Exp. Eye Res. (1992) 54, 1005-1010

Description

of an Acylpeptide K. KRISHNA

Mason

Institute

(Received

of Ophthalmology,

New

York 3 June

SHARMA University

Hydrolase

from

Lens

6. J. ORTWERTH

AND

of Missouri,

7991 and accepted

Columbia,

in revised

MO 65212,

form 74 October

U.S.A.

1991)

Acylpeptide hydrolase, which catalyses the hydrolysis of blocked N-terminal amino acids from peptide substrates, has been identified in the extracts from beef, human, rabbit and rat lens. In bovine lens sections, lower activity was observed in nuclear and inner cortical regions compared to the outer cortical region. The enzyme from bovine lens showed a high molecular weight nature, eluting between CLand p crystailins during Sephadex G-200 chromatography. The activity has a pH optimum around 7.8 when assayed with N-acetyl-Ala-p-NA as substrate. The enzyme was capable of hydrolyzing a variety of blocked peptides including N-acetyl-(Ala),, Me-0-Sue-Ala-Ala-Pro-Val-p-NA, N-Acetyl-Met-Leu-Phe, Acetyl-SerGin-Asn-Tyr and N-formyl-Met-p-NA. In each case the enzyme released an N-blocked amino acid and exposed a free amino group as judged by thin layer chromatography. Neither Ala-p-NA nor N-acetyl-Ala were hydrolysed by the same enzyme preparation. The enzyme activity from human and bovine lens was completely inhibited by DFP, and partially inhibited by PMSF. penicillin-G and ampicillin. These preliminary results show that lens tissue has an active acylpeptide hydrolase, however, a partially purified enzyme preparations was not able to cleave the acetyl-Met- from native aA-crystallin in vitro suggesting that the N-terminus of native crystaliins is not accessible to the enzymes. Key words : lens ; acylpeptide hydrolase ; protein turnover : aminopeptidases : crystallins.

1985). The acetylation of nascent protein chains occurs when they are about 40 residues long

N-acetylated, the failure to remove the blocking Nterminal groups was thought to impede the action of proteases and peptidases on damaged crystalllns (Taylor and Davis, 198 7). This may be one of the reasons for the presence of increased levels of peptides in aged lenses (Srivastava, 1988b). Recently it has been demonstrated that a ubiquitin-dependent cellular

(Tsunasawa and Sakiyama, 1984) and the exact role

protein turnover system is present in bovine and

of this phenomenon is not known. The extent of acetylation of cellular proteins varies from tissue to tissue, with the proteins in Ehrlich ascites cells, mouse

human lens epithelial cells (Jahngen et al., 1986). But it is also known that the presence of a free amino terminal end is essential in the target protein for its

L cells and lens being 7580% acetylated (Brown and Robert, 1976; Brown, 1979: Bloemendal, 1981). It

hydrolysis by the ubiquitin-dependent system (Hersh-

1. Introduction A wide variety of eukaryotic intracellular proteins are found to be acetylated at the amino terminal end (Tsunasawa and Sakiyama, 1984; Driessen et al.,

has been speculated that acetylation may have a protective role (Jornvall, 1975). However, in vitro studies with lens crystallins have shown that Nacetylation

of aA crystallin

is not necessary for

protection against leucineaminopeptidase (Driessen et al., 1983). Lens tissue shows a remarkably slow turnover (Bloemendal, 1981). This is attributed to the low level of functional proteolytic enzymes in the lens.

Several recent studies have demonstrated the presence of appreciable amounts of endopeptidase proteolytic activity in lenses from various species (Srivastava and Ortwerth. 1983; Yoshida, Murachi and Tsukahara, 1984; Ray and Harris, 1985; Srivastava, 1988a;

David and Shearer, 1989). In addition, lens tissue also contains high amounts of leucine aminopeptidase (Hanson and Frohne, 1976) and other peptidases (Swanson et al., 1981; Lafferty, Raducha and Harris, 1984; Sharma and Ortwerth, 1986b). The activity of these enzymes is very high in lens fibres though there is very little protein breakdown. In view of the fact that the bulk of the crystallins are 00144835/92/061005+06 65

