Biomedical subjects

B J Ortwerth

Publications and source records attributed to B J Ortwerth.

At least 55 records · Page 3Linked to original sources

PubMed1992

Glycation of MP26 and MP22 in bovine lens membranes.

Alkali treated membranes were isolated from mature bovine lenses and incubated with different sugars for 3 weeks to study the effect of glycation on the lens intrinsic membrane proteins, MP26 and MP22. The obtained results show that a) [1-14C] ascorbic acid (ASA) was able to glycate the intrinsic membrane proteins as rapidly as soluble lens proteins; b) on 15% acrylamide gels in SDS, glucose, fructose, galactose and ribose exhibited low activity for crosslinking membrane proteins; whereas ASA, dehydroascorbate (DHA), diketogulonate (DKG), xylosone and threose, all showed not only the formation of protein multimers, but also highly crosslinked products, which did not enter the spacer gel; c) except glycated MP22, all of the crosslinks of MP26 or MP22, and also the glycated MP26, showed cross reactivity with polyclonal MP26 antibody; d) the extent of crosslinking correlated with an equal loss of lysine and arginine contents by amino acid analysis.

Animals↗

PubMed1992

Studies on the solubilization of the water-insoluble fraction from human lens and cataract.

Studies were carried out comparing the ability of urea extraction and sonication to solubilize the water-insoluble (WI) protein fraction from human lens tissue. Sonication and urea extraction were able to solubilize greater than 80% of the insoluble protein whether whole lenses or lens nuclei were used. This was true for normal lens and +1 cataracts; however, only 60% solubilization was obtained with the WI fraction from more advanced cataracts. Equal aliquots of a WI fraction from both pooled normal and pooled cataract lens nuclei were solubilized with and without reducing agents. The addition of dithiothreitol (DTT) had no significant effect on solubilization of the normal lens WI fraction. DTT did increase the protein solubilized from the cataract WI fraction by 30% with urea extraction; however, no increase was seen with sonication. When sodium borohydride was used as the reducing agent, essentially the same results were obtained. The solubilized protein populations were identical by SDS-PAGE and amino acid analysis. The addition of reducing agents had no effect on the amino acid content of the solubilized proteins with the single exception of lysine. This amino acid was markedly decreased in the proteins extracted in the presence of 40 mM sodium borohydride, but not with DTT. These data suggest that the borohydride not only increased the amount of protein solubilized, but likely also stabilized glycated lysine residues during the acid hydrolysis. Therefore, sonication readily provides a soluble preparation of the WI proteins from normal and cataract lens nuclei without the need for denaturing agents, however, disulfide-linked and lysine modified crystallins were best solubilized with urea.

Amino Acids↗

PubMed1992

Characterization of the elastase inhibitor properties of alpha-crystallin and the water-insoluble fraction from bovine lens.

Alpha-crystallin exhibits variable inhibition of several members of the chymotrypsin family of proteinases. Complete inhibition of elastase was obtained by the addition of either alpha-crystallin or a sonicated preparation of the water-insoluble fraction from bovine lens. Little or no inhibition was seen, however, with either beta-crystallin or bovine serum albumin under the same conditions. Complete binding of elastase was demonstrated by Sephadex G-100 gel filtration chromatography, and a direct correlation between binding and inhibition was obtained. This observation permitted us to do a Scatchard analysis of the inhibition data. Scatchard plots for the binding of elastase gave a biphasic response suggesting two separate binding sites. These sites had Kd values of 15 and 40 nM for alpha-crystallin and 6 and 42 nM for the bovine water-insoluble fraction. Similarly, a Dixon plot exhibited a Ki value of 3 nM and was consistent with non-competitive inhibition. One mole of alpha-crystallin (8 x 10(5) Da), or an equivalent amount of water-insoluble protein, bound from 13 to 19 mol of elastase which were about equally divided between the higher and lower affinity sites. Saturation studies confirmed 20 and 16 elastase binding sites per 8 x 10(5) Da for alpha-crystallin and water-insoluble protein, respectively. DFP-elastase was capable of binding to alpha-crystallin suggesting that a proteolytic cleavage was not required for complex formation. Stability measurements showed a linear return to 60% of the original activity over a 30-min period. Therefore, the interaction between elastase and alpha-crystallin resembles that of a heterologous protease:inhibitor complex in both binding and stability.

