS, S.-C. JOSEPH FU and B.J. WAGNER Departments of Ophthalmology and Biochemistry, University of Medicine and Dentistry of New Jersey, New Jersey Medical School and Graduate School of Biomedical Sciences, Newark, NJ 07103 (U.S.A.)

(Received December 3rd, 1984) SUMMARY Lens endopeptidase activity and thermal stability have been determined as a function of (1) cell development, (2) cell age, and (3) animal age. Lenses from animals aged 3 months to 15 years (lens weights 1.15 2.80 g) were divided into epithelial (outermost), cortical (peripheral), and nuclear (central) regions. Changes accompanying cell development were determined by measuring specific activity in epithelial (undifferentiated), outer cortical (differentiating), inner cortical (mature) and nuclear (aged) regions of individual lenses. Thermal stability of the enzyme activity obtained from the outer cortical and nuclear regions of the same lenses was also deternrined. Specific activity and thermal stability were found to decrease as a function of lens cell development. Changes with cell development represent the effects of both differentiation and increasing cell age. To determine the effects of cell age alone, activity was determined in the same population of aged, fully differentiated cells in lenses of different ages. Specific activity decreased as a function of cell age alone. Changes with animal age were determined by comparing cells of the same developmental stage from animals of different ages (e.g., differentiating cells of the cortex in animals 3 months to 15 years old). Specific activity for the cortical region increased with animal age while specific activity in the nuclear region appeared to remain constant or decrease slightly with increasing animal age. Thermal stability of the enzyme activity from the cortex was different in young and adult lenses. The change in stability occurred early in the lifespan and was therefore more closely related to animal development than to aging.

*This work was supported in part by a Postdoctoral Fellowship to KRF from Fight for Sight Inc., N.Y.C. and by U.S.P.H.S. grant EY02299 and the Cooperative Cataract Research Group Consortium EY03247. Address reprint requests to. Dr. B.J. Wagner, Department of Biochemistry, UMDNJ-New Jersey Medical School, 100 Bergen Street, Newark, NJ 07103, U.S.A. 0047-6374/85/$03.30 Printed and Published in Ireland

© 1985 Elsevier Scientific Publishers Ireland Ltd.

38

Key words: Endopeptidase; Aging; Development; Bovine; Lens; Stability

INTRODUCTION The lens of the eye provides a suitable system to study aging because of its mode of growth. Lens growth occurs only at the periphery [1] : young cells are laid down over older cells, resulting in a strict segregation by age with the oldest cells at the center of the lens and the youngest at the periphery. Lens growth occurs throughout life so that new cells are continually being formed; since lens cells are not lost during the individual's lifetime, cells of very advanced age are available for the study. The lens can be divided into concentric layers. The epithelium, which covers the anterior surface, consists of undifferentiated cells. The outer cortex consists primarily of differentiating lens fiber cells. These cells lose their nuclei and organelles to become mature lens fiber cells. The inner cortex consists of mature fiber cells. The nuclear region in the center of the lens contains the oldest lens cells, referred to here as "aged." Protein degradation appears to be involved in structural protein changes in the lens. Lens structural proteins (crystallins) constitute the majority of proteins and are essential for transparency. Because protein synthetic capacity is lost as lens cells differentiate, agerelated changes in degradation car~ be expected to alter structural protein integrity. Such changes have, in fact, been described for normal bovine lens crystallins [ 1 - 4 ] . In aged human and animal populations [1,5], opacities are frequently observed with ageing. Observations in human age-related cataractous lenses suggest protein degradation is involved in this loss of transparency [6,7]. Thus, protein degradation appears to be involved in age-related changes in the clear lens and in the loss of transparency observed in aging populations. Endopeptidase activity probably regulates lens protein degradation as it does in many other tissues [8]. Because lens neutral proteinase represents a major endopeptidase activity in the lens [9], we have chosen to study its activity as a function of development and aging. As enzyme molecules age, they may become modified and more susceptible to thermal denaturation. We have, therefore, also measured neutral proteinase thermal stability as a function of development and aging. We have used the bovine lens as a model because the large size of this lens permits measurements on individual lenses and allows isolation of the same cell population for determination of the effects of cell age. We have determined changes in neutral proteinase activity and stability in bovine lens as a function of: (1) cell development; (2) cell age; and (3) animal age. Changes with lens cell development were determined by comparing the epithelial, cortical, and nuclear region from a single lens. Changes occurring with cell development could be the result of both cell differentiation and increasing cell age. Changes with lens cell age alone were determined by comparing the same population of aged cells from lenses of different ages. This cell population was obtained by taking samples of the same wet weight from the