$03.00/O

ko, 1988). Therefore, an enzyme which can effectively remove the blocked amino groups from proteins and peptides may have the pivotal role in determining the turnover of the lens proteins. In recent years several reports have appeared

describing the presence of acylpeptide hydrolase in rat liver (Tsunasawa, Narlta and Ogata, 19 7 5 ; Kobayashi and Smith, 1987), bovine liver (Gade and Brown, 1978), porcine liver (Tsunasawa, Imanaka and Nakazawa, 1983) rat brain (Marks, Lo, Stern and Danho, 198 3). sheep erythrocytes (Witheiler and Wilson, 1972). human erythrocytes (Schonberger and Tsechesche, 1981; Jones and Manning, 1985 ; Jones et al., 1991), rabbit muscle (Radhakrishna and Wold, 1989) and human placenta (Unger, Nagelschmidt and Struck, 1979). The enzyme from rat liver has been studied in detail (Kobayashi et al., 1989) and successfully used to deblock the acetylated peptides for sequencing purpose. In this report we describe the presence of acylpeptide hydrolase in lenses from

various species and some of its properties. 0 1992 AcademicPressLimited EER 54

K. KRISHNA

2. Materials and Methods Materials

Bovine lenses were obtained from a local slaughter house. The rat and rabbit lenses were obtained from animals killed for experiments unrelated to the eye. Normal human lenses were collected from eyes donated to the Lion’s Eye Tissue Bank of Missouri. The lenses were immediately placed in Earle’s basic salts without phenol red and kept frozen until use. Few lenses were analyzed immediately. The substrates, Nacetyl-Ala-p-nitroanilide (NA), N-formyl-Met-p-NA, Nacetyl-Ala-Ala-Ala, Ala-p-NA, Me-O-Suc-Ala-Ala-ProVal, p-NA, N-acetyl-Ser-Gln-Asn-Tyr and N-acetylMet-Leu-Phe were obtained from Sigma Chemical Co. The inhibitors, bestatin, p-hydroxymercuryphenylsulphonate. benzamidine, phenylmethylsulfonylfforide (PMSF). and diisopropylfluorophosphate (DFP) were also obtained from the same source. The antibiotics, ampicillin and penicillin-G were also supplied by Sigma Chemical Co. All other chemicals were the highest grade commercially available. Preparation of Lens Extracts

Decapsulated bovine lenses were stirred with 50 mM Tris-HCl buffer (5 ml per lens), pH 7.8 at 5°C for 1 hr. The soluble proteins from the disrupted lens cells were decanted from the nuclei and centrifuged at 30000 g for 30 min. The supernatant was designated as the crude lens extract and used in further studies. In a separate experiment frozen bovine lenses were sliced and extracted as described eartier (Sharma and Ortwerth, 1986a) using Tris-HCI buffer. The extract from each slice contained about 20 mg of soluble protein. The human and rabbit lenses were individually homogenized with 2-O ml of buffer whereas a pair of rat lenses formed one sample. All the samples were centrifuged at 30000 g for 30 min and the supernatant removed and assayed immediately. Chromatography

of a Bovine Lens Extract on Sephadex

G-200

The crude extract from three bovine lenses (15 ml) was applied to a Sephadex G-200 column (2.5 x 70 cm) previously equilibrated with SO mM Tris-HCl, pH 7.8 and eluted with the same buffer at a flow rate of 20 m1 hr-‘. Ten ml fractions were collected and assayed for acylpeptide hydroiase activity and protein content. The fractions showing maximum enzyme activity (18-21 ; see Fig. 2 below) were pooled and assayed with various substrates and inhibitors. Thin Layer Chromatography

The hydroiysis of N-acetyl-Ala-Ala, N-acetyl-AlaAla-Ala, N-acetyl-Ala-Ala-Ala-Ala, Me-0-sue Ala-Ala-

