Are the proteinase inhibitory activities in lenticular tissues real?

The possibility of proteinase inhibitory activities in lenses measured with synthetic substrates being spurious, due to the effective competition of lens proteins as substrates for the target enzymes, was investigated. Goat, sheep and human cataractous lens proteins were found to be poor substrates for trypsin, elastase and papain compared to casein or bovine serum albumin. Further, the inhibition of elastase catalyzed hydrolysis of succinyl trialanyl p-nitroanilide by casein (500 μg, 53%) and albumin (500 μg, 49%) and of trypsin-catalyzed hydrolysis of benzoyl argininep-nitroanilide by albumin (1 mg, 24%) were significant only at high protein concentrations. These data indicated that the relatively high antielastase and antitryptic activities observed in human cataractous lenses were real. On the other hand, coincident lens protein hydrolysis elevating the true antitryptic and antielastase activities in goat and sheep lenses (that have low activities) could not be ruled out The lesser papain inhibitory activities observed in lenses when albumin was used as substrate compared to activities with benzoyl arginine p-nitroanilide as substrate, appeared to be partly due to lens protein hydrolysis masking the actual inhibition in the former method. Preincubation of goat, sheep and human lens extracts with trypsin for 1 h resulted in complete loss of antitryptic and antielastase activity except in the case of human lens antielastase activity which underwent 50% loss. Papain inhibitory activity was fully stable. Similar papain treatment caused loss of 80–100% of antielastase activity and 45–55% loss of antitryptic activity.     

Increased content of zinc and iron in human cataractous lenses

The purpose of the study was to examine the zinc and iron content of human lenses in different types of cataract and to investigate the possible influence of diabetes on the zinc and iron content of the lens. Iron and zinc of 57 human lenses (28 corticonuclear cataracts and 29 mature cataracts with a mean age of 70.6±16.1 and 74.7±11.1 yr, 41 nondiabetics and 16 diabetics) were determined by atomic absorption spectroscopy. The zinc content of human lenses was significantly increased in mature cataracts compared to corticonuclear cataracts (0.51±0.33 vs 0.32±0.20 μmol/g dry mass, p=0.012). The iron content of mature cataracts was also higher than in corticonuclear cataracts (0.11±0.09 vs 0.07±0.05 μmol/g dry mass, p=0.071). Furthermore, a significant increase of the lens zinc content could be observed with increasing lens coloration (light brown 0.33±0.17 vs dark brown 0.52±0.35 μmol/g dry mass, p=0.032). Diabetic patients seem to have both increased zinc and iron contents in the lens compared to nondiabetic subjects (zinc: 0.45±0.42 vs 0.40±0.22 μmol/g dry mass; iron: 0.12±0.10 vs 0.08±0.05 μmol/g dry mass). These data suggest a possible influence of the lens zinc and iron content on the development of lens opacification. Especially advanced forms of cataract and dark brown colored lenses show significantly increased zinc and iron content.     

Significance of microflora in proteolysis in the colon

Protease activities in human ileal effluent and feces were compared by using a variety of native and diazotized protein substrates. In many cases the diazotized proteins had altered susceptibilities to hydrolysis compared with the native proteins. Proteolytic activity was significantly greater than (P less than 0.001) in small intestinal effluent than in feces (319 +/- 45 and 11 +/- 6 mg of azocasein hydrolyzed per h per g, respectively). Moreover, fecal proteolysis was qualitatively different in that ileal effluent did not hydrolyze the highly globular protein bovine serum albumin, whereas all fecal samples tested degraded this substrate. Inhibition experiments provided further evidence that fecal protease activity differed from that in the small intestine. Physical disruption of fecal bacteria released large quantities of proteases, indicating that the lysis of bacteria in the colon may contribute to the extracellular proteolytic activity in feces. Protease inhibition studies with washed fecal bacteria showed that they produced serine, cystine, and metalloproteases, and experiments with synthetic p-nitroanilide substrates indicated that low levels of trypsin- and chymotrypsin-like activities were associated with whole cells. An elastase-like enzyme was bound to the outer membranes of some fecal bacteria.     