39 center of the lens nuclear region. Changes with animal age were determined by comparing cells of the same developmental stage (e.g. differentiating cells of the cortex) from animals of different ages.

MATERIALS AND METHODS

Chemicals Tris base and Hepes were purchased from Sigma Chemical Co., St. Louis, MO. p-Nitroaniline was obtained from Aldrich Chemical Co., Milwaukee, WI. The substrate, Ncarbobenzoxyglycylglycylleucyl p-nitroanilide, was obtained from the Peptide Institute Inc., Osaka, Japan. All other chemicals were reagent grade. Tissues Bovine eyes from animals aged 3 months to 15 years, were obtained from local slaughterhouses (Bermelon, Inc. Newark, NJ and Greater American Veal Co., Newark, NJ). The lenses were removed within 2 h of death and prepared immediately or stored at --70°C. Lens homogenate preparation Lens homogenate preparation was carried out at 4°C. Individual lenses were weighed and separated into capsule-epithelium (undifferentiated cells and non-cellular capsule), outer cortex (differentiating cells), inner cortex (mature cells) and nuclear region (aged cells). Lens weight was correlated with animal age (see abscissa Fig. 2) using our data and the data of Francois and Rabaey [10], Schmutter [11] and Berstein, Kerrigan and Maisel [12]. The capsule and adherent epithelium was removed and weighed; 500 #1 of 10 mM Hepes with 5 mM MgC12 (pH 7.5) (Hepes buffer) was added and the capsuleepithelium preparation stirred for 30 rain. The outer cortex was separated by gently stirring in 5 vols. (ml/g) of Hepes buffer until 65% of the total lens weight remained. The solubilized outer cortex was decanted and saved. The remaining lens material (inner cortex and nuclear region) was transferred into another 5 vols. of Hepes buffer. The inner cortex and nuclear region were separated by stirring until 30% of the original lens weight remained. The solubilized inner cortex was decanted and saved. The nuclear region was homogenized in 10 vols. of Hepes buffer in a Potter-Elvehjem homogenizer. For experiments to determine the effect of cell age on neutral proteinase activity, the nuclear material was divided into a central and peripheral region using a number 4 cork borer (8 mm diameter). Both the peripheral and central nuclear regions were weighed and homogenized individually in 10 vols. of Hepes buffer as described above. Each preparation was centrifuged at 11 000 g for 25 rain and the pellet washed with Hepes buffer. The pooled supernatant materials for each lens region were stored at --70°C until use; preparations stored for 1 year showed no loss of activity.