SHARMA

AND

t3 J. ORTWERTH

Pro-Val-p-NA, N-acetyl-Ala, Ala-p-NA. acetyl-SerGln-Asn-Tyr, N-acetyl-Met-Leu-Phe was confirmed as follows. The reaction mixture containing 10 mM TrisHCl pH 7.8, 1 mM substrate, 0.1 mm bestatin and an aliquot of the Sephadex G-200 enzyme fraction were incubated for 6 hr at 37°C. The samples were then passed through a Sephadex G-10 column ( 1 .O x 30 cm) and l-ml fractions were collected. The fractions were concentrated and the products of enzyme reactions were identified by thin layer chromatography on silica gel plates. The plates were developed with butanol : acetone : acetic acid : water ( 7 : 7 : 2 : 4) and stained with ninhydrin. Appropriate control lanes with standard peptides were run simultaneously. Digestion of‘ a-Crystallin with Acylpeptide Hydrolase and N-terminal Sequencing

Purified ol-crystallin (500 ,ug) was incubated with 100 units of partially purified lens enzyme in the presence of 10 pmoles of Tris-HCl, pH 7.8 at 37°C for 12 hr. Aliquots were subjected to SDS gel electrophoresis and transferred (Matsudira, 1989) to PVDF membranes (Applied Biosystems ProBlot PVDF). The protein band corresponding to aA chain was subjected to sequencing on an Applied Biosystems, 470A gasphase protein sequencer coupled to an on-time PTH microbore analyzer. Enzyme Assay and General Methods

Acylpeptide hydrolase activity in the crude extract and Sephadex fractions was measured by continuously monitoring the hydrolysis of synthetic substrates. Aliquots of each extract, 50-200 ,ul, were mixed with 50 mM/Tris-HCl buffer, pH 7.8 in a total volume of 0.95 ml and incubated at 3 7°C in a water jacketed cuvette holder. Reactions were initiated by the addition of SO ,~l of substrate (1.2 pmol). The liberation of p-nitroaniline was continuously monitored in a Perkin Elmer Lambda 3 Spectrophotometer at 405 nm. One unit of activity corresponds to the amount of enzyme which catalyzes the hydrolysis of 1 nmol of substrate per minute. Protein concentration was determined by the BCA method as described by the supplier (Pierce) using bovine serum albumin as standard. The elution of protein during Sephadex G-200 chromatography was monitored by reading the absorbance at 280 nm. 3. Results The acylpeptide hydrolase activity was observed in the water-soluble fraction isolated from bovine, human, rabbit and rat lenses when assayed at pH 7.8. The data shown in Table I presents the relative enzyme activity with N-acetyl-Ala-p-nitroanilide as substrate. The rat lenses showed the highest specific activity whereas the bovine lenses exhibited the lowest. The ratio of activity

ACYLPEPTIDE

HYDROLASE

FROM

LENS

TABLE I

Activity of acylpeptide hydrolase in normal lenses of various species

Species Beef (n = 9)

Activity (units per mg protein) Meanf S.D.

Age

I__-_.-~---

Human (n = 10) 19-60 years Rabbit (n = 5) 1 year Rat (n = 9) 2 month Activity was measured using described under the methods.

-

0.38 kO.07 0.66&0.11 1~05+0~17 1.26 kO.04

6 years

N-acetyl-Ala-p-NA as substrate as

I

Fractlan

number

FIG. 2. SephadexG-200 gel filtration chromatography of bovine lens cortical extract. A 15ml sampleof cortical extract was applied to a 2.5 x 90 cm Sephadex column and eluted with 50 mM Tris-HCl, pH 7.8 at a flow rate of

20 ml h-l. Fractionsof 10 ml were collectedand assayedfor protein at 280 nm (0-O) and for acylpeptidehydrolase activity with N-acetyl-Ala-p-NA (0-O) at pH 7.8 as describedunder methods.

0

I

I

I

3

6

9

Slice

I

number

1. Distribution of acylpeptidehydrolase activity in bovine lens.Enzymeactivity wasdeterminedin each sliceof bovine lens(age6 years) asdescribedunder methodsusing N-acetyl-Ala-p-NA as substrate. Slice one representsthe outermost region while slice nine representsthe nuclear region. FIG.

shown). The release of p-nitroaniline and acetylalanine was also confirmed by thin layer chromatography indicating the cleavage of the peptide bond. In bovine lens, the maximal activity was located in the outer cortical region. Localization of enzyme activity showed a gradual decline in acylpeptide hydrolase activity from the equator to the lens nucleus. The nuclear region, however, still exhibited about 25 % of the activity seen in the outermost cortical layer. The results are presented in Fig. 1. Such a distribution study was not carried out with lenses from other sources. Chromatography of Crude LensExtract on SephadexG-