Significance of microflora in proteolysis in the colon.

Protease activities in human ileal effluent and feces were compared by using a variety of native and diazotized protein substrates. In many cases the diazotized proteins had altered susceptibilities to hydrolysis compared with the native proteins. Proteolytic activity was significantly greater than (P less than 0.001) in small intestinal effluent than in feces (319 +/- 45 and 11 +/- 6 mg of azocasein hydrolyzed per h per g, respectively). Moreover, fecal proteolysis was qualitatively different in that ileal effluent did not hydrolyze the highly globular protein bovine serum albumin, whereas all fecal samples tested degraded this substrate. Inhibition experiments provided further evidence that fecal protease activity differed from that in the small intestine. Physical disruption of fecal bacteria released large quantities of proteases, indicating that the lysis of bacteria in the colon may contribute to the extracellular proteolytic activity in feces. Protease inhibition studies with washed fecal bacteria showed that they produced serine, cystine, and metalloproteases, and experiments with synthetic p-nitroanilide substrates indicated that low levels of trypsin- and chymotrypsin-like activities were associated with whole cells. An elastase-like enzyme was bound to the outer membranes of some fecal bacteria.     

晶状体生化的研究:正常和亚硒酸钠诱发白内障大鼠晶状体中与谷胱甘肽代谢有关的三种酶活性的测定

测定了用亚硒酸钠诱发的大鼠白内障晶状体中谷胱甘肽过氧化物酶(GSH-Px)、谷胱甘肽还原酶(GSSG-R)和谷胱甘肽硫转移酶(GSH-S)的活性,并与正常晶休中这三种酶的活性作了比较。结果表明,核浊浑期晶状体中GSH-Px的活性比正常晶状体的高一倍,但在整个晶状体浑浊时降低,GSSG-R的活性变化与GSH-PX相似,这两种酶在代谢上是相关的。GSH-S的活性在核浑浊期不改变,但在完全浑浊后降低。     

Post-translational modifications in the nuclear region of young, aged, and cataract human lenses

The urea-soluble proteins from the nucleus of two young, two aged, and two early-stage nuclear cataract lenses were subjected to tryptic digestion and analysis by 2D LC-MS/MS. Several novel post-translational modifications were identified. Deamidation was, by far, the most common modification. A number of differences were found in cataract compared to normal lenses, most notably an increase in the number of oxidized tryptophan residues. Semiquantitative analysis revealed that there appeared to be a trend toward increased levels of deamidation with age; however, there was no apparent increase upon the onset of nuclear cataract. This is in contrast to Trp oxidation, where an increase in the extent of modification was apparent in cataract lenses when compared to aged normal lenses. These findings suggest Trp oxidation may be involved in nuclear cataract development.     

Decomposition of H2O2 by human cataractous lenses

It was shown that human lens opacity was accompanied by the decrease in the lens ability to cleave H2O2 (10(-4) M), added to the lens-surrounding medium. The rate of peroxide decomposition at the stage of mature cataract in isolated human lenses was 3.5 times lower than that of the control human lenses (transparent lens, initial cataract). Specific catalase inhibitor--3-amino,IH-1,2,4-triazole showed no significant influence on the rate of H2O2 cleavage. Reduced glutathione (10 microM) added to the lens incubation medium induced a sharp increase in the rate of H2O2 detoxication. The results indicate that reduced glutathione metabolism is of primary importance in the maintenance of anti-peroxide activity in the lens.     

Heat shock proteins of adult and embryonic human ocular lenses.