40 Neutral proteinase assay Lens endopeptidase activity was measured by incubation with the chromogenic synthetic substrate, N-carbobenzoxyglycylglycylleucyl p-nitroanilide, in Hepes buffer for 1 h at 37°C as previously described [13]. Activity is expressed as nmolesp-nitroaniline (pNA) produced/h/mg soluble protein. Soluble protein was determined by measurement of absorbance at 280 nm. To determine if lens protein absorbance at 280 nm was influenced by age, soluble protein from the cortex and nuclear region of lenses weighing 1.15-2.75 g was also determined using a biuret procedure (absorbance measured at 550 nm; Colowick and Kaplan [14]) with bovine serum albumin as the standard. The ratio A2ao/A sso was constant over this age range. A total of 28 lenses weighing 1.15-2.80 g was prepared and assayed individually to determine the effects of cell development, cell age, and animal age on neutral proteinase activity and thermal stability. For experiments on thermal stability, lens homogenates were preincubated at 60°C for 0 - 6 0 min. Samples were cooled on ice for at least 15 rain prior to determination of neutral proteinase activity. The neutral proteinase activity at 37°C was determined as described above. Results are plotted as the log percent of activity remaining vs. preincubation time at 60°C. Neutral proteinase thermal stability was determined as a function of cell development (7 experiments), cell age (5 experiments) and animal age (5 experiments). Data analysis was performed on the Prophet system computer, a national computer resource sponsored by the Division of Research Resources, N1H. This included regression analysis using a least squares method to determine the effects of animal age on neutral proteinase activity. RESULTS The effect of cell development on neutral proteinase activity was determined by comparing the specific activity in the epithelium, outer cortex, inner cortex and nuclear region of individual bovine lenses. A total of 28 experiments with lenses aged 3 months to 15 years old were conducted. Results of a representative experiment are shown in Fig. 1 for a 5-year-old (2.45 g) lens. The rate of hydrolysis in extracts from each lens region is linear with time for at least 1 h. The specific activities in the epithelium, outer cortex, inner cortex and nuclear region are 13, 1 l, 4, and 1 l~mol pNA/mg protein/h, respectively. Comparison of specific activity in the whole homogenate (soluble and insoluble protein) and the supernatant material (soluble protein)demonstrates that neutral proteinase activity is found in the soluble fraction in all lens regions. Therefore, decreased specific activity is not due to differences in partitioning of active enzyme molecules between soluble and insoluble fractions. These data demonstrate that specific activity declines from epithelium to nuclear region, that is, with increasing cell development. The effects of cell age were determined by comparing the specific activity of the same fully differentiated cell population from lenses of different ages. In 4-month-old lenses,

41

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Fig. 1. Neutral proteinase activity vs. incubation time for a 5-year-old (2.45 g) lens: (o) epithelium; (-) outer cortex; (o) inner cortex; (m) nuclear region. Each point represents the mean of duplicate assays. Assay conditions are described in the text.

this cell population represents the entire nuclear region. As the lens ages, this cell population represents only a portion o f the nuclear region. In the oldest lenses studied, this cell population represented the inner 50% o f the nuclear region. The results o f these experiments are shown in Table I. Specific activity is unchanged in this cell population in lenses < 2 years old (lens weights < 2.15 g). Specific activity is lower in the population from 7- to 9-year-old lenses (lens weights 2 . 5 4 - 2 . 6 0 g) and is even lower in lenses > 11 years old (lens weights > 2.70 g). Therefore, specific activity in the same cell population declines as a function o f cell age in adult lenses. The effects o f animal age on lens neutral proteinase activity were determined b y comparing activity in cells at the same developmental stage from animals o f different TABLE I NEUTRAL PROTEINASE ACTIVITY IN WEIGHT-MATCHED BOVINE CENTRAL NUCLEAR REGION a Animal age

Lens weight {g}

nmoles pNA/mg/h

< 6 months

1.15-1.40

10 months to 2 years

1.67-2.1

6-8 year

2.54-2.60

> 11 years

> 2.70

2.21 (range: 1.15-3.77) 2.47 (range: 0.7-4.31) 0.82 (range: 0-1.51) 0.06 (range: 0-0.10)

aln lenses > 11 years old, this constitutes the central 50% of the total nuclear region.