Animals↗

PubMed1992

The glycation and cross-linking of isolated lens crystallins by ascorbic acid.

Individual lens crystallins were isolated from calf lens extracts and incubated in the presence of ascorbic acid for 3 weeks under aerobic conditions. Both alpha-crystallin and beta H-crystallin rapidly cross-linked to form high molecular weight proteins, which did not enter the resolving gel on SDS-PAGE. Beta L-crystallin was somewhat less reactive, but gamma-crystallin showed little or no crosslinking. Gamma-crystallin, however, was almost equivalent to the other crystallins as a substrate for glycation. This was measured by: (a) the binding of protein to a boronate affinity column; (b) the incorporation of 3H from NaB3H4 into protein; (c) amino acid analysis of the modified proteins to estimate the extent of lysine modification; and (d) the incorporation of [1-14C]ASA into individual crystallins. When the separated crystallins were combined with [125I]gamma-crystallin and incubated with ascorbic acid, radioactivity was readily incorporated into the cross-linked products with other crystallins, but again not with gamma-crystallin itself. Gel filtration chromatography of a mixture of [125I]gamma-crystallin and alpha-crystallin showed the formation of a complex between gamma- and alpha-crystallins. These data suggest that all crystallins are glycated, but that cross-linking occurs preferentially between proteins, which are already bound together non-covalently.

Amino Acids↗

PubMed1992

The effect of urea on the aggregate state and elastase inhibitor activity of the water-insoluble fraction from bovine and human lens.

Preparations of alpha-crystallin from bovine and human lens exhibited elastase inhibitor activity with a specific activity of 100-250 U mg-1 protein. A washed water-insoluble fraction from bovine, human and cataractous lens nuclei, when solubilized by sonication, gave specific activities of 910, 950 and 1270 U mg-1, respectively. Disaggregation of these water-insoluble fractions in 8.0 M urea, with subsequent reaggregation by urea removal, resulted in a decrease in inhibitor activity. Agarose A-5m gel filtration chromatography after the urea treatment resolved a residual high molecular weight (HMW) fraction and a peak which eluted at the position of water soluble alpha-crystallin. Assays showed that the urea-induced 'alpha-crystallin' peaks from all three preparations had specific activities, equivalent to native alpha-crystallin, whereas the HMW fractions retained their original high specific activity. We conclude that the increased elastase inhibitor activity of the water-insoluble fraction is a property of the aggregate state of the component alpha-crystallin molecules, which is lost upon reaggregation to an 800-kDa alpha-crystallin. Amino acid analysis of the bovine water-insoluble fraction suggested a content of 85-90% alpha-crystallin and 10-15% beta H-crystallin, which was confirmed by SDS-PAGE.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

PubMed1992

Description of an acylpeptide hydrolase from lens.

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 alpha and beta crystallins 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)2, Me-O-Suc-Ala-Ala-Pro-Val-p-NA, N-Acetyl-Met-Leu-Phe, Acetyl-Ser-Gln-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 alpha A-crystallin in vitro suggesting that the N-terminus of native crystallins is not accessible to the enzymes.

Adult↗

PubMed1991

High correlation between pentosidine protein crosslinks and pigmentation implicates ascorbate oxidation in human lens senescence and cataractogenesis.

Pentosidine is a recently discovered protein crosslink, involving lysine and arginine residues linked together in an imidazo [4,5,6] pyridinium ring formed by a 5-carbon sugar during nonenzymatic browning (Maillard reaction). The presence of high ascorbate levels in the human lens and its ability to undergo nonenzymatic browning led us to investigate pentosidine formation in the aging human lens. Incubation of lens crystallins with ascorbate and its oxidation products dehydroascorbate and 2,3-diketogulonate leads progressively to the formation of pentosidine crosslinks in the presence of oxygen. Under nitrogen, however, pentosidine forms only from 2,3-diketogulonate or xylosone, a degradation product of 2,3-diketogulonate. A high correlation between pentosidine crosslinks and the degree of lens pigmentation is noted in cataractous lenses. Pentosidine is found to be primarily associated with alpha-crystallin fractions of 300-5000 kDa. These results suggest that redox imbalance in cellular senescent systems such as the ocular lens may lead to irreversible ascorbate oxidation and protein crosslinking by xylosone. This mechanism may play an important role in the pathogenesis of "brunescent" cataracts.