We investigated the presence and distribution of heat shock proteins, HSP-70 [Horwitz, J. 1992. Proc Natl Acad Sci 89:10449-10453], HSP-40, HSc-70, HSP-27, and alphabeta-crystallin in different regions of adult and fetal human lenses and in aging human lens epithelial cells. This study was undertaken because heat shock proteins may play an important role in the maintenance of the supramolecular organization of the lens proteins. Human adult and fetal lenses were dissected to separate the epithelium, superficial cortex, intermediate cortex, and nucleus. The water soluble and insoluble protein fractions were separated by SDS-PAGE, and transferred to nitrocellulose paper. Specific antibodies were used to identify the presence of heat shock proteins in distinct regions of the lens. HSP-70 [Horwitz, 1992], HSP-40, and HSc-70 immunoreactivity was mainly detected in the epithelium and superficial cortical fiber cells of the adult human lens. The small heat shock proteins, HSP-27 and alphabeta-crystallin were found in all regions of the lens. Fetal human lenses showed immunoreactivity to all heat shock proteins. An aging study revealed a decrease in heat shock protein levels, except for HSP-27. The presence of HSP-70 [Horwitz, 1992], HSP-40, and HSc-70 in the epithelium and superficial cortical fiber cells imply a regional cell specific function, whereas the decrease of heat shock protein with age could be responsible for the loss of optimal protein organization, and the eventual appearance of age-related cataract.     

Proteomic analysis of the oxidation of cysteine residues in human age-related nuclear cataract lenses

Loss of protein thiols is a key feature associated with the onset of age-related nuclear cataract (ARNC), however, little is known about the specific sites of oxidation of the crystallins. We investigated cysteine residues in ARNC lenses and compared them with age-matched normal lenses. Proteomic analysis of tryptic digests revealed ten cysteine residues in older normal lenses that showed no significant oxidation compared to foetal counterparts (Cys 170 in betaA1/3-crystallin, Cys 32 in betaA4-crystallin, Cys 79 in betaB1-crystallin, Cys 22, Cys 78/79, C153 in gammaC-crystallin and Cys 22, Cys 24 and Cys 26 in gammaS-crystallin). Although these thiols were not oxidised in normal lenses past the 6th decade, they were present largely as disulphides in the ARNC lenses. By contrast, two cysteine residues, Cys 41 in gammaC-crystallin and Cys 18 in gammaD-crystallin, were not oxidised, even in advanced ARNC lenses. These cysteines are buried deep within the protein and any unfolding associated with cataract must be insufficient to expose them to the oxidative environment present in the centre of advanced ARNC lenses. The vast majority of the loss of protein thiol observed in such lenses is due to disulphide bond formation.     

Accumulation of lipid peroxidation products in cataractous lenses

A study was made of the content of lipid peroxidation products (LPP) in the lenses extracted during operations for cataract as well as in transparent human lenses. In a mature cataract, the elevated content of primary, secondary and end products of lipid peroxidation (diene and triene conjugates, Schiff bases) was revealed. The content of LPP was identical in different clinical patterns of a mature cataract (senile, traumatic, complicated), which points to the universal role of lipid peroxidation in lenticular opacity.     

Comparison of d-aspartic acid contents in alpha A-crystallin from normal and age-matched cataractous human lenses

We have previously shown that biologically uncommon d-beta-aspartic acids (Asp) were localized with very high contents at Asp-151 and Asp-58 of alpha A-crystallin from aged human lenses. The amounts increased with age, and we have proposed the mechanism of this reaction. In the present study, in order to elucidate the possible relationship between the formation of d-beta-aspartic acids in alpha A-crystallin and cataract formation, we measured the d/l ratio of beta-Asp-151 of alpha A-crystallin from both cataractous and age-matched normal human lenses. alpha A-crystallin from total proteins of cataractous and age-matched normal lenses was prepared, followed by tryptic digestion and quantification of d/l ratios for tryptic fragments containing the alpha- and beta-aspartate forms of Asp-151 residues. The results demonstrate that the d/l ratio of beta-Asp-151 of alpha A-crystallin from normal lenses is not statistically significant from that of alpha A-crystallin from cataractous lenses, suggesting that formation of this biologically uncommon amino acid may not play a role in human cataractogenesis.     

Purification and partial characterization of an elastolytic serine protease of Prevotella intermedia.