(n - 11) (n = 6) (n = 5) (n = 3)

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Fig. 2. Neutral proteinase activity vs. lens weight (A) outer cortex, regression line: y = 5.993x --0.695 (B) inner cortex, regression line: y = 2.143x + 2.108 (C) nuclear region regression line: y = 1.085x + 3.910. The regression coefficients are significant at the following levels: outer cortex P < 0.001, inner cortex P < 0.03, and nuclear region P < 0.10. The coefficients of determination were outer cortex 0.36, inner cortex 0.16, and nuclear region 0.21. Assay conditions are described in the text.

ages. The results of these experiments are shown in Figs. 2A-C. Regression analysis using a least squares method was performed on the Prophet computer system. Specific activity for the outer cortex, shown in Fig. 2A, increases with animal age. In this lens region, specific activity ranged from 3.7 to 12.7 nmol pNA/mg protein/h in lenses from young animals < 2 years age (lens weight < 2.15 g) and from 7.5 to 29.4 nmol pNA/mg protein/h in older animals > 2 years age (lens weight > 2.15 g). Specific activity in the inner cortex, shown in Fig. 2B, also increases with animal age (lens weight). The range in specific activities in the inner cortex is 1 . 5 - 1 6 nmol pNA/mg protein/h with median activity in the 4 - 8 nmol pNA/mg protein/h range. Specific activity in the nuclear region, shown in Fig. 2C, is low but appears to be maintained with animal age. Activity is detectable in the oldest animals studied. The decrease in activity indicated by the regression line is not significant. Specific activity was more varied in the epithelium than in other lens regions and showed no correlation with lens weight. Specific activities in the epithelium range from 3 to 30 nmol pNA/mg protein/h with the median in the 1 1 - 1 5 nmol pNA/ protein/h range. The same age-related pattern is observed for each lens region whether data were expressed per mg protein, per mg wet weight, or per lens. The variation in specific activities within each lens region appears to reflect animal-toanimal variation. Lenses were obtained from livestock animals: the breed of cattle and the

43 conditions under which the animals were maintained could not be controlled. Contralateral lenses have similar specific activities. Duplicate assays of the same sample varied by less than 8%. Thermal stability of neutral proteinease was determined as a function of lens cell development, cell age, and animal age. Thermal stability as a function of cell development was determined by comparing activity remaining in the outer cortex and central nuclear regions after preincubation at 60°C. Results in Fig. 3A are for the outer cortex and central nuclear region o f a 5-year-old (2.45 g) lens. The time for 50% inactivation is > 60 min for the outer cortex and 5 min for the central nuclear region. In all lenses, thermal stability of the outer cortex is considerably greater than that of the nuclear region. The thermal stability curve for the outer cortex is biphasic, indicating that more than one form of the activity is present. Thermal stability in the central nuclear region is linear, suggesting one form o f the enzyme. Neutral proteinase thermal stability was determined as a function of cell age by comparing stability in the same aged cell population from lenses of different ages. Results, shown in Fig. 3A, are for 5-year-old lens cells (2.45 g lens) and for 4-month-old lens cells (1.27 g lens). The time for 50% inactivation is 10 min for the young cells and 5 min for the older cells. Although the difference in inactivation time is small, the same pattern was observed in all experiments

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Fig. 3. Neutral proteinase thermal stability vs. pre-incubation time: (A) (A) outer cortex of 5 year old (2.45 g) lens, original activity 11.4 nmol pNA/mg protein/h; (=) central nuclear region of 5-year-old (2.45 g) lens, original activity 1.2 nmol pNA/mg protein/h; (=) nuclear region of 4-month-old (1.27 g) lens, original activity 1.2 nmol pNA/mg protein/h. (B) (A) outer cortex of 2-year-old (2.14 g) lens, original activity 7.3 nmol pNA/mg protein/h; (:~) outer cortex of 4-month-old (1.25 g) lens, original activity 4.0 nmol pNA/mg protein/h.