TABLE II

Relative activities of acylpeptide hydrolase from bovine lens towards various substrates Substrate

Relative rate of hydrolysis

N-Acetyl-Ala-p-NA N-Acetyl-Phe-p-NA N-Acetyl-Leu-p-NA N-Acetyl-Ala Ala-p-NA + Bestatin N-Formyl-Met-p-NA

100 2 1 0 0 42

The relative rate of hydrolysis was determined as described under experimental procedures using 1.0 mM concentration of each substrate except N-acetyl-Phe-p-NA which was used at 025 mM due to lower solubility.

was 1: 1.7 : 2.7 : 3.3 for bovine, human, rabbit and rat lens extracts respectively. This ratio was unaltered by the presence of EDTA and bestatin to inactivate any amino peptidases present in the extract (data not

200

The elution profile of a crude bovine lens extract on a Sephadex G-200 column is shown in Fig. 2. All fractions were assayed for acylpeptide hydrolase activity with N-acetyl-Ala-p-NA as substrate, and the enzyme activity was found to elute between the CL-and /?-crystallin peaks. The activity peak indicated a molecular weight of about 300 000 Da for the enzyme. The enzyme was stable at room temperature for 48 hr at neutral PH. Thereafter a gradual loss in the activity was observed. In crude preparations the maximal activity was observed at pH 7.5-8.0 (data not shown). As shown in Table II, the enzyme effectively cleaved N-acetyl and N-formyl substrates. Highest activity was observed when N-acetyl-Ala-p-NA was used as substrate. In addition, it was observed that Me-O-Suc-AlaAla-Pro-Val-p-NA was also cleaved to liberate a free amino group as judged by thin layer chromatography.

1008

K. KRISHNA TABLE

III

Effect oj various reagents on lens acylpeptide hydrolasr activity Relative activity Reagent None DFP PMSF p-OH mercuryphenyl sulfonate Penicillin-G Ampicillin Benzamidine Bestatin DTT EDTA

Concentration (mM) 0.5

Bovine

Human

100

100

0

0

1.0 1.0

51 67

48

1.0 1.0 1.0 1.0 1.0 I.0

17 38

28 19

100 100 100 100

100 100 100 100

Enzyme activity was measured after 15 min of incubation various reagents at room temperature using N-acetyl-Ala-pNA substrate.

6

with as

The hydrolysis of N-acetylated di, tri and tetra alanine in the presence of bestatin produced free alanine, alanylalanine and ala-ala-ala respectively. These products were identified in the reaction mixture by thin layer chromatography with appropriate standards. The relative rate of hydrolysis of these substrates was not determined. The lens extract did not hydrolyse either N-acetyl-Ala or Ala-p-NA under similar assay conditions indicating that the acylpeptide hydrolase activity did not exhibit deacylase or classical aminopeptidase activity. When the native a-crystallin was subjected to digestion by the lens acylpeptide hydrolase, a free Nterminus was not formed as evidenced by the failure to sequence aA chains by Edman degradation in a gas phase sequencer. However, under the same assay conditions, N-acetyl-Met-Leu-Phe as well as N-acetylSer-Gln-Asn-Tyr were readily cleaved to liberate free amino termini suggesting that the N-terminus in the intact protein is not available to the enzyme.

Eflect of Various Compounds on Enzyme Activity The bovine and human lens enzymes were preincubated for 30 min at 25°C in the presence of various reagents at 0.5 or 1.0 mM concentration. All assays were carried out with N-acetyl-Ala-p-NA as substrate and the data are shown in Table III. EDTA, bestatin and benzamidine did not inhibit the enzyme activity. A partial inactivation of the enzyme was observed with PMSF whereas DFP completely abolished the hydrolytic activity indicating an active site serine residue. Sulfhydryl blocking agents inhibited the enzymes from human and bovine sources to varying degrees but addition of dithiothreitol (DTT) did not alter the activity of native enzyme. Of the two antibiotics tested, penicillin-G was more effective as an inhibitor of the