Adult↗

PubMed1991

Transfer RNA(4lys) and cell cycle progression: characterization of ts-694 cells.

Lysine tRNA modification has been studied in mammalian ts-694 cells with respect to cell cycle progression in temperature downshift and upshift experiments. The modification of tRNA(lys) measured in temperature downshift experiments showed that tRNA(4lys) levels start to increase 6 h following the temperature shift, approximately 10-12 h prior to the cells entry into S phase. Ts-694 cells showed a gradual decrease in the level of tRNA(4lys) and the rates of DNA synthesis following a temperature upshift. The cells became growth arrested following incubation for 36-45 h at the rt. Cell cycle mapping of the temperature restriction point suggests a G1 block prior to the serum deprivation restriction point. Depletion of cellular tRNA(4lys) by serum deprivation followed by simultaneously shifting cells to the rt and feeding medium containing 10% serum showed that cells with low tRNA(4lys) levels and no mechanism for the synthesis of tRNA(4lys) could not enter S phase and synthesize DNA. Blocking of ts-694 at the G1/S boundary with aphidicolin indicates that cells that have passed through G1 are capable of entering S phase and synthesizing DNA independent of the incubation temperature. These results indicate that tRNA(4lys) is not needed during S phase for DNA replication but suggests that tRNA(4lys) is required for cells to progress through G1.

Animals↗

PubMed1991

The glycation-associated crosslinking of lens proteins by ascorbic acid is not mediated by oxygen free radicals.

The reaction by which ascorbic acid (ASA) causes the glycation and crosslinking of lens proteins displays a rigid requirement for the presence of oxygen, and is inhibited by the presence of glutathione. Oxygen is required to oxidize ASA to dehydroascorbic acid (DHA) and other products which are the active glycating species. No evidence could be found to support a role for oxidative protein crosslinking by a free radical mechanism. Crosslinking was not inhibited by blocking protein sulfhydryl groups with iodoacetamide, nor were the protein crosslinks dissociated by boiling with 2% mercaptoethanol prior to SDS-PAGE. The addition of a variety of oxygen free radical quenchers had no effect on the extent of protein crosslinking. In fact, the removal of oxygen from the reaction mixture had no effect on either protein glycation, protein crosslinking or the modification of lysine residues, provided DHA was used as the glycating agent. All of these activities were inhibited, however, if ASA was the glycating agent. This confirms that oxygen is required only to convert ASA to DHA.

Animals↗

PubMed1990

Glycation of lens proteins by the oxidation products of ascorbic acid.

Bovine lens water-soluble proteins were incubated with [I-14C]ascorbic acid (ASA) for 6 days, and the incorporation into protein was measured at daily intervals. Aliquots were also withdrawn to determine the distribution of label among the various ASA oxidation products. A linear incorporation into protein was observed in the presence of NaCNBH3, however, little or no incorporation was seen in its absence. TLC analysis showed a complete loss of ASA by day 3, whereas both dehydroascorbate (DHA) and diketogulonic acid (DKG) remained constant for 6 days, consistent with the linear incorporation into protein. The amino acid composition of the proteins glycated in the presence of NaCNBH3 was identical to controls except for a 70% reduction in lysine residues and a corresponding increase in an unknown product which eluted slightly earlier than methionine. In the absence of NaCNBH3 lysine decreased linearly to 20% with an additional decrease in arginine and histidine at later times concurrent with protein crosslinking. DHA and DKG were prepared and incubated directly with lens proteins for an 8 day period. Both compounds glycated lens protein as evidenced by an increased binding to a boronate affinity column. SDS-PAGE showed that both compounds were also capable of causing protein crosslinking. DHA is apparently capable of reacting directly with protein since glycation was observed with the ASA analog, reductic acid, which can be oxidized to dehydroreductic acid, but which cannot be hydrolyzed to an open chain structure. DHA also produced a lysine adduct which was not obtained with DKG, supporting the idea that both species have glycating ability.