44 The influence of animal age on neutral proteinase thermal stability was also determined. In these experiments, enzyme stability in the lens outer cortex was compared. Figure 3B shows a representative experiment using the outer cortex of a 4-month-old lens (1.25 g) and 2-year-old lens (2.14 g). The time for 50% inactivation is 11 min for the 4-month-old lens and 28.5 min for the 2-year-old lens. The linear curve for the younger lens suggests a single form of the activity is present, while the biphasic curve for the older lens indicates that more than one form is present. In animals 4 - 5 months old (lens weight 1.15-1.37 g), two thermal stability patterns were obtained. In 3 out of 5 experiments (lens weights: 1.18, 1.25, 1.32 g) the decrease in activity vs. time was linear, while in the remaining 2 experiments the decrease was biphasic (lens weights: 1.25, 1.37 g). In these two experiments, the biphasic curves were similar to curves obtained from older lenses. In all experiments with lenses from animals > 2 years old (.lens weights > 2.15 g), the decrease in thermal stability was biphasic. In these lenses, the 50% inactivation times varied from lens to lens. Contralateral lenses had similar inactivation times. The lens-tolens variability was not age-related. These data indicate that thermal stability curves are linear in young animals. At 4 5 months of age, the stability curves shift to a biphasic pattern characteristic of adult animals. In adults, there is no age-related decrease in thermal stability. DISCUSSION Lens neutral proteinase activity and thermal stability have been determined as a function of lens cell development, cell age and animal age. Although bovine lenses > 6 months old have more than one endopeptidase activity [13,14], results from HPLC analysis and inhibitor studies using partially purified neutral proteinase preparations indicate that only one enzymatic activity is responsible for hydrolyzing N-carbobenzoxyglycylglycylleucyl p-nitroanilide [ 15,16]. Neutral proteinase specific activity declines as a function of lens cell development, that is from epithelium to nuclear region. This is an expected result; the activity of most lens enzymes declines with cell development [ 1]. Lens structural proteins in the epithelium and nuclear regions partition differently between the soluble and unsoluble fractions with cell development [1]. However, these studies show that decreased neutral proteinase activity is not due to differences in partitioning of catalytically active enzyme molecules between the soluble and insoluble fractions. Soluble structural protein concentration increases from epithelium to outer cortex [1]. Therefore, the decrease in neutral proteinase specific activity from epithelium to outer cortex can be accounted for, at least in part, by the increased structural protein concentration of the outer cortex. From the outer cortex to the nuclear region, the soluble protein concentration does not increase. Therefore, the decrease in neutral proteinase specific activity from outer cortex to nuclear regions is not the result of increased structural protein concentration. The decrease could represent loss of catalytic activity. Thermal stability of the enzyme from the nuclear region is lower than that m

45 the outer cortex. This is consistent with a decrease in enzymatic activity in the nuclear region compared to the outer cortex. Decreased activity and thermal stability with cell development has been reported for rat lens neutral proteinase [18] and for other lens enzymes [19,20]. Neutral proteinase activity also decreases solely as a function of cell age. The cell populations used for these experiments are fuUy differentiated and are the same age as the eye donor. In this study the cells were from 3 months to 15 years of age. These cells are not known to synthesize protein [ 1]. Consequently, enzyme molecules which become inactive cannot be replaced. In this cell population, the decrease in activity with cell age reflects such enyzme inactivaton. Decreased enzyme activity with increased cell age has been reported in other tissues [21 ]. Changes in neutral proteinase activity with animal age were determined by comparing specific activity for the same lens region (i.e. same stage of differentiation) from animals of different ages. Specific activity in the outer and inner cortex increased with increasing lens weight. In the nuclear region, specific activity was unchanged with animal age. The same age-related patterns of activity were observed for each lens region whether activity was expressed per unit protein, per unit wet weight, or per lens. Because the amount of insoluble lens protein increases substantially with animal age, an increase in activity (outer cortex) expressed per mg soluble protein does not necessarily indicate increased activity. That the specific activity increases when data is expressed per mg wet weight or per lens indicates neutral proteinase activity in the outer cortex does increase with age. Regression analysis of the data indicates that the increase in neutral proteinase activity in both the outer and inner cortex is related to animal age. The coefficients of determination indicate that age is not the only factor involved. Many lens enzymes decrease in activity with increasing age [19]. The increased activity in the outer cortex of lenses from older animals was unexpected and suggests that neutral proteinase activity has an important role in the older lens. Rat lens neutral proteinase shows a similar age-related increase [18]. Another proteinase activity, hydrolyzing azocasein with a neutral pH optimum, also increases with age [13]. The proteinase activity studied here does not hydrolyze azocasein [9]. Enzymatic activity is maintained in the nuclear region. This was unexpected because activity was shown to decline in the same cell population in the central nuclear region with cell age. This means that the majority of activity is localized in the younger cells of the nuclear region. In experiments where the activity was measured in the central and peripheral nuclear region (representing the younger cells of the nuclear region), the majority of the activity was, in fact, located in the peripheral nuclear region [Fleshman and Wagner, unpublished observations]. Thermal stability curves for neutral proteinase in the lens outer cortex were found to be linear in young lenses and biphasic in older lenses. The data suggest that a transition from a single enzymatic form to multiple forms occurs. Data obtained from homogenates from lenses with biphasic stability curves suggests that the transition occurs at about age 4 months. There is no change in thermal stability with age for animals > 2 years old. In all lenses with biphasic thermal stability curves, the new form(s) of the enzyme is