SHARMA

AND

6. J. ORTWERTH

bovine enzyme than the human enzyme. An opposite pattern was observed with ampicillin. 4. Discussion The data presented here clearly demonstrate the presence of an enzyme which can liberate N-protected amino acids from the corresponding N-protected peptides. Such an enzyme has been isolated from various other mammalian tissues. The best substrate for these enzymes are N-acetyl derivatives of most commonly found N-terminal amino acid derivatives (Kobayashi and Smith, 1987 ; Radhakrishna and Weld. 1989). In our study the crude enzyme preparations showed higher activity against N-acetyl-Ala-pNA than N-formyl-Met-pNA. The enzyme was active on short peptide derivatives indicating that it may be active under in vivo conditions on peptides derived by proteolysis. The activity present in rat lens extracts is much higher than that present in bovine and human lens extract, but it is about three to four-fold lower than that reported for a rat liver extract (Kobayashi and Smith, 198 7). These differences in specific activity may be due more to changes in protein content than in absolute enzyme levels. The lower activity in human and bovine lens could also be due to the inactivation of the enzyme due to the longevity of the tissue or a lower initial activity. The enzyme activity distribution study with bovine lens supports the idea that the older lens fibre cells have a lower activity (Fig. 1). The enzyme activity retained in the nuclear region, however, is far greater compared to the residual aminopeptidase III in the nuclear region (Sharma and Ortwerth, 1986a). Only about 5% activity was seen in nuclear region compared to outer cortical region in the case of aminopeptidase III whereas nearly 2 5 y0 of acylpeptide hydrolase activity is still active in the nuclear region of bovine lenses. Loss of leucineaminopeptidase (Taylor et al., 1982-83) and neutral protease activity in nuclear region of lenses (Fleshman et al., 198 5) has been reported by others. A decrease in the activity of calpain I and II in the nuclear region of bovine lens extract has also been reported (Yoshida, Murachi and Tsukahara. 198 5). The elution profile of the enzyme from a Sephadex G-200 column and its inactivation by DFP and PMSF suggest that the lens enzyme is similar to the enzyme isolated from rat liver (Kobayashi and Smith, 1987). The inhibition studies with sullhydryl blocking agents indicates that a sullhydryl group may be required for the stabilization of the enzyme. The enzyme from human and bovine lenses was found to be sensitive to lactam antibiotics. The mode of interaction of the antibiotics with the enzyme is not known at present. Human liver alanine aminopeptidase is also inhibited by the same antibiotics (Starnes, Szechinsli and Behal, 1982). The susceptibility of the lens acylpeptide hydrolase to the antibiotics suggests that the pro-

ACYLPEPTIDE

HYDROLASE

FROM

LENS

longed use of these antibiotics to control infections of the eye may lead to an impairment of the normal function

of these enzymes. Further

studies are needed

to confirm this hypotheses. The inability of the lens enzyme to cleave N-acetylMet- from native a-crystallin under the conditions of successful

cleavage of most peptides suggests that the

enzyme may only be active on a-crystallin fragments in vivo. In fact, Radhakrishna, Chin and Wold (199 1) have shown that rabbit muscle acylpeptide hydrolase is active with a-crystallin derived fragments. These data together with the previous observation (Driessen et al., 1983) show that the tertiary structure of a-

crystallin

prevents the action of either acylpeptide

hydrolase or aminopeptidases on its N-terminus. The physiological function of acylpeptide hydrolase in lens, which is rich in N-acetylated proteins, is not obvious at present. Perhaps the enzyme has its principal role in epithelial cells or during cell differentiation to lens fibre ceils which involves rapid removal of enzymes and proteins no longer needed in metabolic processes. It is possible, however, that the acylpeptide hydrolase together with other proteolytic systems could as well be part of an intracellular protein

degrading

system, with

proteases and peptidases

designed to clear the damaged structural proteins in lens. The loss of enzyme activity in older fiber cells may