Elastolytic strains of Prevotella intermedia were isolated from pus samples of adult periodontal lesions. Elastase was found to associate with envelope, and it could be solubilized with guanidine-HCl. The enzyme was purified to homogeneity by sequential procedures including ion-exchange chromatography, gel filtration, and hydrophobic interaction chromatography. This elastase was a serine protease, and its mass was 31 kDa. It hydrolyzed elastin powder, but collagen and azodye-conjugated proteins were not degraded by this enzyme. Both synthetic substrates for human pancreatic (glutaryl-L-alanyl-L-alanyl-L-prolyl-L-leucine p-nitroanilide) and leukocyte elastase (methoxy succinyl-L-alanyl-alanyl-L-prolyl-L-valine p-nitroanilide) were hydrolyzed.     

Further studies on elastase and trypsin inhibitory activities in mammalian lenses

Elastase and trypsin inhibitory capacities increased significantly on heat treatment of the lens extract for 15 min at 60 degrees C in human infant (mean increase 290 and 335%), human adult (130 and 245%), ovine (90 and 140%), and bovine (70 and 90%) lenses. No increase was observed in human cataractous lenses. Preincubation with target enzymes in the absence of substrate abolished the antitryptic activity in lenses whereas antielastase activity was more resistant. No decrease in antielastase activity in human adult and cataractous lenses was observed on 15-min preincubation whereas about 50% of activity was abolished in human infant lenses. The differences were attributed to the changes in the levels of endogenous proteinases and proenzymes during cataractogenesis and aging.     

Thermostable leukocyte alpha-glycoprotein: immunochemical study and enzyme activity research

Using immunochemical analysis with standard antisera, leukocyte thermostable alpha-glycoprotein (LT alpha G) was shown to be distinct from lactoferrin, lysozyme, and fibronectin. The determination of peroxidase and nonspecific elastase in immune precipitates of LT alpha G gave negative results. Affinity sorption of LT alpha G onto the pus protein component was revealed. Purified LT alpha G had amidolytic activity in response to a substrate for elastase (p-nitroanilide succinyl-trialanyl). The ability of LT alpha G to cause the hydrolysis of substrates for thrombin, kallikrein, plasmin was investigated. The identity of LT alpha G and granulocyte elastase is suggested.     

Study of soluble proteins of mouse crystalline lenses (normal and in cataract)

The water-soluble proteins from mice lenses (normal and cataract lenses) were investigated by methods of absorption spectrophotometry and kinetics of UV-induced radical decay. General characteristic of internal structure of extraction proteins was investigated by recombination kinetic method. It was shown that concentration of water-soluble proteins lowered ten times in lenses of mature cataract, i. e. 90% protein molecules were connected in lenses of mature cataract.     

Determination of esterase activity of human and animal serine proteinases using fluorogenic esters of amino acids as substrates]

Two fluorogenic derivatives of amino acids are proposed as substrates for the purpose of enzymatic assay: N-benzyloxycarbonyl-phenylalanine-4-methyl umbelliferyl ester (substrate-1) and tert-butyloxycarbonyl-alanine-4-methyl-umbelliferyl ester (substrate-II). Chymotrypsin-like (hydrolysis of substrate-1), elastase-like (hydrolysis of substrate-II) esterase activity of bovine pancreatic chymotrypsin, activities of cathepsin G and elastase from human, porcine and rat neutrophils and esterase activity of human, porcine and rat serum were assayed. Differences in the level of chymotrypsin-like and elastase-like activities of human, porcine and rat serum were established. Activities of purified elastase and cathepsin G from human and animal neutrophils were shown to have no significant distinctions.     