2,3-Diketogulonic Acid↗

PubMed1989

Studies on the nature of the water-insoluble fraction from aged bovine lenses.

The water-insoluble fraction from mature bovine lens was solubilized to the same extent either by extraction with 6.0 M urea, by sonication of the suspended proteins or by a brief adjustment of the pH to 3.0 or 11.0. Sonication gave soluble protein levels of 50 mg ml-1 or greater with water or dilute buffers, but the presence of salt markedly diminished the solubility of the sonicated proteins. The sonicated proteins remained soluble upon storage at 5 degrees C, but were readily precipitated by either freezing or by the addition of salt. These re-precipitated proteins were once again insoluble when suspended in dilute aqueous buffers. Water-soluble alpha-crystallin at the same concentrations was unaffected by either high salt or freezing. The sonication-solubilized proteins were shown to be similar in aggregate size and polypeptide composition to the water-soluble HMW fraction isolated from the same lenses. An [125I]-labeled soluble HMW fraction was precipitated to the same extent as [125I]-labeled sonication-solubilized proteins upon freezing. The distribution of HMW aggregated protein between water-soluble aggregates and the water-insoluble fraction was unaltered by the presence of either dithiothreitol (DTT) or high levels of salt during the homogenization. The presence of either [125I]-labeled water-soluble HMW aggregates or [125I]-labeled water-insoluble sonicate supernatant during lens homogenization did not result in a significant incorporation of radioactivity into the water-insoluble fraction. These data argue that the water-insoluble fraction represents coalesced HMW aggregates which had already formed in the lens prior to homogenization. When the sonication-solubilized fraction was disaggregated in 6.0 M urea and then reaggregated by urea removal, the proteins no longer precipitated on freezing, and 85-90% of the protein eluted in the region of alpha-crystallin from an Agarose A-5m column. Only 3-6% of the original protein remained as a void volume peak, and was composed almost exclusively of highly crosslinked proteins. The limited solubility of the HMW proteins may therefore reflect the aggregate state of the alpha-crystallin rather than an inherent insolubility of the subunits.(ABSTRACT TRUNCATED AT 400 WORDS)

Aging↗

PubMed1989

The effects of aging and cataract formation on the trypsin inhibitor activity of human lens.

Assays were carried out to determine the trypsin inhibitor activity present in the water-soluble and water-insoluble fractions of human lenses of various ages. Little change was seen in the inhibitor activity of the water-soluble protein fraction. When this fraction was chromatographed on an Agarose A-1.5 m column, however, the inhibitor activity was increasingly associated with the high molecular weight (HMW) protein fraction with age. A gradual increase in water-insoluble inhibitor was seen up to age 60, which correlated with the increase in protein in this fraction. After age 60, a marked increase in the water-soluble inhibitor activity was observed. In 80-90-yr old lenses, 1 mg of water-insoluble protein was able to inhibit 200 micrograms of crystallin trypsin by 50%. Similar assays on a collection of cortical and brunescent cataracts also showed very high levels of water-insoluble inhibitor activity. In most cases, these values were higher than those for the age-matched control lenses. Fractionation of the water-insoluble proteins showed that the bulk of the activity remained with the urea-insoluble fraction in cataractous lenses. A low molecular weight trypsin inhibitor was isolated from the water-soluble and water-insoluble fractions of human lenses. An age-dependent increase in this inhibitor was observed by activity measurements and electrophoretic analysis.

Adolescent↗

PubMed1988

Ascorbic acid-induced crosslinking of lens proteins: evidence supporting a Maillard reaction.