result in a lower degree of removal of N-terminally blocked

amino

proteins,

making

acids from

the degraded

these resistant

crystallin

to exopeptidase

action. This may lead to the increased accumulation

of

peptides observed in older fiber cells (Srivastava, 1988b). Acknowledgements The authors would like to express their appreciation to MS Sherry DiMaggio for typing this manuscript. This work was supported in part by NIH grant EY02035 and in part by Research to Prevent Blindness Inc. References Bloemendal, H. (Ed.) (1981). Molecular and Cellular Biology of the Eye Lens. Wiley: New York. Brown, J. L. and Robert, W. K. (1976). Evidence that approximately eighty percent of the soluble proteins from Ehrlich Ascites Cells are N-acetylated. J. Bioi. Chem. 251. 1009-14. Brown, J. L. (1979). A comparison of the turnover of d-Nacetylated and non-acetylated mouse L. cell proteins. 1. Biol. Chem. 254. 1447-9. David, L. L. and Shearer, T. R. (1989). Role of proteolysis in lenses: a review. Lens Toxicol. Res. 6(4), 725-47. Driessen. H. P. C., Ramaekers, P. C. S., Vree Egberts, W. T. M. et al. (1983). The function of Na-acetylation of the eye lens crystallins. Eur. 1. Biochem. 136, 403-6. Driessen. H. P. C.. de Jong, W. W., Tesser. G. I. and Bloemendai, H. (1985). The mechanism of N-terminal acetylation of proteins. CRC Crit. Rev. Biochem. 18(4), 281-325.

Fleshman. K. R.. Margolis, J. W., Fu, S.-C. J. and Wagner, B. J. (1985). Age changesin bovine lens endopeptidase activity. Mech.Aging Develop.31, 3747.

Gade,W. and Brown, J. L. (1978). Purification and partial characterization of a-N-acylpeptide hydrolase from Bovine Liver. J. Biol. Chem.253, 5012-8. Hanson, H. and Frohne, M. (1976). Crystalline leucineaminopeptidasefrom lens. In Methodsin Enzfimology, vol. 45. (Ed. Lorand L.). Pp. 504-21. AcademicPress: New York. Hershko, A. (1988). Ubiquitin-mediated protein degradation. I. Biol. Chem. 263. 1523740. Jahngen. J. H., Haas. A. L.. Ciechanover, A., Blondin, J.. Eissenhauer, D. andTayler. A. (1986). The eyelenshas an active ubiquitin-protein conjugationsystem.J. Biol. Chem.261, 13760-7. Jones. W. M. and Manning, J. M. (1985). Acylpeptide hydrolaseactivity from erythrocytes. Biochem.Biophys. Res.Commun.126, 93340. Jones, W. M., Scaloni, A., Bossa. F., Popowicz, A. M., Schneedwind,0. and Manning, J. M. (1991). Genetic relationshipbetweenacylpeptidehydrolaseand acylase, two hydrolytic enzymes with similar binding but differentcatalytic specificities.Proc.Natl Aced.Sci.U.S.A. 88, 2194-8. Jornvall, H. (1975). Acetylation of protein N-terminalamino groups. Structural observationson amino acetylated proteins.J. Theor.Biol. 55, 1-12. Kobayashi, K. and Smith, J. A. (1987). Acyl-peptide hydrolase from rat liver: Characterization of enzyme reaction. I. Biol. Chem. 262, 11435-45. Kobayashi.K.. Lin, L. W., Yeadon,J. E., Klickstein,B. L. and Smith, J. A. (1989). Cloningand sequenceanalysisof a rat liver cDNA encodingacyi-peptidehydrolase.J. Biol. Chem. 264, 8892-9.