46 (are) more heat stable than the original form. It is not known whether the new form(s) of the activity in the cortex represents post-translational modification, synthesis of new gene products, or a change in the interaction of the enzyme with other molecules of the cytoplasm, such as has been demonstrated for glucose-6-phosphate dehydrogenase [22]. A similar change in neutral proteinase thermal stability was observed in rat lenses [18]. Increased thermal stability has been reported for human lens glutathione reductase [23]. Bovine lens growth (wet weight) is linear for the first 3 months after birth but begins to slow to the lower adult rate at about 4 months o f age. The appearance of the more heat stable form(s) of endopeptidase activity correlates with this slowing of lens growth. In fact, the more heat stable form(s) of neutral proteinase activity appears at the same time in the lifespan in both bovine and rat lenses. Thus, in the bovine lens the more heat stable form(s) appears at about 4 months of age (0.33 years) or at 1.1% of the lifespan (30 years). In the rat, the more heat stable form(s) appears at 15 days (0.04 years) or at 1.3% o f the lifespan (3 years). Since this occurs early in the lifespan (betore sexual maturity is reached), the appearance o f more stable forms o f the activity is correlated with animal development rather than with aging. Altered heat stability associated with maturation has been reported for mouse liver glucose-6-phosphate dehydrogenase [24]. The physiological function(s) o f lens neutral proteinase has not been determined. The appearance of more heat stable forms o f the activity during development is different from that o f many lens enzymes and may be related to its function. The pattern of maintained activity suggests neutral proteinase has an important role in the older lens. The presence of activity in the nuclear region o f the lens is particularly significant since protein synthesis probably does not occur in these cells. The presence of neutral proteinase activity in this region where protein synthesis is absent supports the proposal that protein degradation has a role in lens cell development and in the aging lens. REFERENCES 1 H. Bloemendal, Lens Proteins. Molecular and Cellular Biology o l the Eye Lens, John Wiley and Sons, New York, 1981. 2 D. Roy and A. Spector, Absence of low-molecular-weight alpha crystallin in the nuclear region of old human lenses. Proc. Natl. Acad. Sci. USA, 73 (1976) 3484-3487. 3 J.S. Zigler, Jr., Age-related changes in the polypeptide composition of #-crystallin from bovine lens. Exp. Eye Res., 26 (1978) 537-546. 4 W.W. de Jong, F.S.M. van Kleef and H. Bloemendal, IntraceUular carboxy-terminal degradation of the A chain of a-cyrstallin. Eur. J. Biochern., 48 ( 1974) 237- 247. 5 K.N. Gelatt and N.D. Das, Animal models for inherited cataracts: a review. Curr. Eye Res., 3 (1984) 765-778. 6 G. Marianni and R. Mangili, Differences in proteins and in the water balance of the lens in nuclear and cortical types of senile cataract. In Ciba Foundation Symposium 19, The Human Lens - In Relation to Cataract, Associated Scientific Publishers, New York, 1973, pp. 79-97. 7 J. Horwitz, J.S. Hansen, C.C. Cheung, L.-L. Ding, B.R. Straatsma, D.O. Lightfoot and L.J. Takemoto, Presence of low molecular weight polypeptides in human brunescent cataracts. Biochem. Biophys. Res. Commun., 113 (1983) 65-71. 8 A.J. Barrett, The many forms and functions of cellular proteases. Fed. Proc., 39 (1980) 9-14. 9 A.M.J. Blow, R. vanHeyningen and A.J. Barrett, Metal-dependent proteinase of the lens. Biochem. J., 145 (1975) 591-599.