The incubation of calf lens extracts with 20 mM ascorbic acid under sterile conditions for 8 weeks caused extensive protein crosslinking, which was not observed with either 20 mM sorbitol or 20 mM glucose. While no precipitation was observed, ascorbic acid did induce the formation of high-molecular-weight protein aggregates as determined by Agarose A-5m chromatography. Proteins modified by ascorbic acid bound strongly to a boronate affinity column, however, crosslinked proteins were present mainly in the unbound fraction. These observations suggest that the cis-diol groups of ascorbic acid were present in the primary adduct, but were either lost during the crosslinking reaction or sterically hindered from binding to the column matrix. The amino acid composition of the ascorbic acid-modified proteins was identical to controls except for a 15% decrease in lysine. Amino acid analysis after borohydride reduction, however, showed a 25% decrease in lysine, a 7% decrease in arginine and an additional peak which eluted between phenylalanine and histidine. Extensive browning occurred during the ascorbic acid-modification reaction. This resulted in protein-bound chromophores with a broad absorption spectrum from 300 to 400 nm, and protein-bound fluorophores with excitation/emission maxima of 350/450 nm. A 4 week incubation of dialyzed crude lens extract with [1-14C]ascorbic acid showed increased incorporation for 2 weeks, followed by a decrease over the next 2 weeks as crosslinking was initiated. The addition of cyanoborohydride to the reaction mixture completely inhibited crosslinking and increased [1-14C]ascorbic acid incorporation to a plateau value of 180 nmol per mg protein. Amino acid analysis showed a 50% loss of lysine, and 8% decrease in arginine and the presence of a new peak which eluted slightly earlier than methionine. These data are consistent with the non-enzymatic glycation of lens proteins by either ascorbic acid or an oxidation product of ascorbic acid via a Maillard-type reaction.

Amino Acids↗

PubMed1988

The precipitation and cross-linking of lens crystallins by ascorbic acid.

Bovine lens beta-crystallin was incubated with increasing concentrations of sugars and sugar derivatives for a period of 2 weeks in the dark at 37 degrees C. Marked protein precipitation and a browning reaction was observed with both ascorbic acid (ASA) and dehydroascorbic acid (DHA), but little or no reaction was seen with several other sugars and sugar analogs. Similar incubations were carried out with 20 mM ASA, 20 mM DHA and 20 mM glucose, but with increasing amounts of the individual crystallins. Glucose was capable of precipitating gamma-crystallin in the presence of air, but this reaction was decreased if dithiothreitol and a chelating agent were added prior to incubation. ASA and DHA produced precipitation and browning with gamma- and beta-crystallin, but not with alpha-crystallin or lens soluble proteins. Similar reactivities were observed both in air and under reducing conditions. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis of these reaction mixtures showed little or no cross-linking with any of the lens proteins by glucose. ASA and DHA caused detectable dimer formation with gamma-crystallin, but produced the formation of dimers as well as highly polymerized proteins at the top of the gel with all the other crystallins and with lens soluble proteins. A time-course experiment with alpha-crystallin in the presence of air showed no cross-linking with 100 mM glucose over a 6-week period; however, 10 mM ASA caused definite cross-linking at 2 weeks, and at 6 weeks a dark smear of protein was visible throughout the gel. ASA was still capable of inducing cross-linking under low oxygen conditions but the protein smearing was markedly diminished. Further, the cross-linking pattern was similar to that seen in the water-insoluble fraction from older human lenses and cataracts. This reaction may be significant in vivo because cross-linking was observed under low-oxygen conditions with as little as 2 mM ASA, which is the level of ASA normally present in human lenses.

Animals↗

PubMed1988

Glutathione inhibits the glycation and crosslinking of lens proteins by ascorbic acid.

The incubation of crude extracts of bovine lens with 20 mM ascorbic acid leads to the formation of covalent adducts even in the presence of saturating levels of a metal chelator. When dialysed lens extracts were used both ASA-protein adducts and highly crosslinked lens proteins were observed which are similar to those found in the water insoluble fraction from cataractous lenses. Both adduct formation and protein crosslinking, however, were markedly inhibited if undialysed lens extracts were used or if increasing concentrations of glutathione were added to the incubation mixture. Similar inhibition was seen with cysteine, dithiothreitol and sodium bisulfite, but little effect was observed with the glutathione analog ophthalmic acid or with free radical quenchers. Glutathione was readily oxidized during the incubation and no oxidation of ascorbic acid was observed until all the reduced glutathione was exhausted. No loss of ascorbic acid and no protein crosslinking were observed when oxygen was completely removed from the reaction mixture. These data strongly suggest that the glycating species was an oxidized form of ascorbic acid. Ascorbic acid solutions displayed a rapid oxidation in vitro, which was decreased 80-fold upon the addition of 1 mM chelator and was completely inhibited by both glutathione and chelator. A rapid decrease in the level of dissolved oxygen was seen in the presence of ascorbic acid or ascorbic acid and glutathione, but not with glutathione alone. These data argue that glutathione inhibits glycation by rapidly reducing dehydroascorbic acid back to ascorbic acid, which is not active in protein glycation

Animals↗

PubMed1987

The binding and inhibition of trypsin by alpha-crystallin.