Lafferty. M. A., Raducha.M. and Harris, H. (1984). Soluble exopeptidasesof bovine and human lens. Chnracteri:ation by electrophoresis. Cut-r. Eye Res. 3, 1017-31. Marks, N.. Lo. E. S., Stern, F. and Danho. W. (1983). Observationson N-deacetylationof modelamino acids peptides:Distribution and purification of a specificNacetyl amino acid releasingenzyme in rat brain, 1. Neurochem. 41. 201-8. Matsudaira, P. T. (Ed.) 1989. A practical guide to protein andpeptidepurification for microsequencing. Academic PressInc: San Diego. Radhakrishna,G. and Weld. F. (1989). Purification and characterization of an N-acylaminoacyl-peptidehydrolasefrom rabbit muscle.J. BioI. Chem.264, 1107681. Radhakrishna,G., Chin, C. C. 0. and Wold, F. (1991). NTerminal sequenceanalysisof blockedproteins using Nd-acylaminoacyl-peptidehydrolase. FASEB 1. S(4), A443. Ray, K. and Harris, H. (1985). Purification of neutral lens endopeptidase:Closesimilarity to a neutral proteinase in pituitary. Proc. Nutl Acad. Sci. U.S.A. 82, 7545-9. Schonberger,0. L., and Tschesche,H. (1981). N-Acetylalanine aminopeptidase,A new enzyme from human erythrocytes. Hoppe-Seylers Z. Physiol. Chem.362, S 865-73.

Sharma, K. K. and Ortwerth. B. J. (1986a). Aminopeptidase III activity in normal and cataractouslenses.Curr. Eye Res.5, 373-80.

Sharma. K. K. and Ortwerth, B. J. (1986b). Isolation and characterizationof a new aminopeptidase from bovine lens.1. Biol. Chem.261. 4295-301. Srivastava. 0. P. (1988a). Characterization of a highly purified membraneproteasefrom bovine lens.Exp. Eye Res. 46, 269-83.

Srivastava, 0. P. (1988b). Age related increase in concentration and aggregationof degradedpolypeptidesin human lenses.Exp. Eye Res.47. 52543.

1010

Srivastava, 0. P. and Ortwerth, B. J. (198 3). Isolation and characterization of a 2 SK serine proteinase from bovine lens cortex. Exp. Eye Res. 37, 597-612. Starnes, W. L., Szechchinski, J. and Behal, F. (1982). Human liver alanine aminopeptidase : A kinin-converting enzyme sensitive to @actam antibiotics. Eur. 1. Biochem. 124, 363-70. Swanson, A. A., Davis, R. M., Jackson, B. A. and McDonald, J. K. (1981). Lens exopeptidases. Exp. Eye Res. 32, 163-73. Taylor, A., Daimes, A. M., Lee, J. and Surgenor. T. (1982-83). Identification and quantification of Ieucineaminopeptidase in aged normal and cataractous human lenses and ability of bovine lens leucineaminopeptidase to cleave bovine crystallins. Cut-r. Eye Res. 2, 45-56. Taylor. A. and Davies, J. A. (1987). Protein oxidation and loss of protease activity may lead to cataract formation in the aged lens. Free Radical Biol. Med. 3. 371-77. Tsunasawa, S., Imanaka, T. and Nakazawa, T. (1983). Apparent diepetidyl peptidase activities of acyl-amino acid releasing enzymes. J. Biochem. (Tokyo) 3. 12 17-20.

K. KRISHNA

SHARMA

AND

B. J. ORTWERTH

Tsunasawa. S. and Sakiyama, F. (1984). Aminoterminal acetylation of proteins: An overview. In Methods in Enzymology, Vol. 106. (Eds Weld. F. and Moldave, Ii.). Pp. 165-70. Academic Press: New York. Tsunasawa, S., Narita. K. and Ogata. K. ( I 9 7 5 ). Purification and properties of acylamino acid-releasing enzyme from Rat Liver. 1. Biochem. 77, 89-102. Unger. T.. Nagelschmidt. M. and Struck, H. (1979). Nacetyiaminoacyl-p-nitranilidase from human placenta. Purification and some properties. Eur. 1. Bioc’hem. 97, 205-11. Witheiler. J. and Wilson, D. B. (19 72). The purification and characterization of a novel peptidase from sheep red cells. /. Biol. Chem. 247, 2217-2 1. Yoshida, H.. Murachi, T. and Tsukahara, I. ( 1984). Limited proteolysis of bovine lens crystallin by calpain. a Ca’+dependent cysteine proteinase. isolated from the same tissue. Biochem. Biophys. Acta 798, 252-9. Yoshida, H., Murachi, T. and Tsukahara. I. (1985). Distribution of calpain I, calpain II, and calpastatin in bovine lens. Invest. Ophthalmol. Vis. Sci. 26, 953-6.