47 10 J. Francois and M. Rabaey, On the existence of an embryonic lens protein. A.M. Archiv. Ophthal., 57 (1957) 6 7 2 - 6 8 0 . 11 J. Schmutter, Untersuchungen uber die altersabhangigkeit des gewiehtes und volumens yon rinderlinsen. Doctoral dissertation University of Bonn, 1961. 12 L. Bernstein, M. Kerrigan and H. Maisel, Lactic dehydrogenase isozymes in lens and cornea. Exp. EyeRes., 5 (1966) 309-314. 13 U. Hahn, A.A. Swanson and O. Hockwin, Age-related changes in the proteolytic enzyme of mammalian lens. Albrecht yon Graefes Arch. Klin. Exp. OphthalmoL, 199 (1976) 1 9 7 - 206. 14 O.P. Srivastava and B.J. Ortwerth, Purification and characterization of phacolysin B t and B 2, two trypsin like proteinases, from bovine lens cortex. Fed. Proc., 40 (1981) 1719. 15 B.J. Wagner, S.-C.J. Fu, J.W. Margolis and K.R. Fleshman, A synthetic endopeptidase substrate hydrolyzed by the bovine lens neutral proteinase preparations. Exp. Eye Res., 28 (1984) 4 7 7 - 4 8 3 . 16 B.J. Wagner, J.W. Margolis, S.-C.J. Fu, A.S. Abramovitz and K.R. Fleshman, A new component of the bovine lens neutral proteinase family. Invest. Opthal. Vis. ScL, 25 (1984) 135. 17 S. Colowick and N. Kaplan, Methods in Enzymology, Voi. III, Academic Press, New York, 1957, pp. 4 5 0 - 4 5 1 . 18 K.R. Fleshman and B.J. Wagner, Changes during aging in rat lens endopeptidase activity. Exp. Eye Res., 39 (1984) 5 4 3 - 5 5 1 . 19 C. Ohrloff, Age changes of enzyme properties in crystailin lens. Interdiscipl. Top. GerontoL, 12 (1978) 158-179. 20 C. Ohrloff, U. Teimann andO. Hockwin, Post-synthetic alterations of bovine lens enzymes demonstrated by heat lability measurements. Doc. Ophthal. Proc., 18 (1979) 205-217. 21 M. Rothstein, Recent developments in the age related alterations of enzymes: a review. Mech. AgeingDev., 6 (1977) 241-257. 22 A. Kahn, A. Guillouzo, M.-P. Leibgvitch, D. Cottreau, M. Bourel and J.-C. Dreyfus, Heat lability of glucose-6-phosphate dehydrogenase in some senescent human cultured cells. Evidence for its post-synthetic nature. Biochem. Biophys. Res. Commun., 77 (1977) 760-766. 23 J.J. Harding, Altered heat-lability of a fraction of glutathione reductase in ageing human lens. Biochem. J., 134 (1973) 995-1000. 24 J.D. Schofield and J.M. Handfield, Age-related alterations in the heat-lability of mouse liver glucose-6-phosphate dehydrogenase. Exp. Gerontol., 13 (1978) 147-157.