One of the major lens-structural proteins, alpha-crystallin, is a multimeric protein containing 40 subunits of approx. 20 kDa each. There are two subunit types with distinct but similar structures. This protein was capable of inhibiting trypsin, chymotrypsin and elastase, but had no effect on thrombin or kallikrein. Complete inhibition was not observed, but rather plateau levels of inhibition were obtained in each case. Maximum inhibition was observed at a ratio of 1 mol of alpha-crystallin for every 9-10 mol of trypsin. alpha-Crystallin also inhibited the labeling of the active site of trypsin by [3H]diisopropyl fluorophosphate (DFP). Greater than 90% inhibition of DFP labeling was observed at a ratio of 1 mol of alpha-crystallin for every 7-8 mol of trypsin. Both trypsin and [3H]DFP-labeled trypsin formed a complex with alpha-crystallin, as demonstrated by gel-filtration chromatography. The active site of trypsin when bound to alpha-crystallin was still capable of reacting with p-nitrophenyl p-guanidobenzoate and soybean trypsin inhibitor, but was inaccessible to alpha 1-antitrypsin. These data suggest that alpha-crystallin acts as a multivalent modified inhibitor which is consistent with the proposed quaternary structure of alpha-crystallin.

Animals↗

PubMed1987

A preferential role for lysyl-tRNA4 in the synthesis of diadenosine 5',5'''-P1,P4-tetraphosphate by an arginyl-tRNA synthetase-lysyl-tRNA synthetase complex from rat liver.

The synthesis of diadenosine 5',5'''-P1,P4-tetraphosphate (Ap4A) can be catalyzed in vitro by a tetrameric tRNA synthetase complex from rat liver containing two lysyl-tRNA synthetase and two arginyl-tRNA synthetase subunits. This reaction required ATP, AMP, 50-100 microM zinc, and inorganic pyrophosphatase. We show here that AMP can be omitted from the reaction and that the zinc levels can be markedly reduced provided catalytic amounts of tRNA(Lys) are added to the reaction mixture. Ap4A synthesis with purified tRNA(Lys) isoacceptors showed that the minor species, tRNA(4Lys), was 3-fold more active than either of the two major tRNA(Lys) species, tRNA(2Lys) and tRNA(5Lys). No activity could be demonstrated with tRNA(Lys) from Escherichia coli or with tRNA(Lys) or tRNA(Phe) from yeast. Aminoacylation of tRNA(4Lys) was strictly required as determined by the fact that Ap4A synthesis was not observed until aminoacylation was nearly complete, inhibitors of aminoacylation blocked Ap4A synthesis, and there was a strict requirement for added lysine. None of the above observations could be demonstrated, however, when lysyl-tRNA(Lys) was directly supplied to the reaction mixture. Optimum Ap4A synthesis was obtained by the addition of 1 mol of tRNA(Lys)/mol of the synthetase complex. This reaction is unique because it does not require the prior formation of an aminoacyl-AMP intermediate and because it can actively synthesize Ap4A at physiological zinc concentrations. The preferential role for tRNA(4Lys) in Ap4A synthesis is consistent with its prior implication in cell division.

Adenine Nucleotides↗

PubMed1987

Purification and characterization of an aminopeptidase from bovine cornea.

An aminopeptidase from bovine cornea has been extensively purified by gel filtration and ion-exchange chromatography. The purified enzyme with a molecular weight of 96,000 showed broad substrate specificity. All of the various aminoacyl bonds were hydrolyzed optimally at pH 6.5. The purified enzyme showed hydrolytic activity towards bioactive peptides such as enkephalins, bradykinin, and angiotensin-II. The enzyme was inhibited by bestatin, amastatin, puromycin, bacitracin, sulfhydryl reagents and metal chelators. Only Co2+ stimulated the enzyme whereas other heavy metal ions were toxic. The gross properties of the corneal enzyme resemble those of an aminopeptidase III isolated from bovine lens.

Aminopeptidases↗