2-ammonio-6-(3-oxidopyridinium-1-yl)hexanoate (OP-lysine) is a newly identified advanced glycation end product in cataractous and aged human lenses

Post-translational modifications of proteins take place during the aging of human lens. The present study describes a newly isolated glycation product of lysine, which was found in the human lens. Cataractous and aged human lenses were hydrolyzed and fractionated using reverse-phase and ion-exchange high performance liquid chromatography (HPLC). One of the nonproteinogenic amino acid components of the hydrolysates was identified as a 3-hydroxypyridinium derivative of lysine, 2-ammonio-6-(3-oxidopyridinium-1-yl)hexanoate (OP-lysine). The compound was synthesized independently from 3-hydroxypyridine and methyl 2-[(tert-butoxycarbonyl)amino]-6-iodohexanoate. The spectral and chromatographic properties of the synthetic OP-lysine and the substance isolated from hydrolyzed lenses were identical. HPLC analysis showed that the amounts of OP-lysine were higher in water-insoluble compared with water-soluble proteins and was higher in a pool of cataractous lenses compared with normal aged lenses, reaching 500 pmol/mg protein. The model incubations showed that an anaerobic reaction mixture of Nalpha-tert-butoxycarbonyllysine, glycolaldehyde, and glyceraldehyde could produce the Nalpha-t-butoxycarbonyl derivative of OP-lysine. The irradiation of OP-lysine with UVA under anaerobic conditions in the presence of ascorbate led to a photochemical bleaching of this compound. Our results argue that OP-lysine is a newly identified glycation product of lysine in the lens. It is a marker of aging and pathology of the lens, and its formation could be considered as a potential cataract risk-factor based on its concentration and its photochemical properties.     

A study of chromium in human cataractous lenses and whole blood of diabetics,senile, and normal population

Chromium (Cr) is of known biological importance, necessary for the maintenance of normal glucose metabolism. There is a lower level of blood Cr concentrations in cases of diabetes. Diabetes carries a risk of cataract development, so the potential effects of Cr on the eye may need to be studied in more depth. The presence of this trace element in both normal and cataractous human lenses has to our knowledge not been investigated so far. The concentration of total Cr in 61 human lenses and 38 blood samples was determined by electrothermal atomic absorption spectrometry with Zeeman effect (EAASZ). Analysis of the levels of Cr in human lenses shows a significant difference between normal and diabetic populations, and an absence of difference between senile and diabetic populations.     

Accumulation of the hydroxyl free radical markers meta-, ortho-tyrosine and DOPA in cataractous lenses is accompanied by a lower protein and phenylalanine content of the water-soluble phase

Post-translational modifications of lens proteins play a crucial role in the formation of cataract during ageing. The aim of our study was to analyze protein composition of the cataractous lenses by electrophoretic and high-performance liquid chromatographic (HPLC) methods. Samples were obtained after extracapsular cataract surgery performed by phacoemulsification technique from cataract patients with type 2 diabetes mellitus (DM CAT, n = 22) and cataract patients without diabetes (non-DM CAT, n = 20), while non-diabetic non-cataractous lenses obtained from cadaver eyes served as controls (CONTR, n = 17). Lens fragments were derived from the surgical medium by centrifugation. Samples were homogenized in a buffered medium containing protease inhibitor. Soluble and insoluble protein fractions were separated by centrifugation. The electrophoretic studies were performed according to Laemmli on equal amounts of proteins and were followed by silver intensification. Oxidized amino acid and Phe content of the samples were also analyzed by HPLC following acid hydrolysis of proteins. Our results showed that soluble proteins represented a significantly lower portion of the total protein content in cataractous lenses in comparison with the control group (CONTR, 71.25%; non-DM CAT, 32.00%; DM CAT, 33.15%; p < 0.05 vs CONTR for both). Among the proteins, the crystallin-like proteins with low-molecular weight can be found both in the soluble and insoluble fractions, and high-molecular weight aggregates were found mainly in the total homogenates. In our HPLC analysis, oxidatively modified derivatives of phenylalanine were detected in cataractous samples. We found higher levels of m-Tyr, o-Tyr and DOPA in the total homogenates of cataractous samples compared to the supernatants. In all three groups, the median Phe/protein ratio of the total homogenates was also higher than that of the supernatants (total homogenates vs supernatants, in the CONTR group 1102 vs 633 micromol/g, in the DM CAT group 1187 vs 382 micromol/g and in the non-DM CAT group 967 vs 252 micromol/g; p < 0.05 for all). In our study we found that oxidized amino acids accumulate in cataractous lenses, regardless of the origin of the cataract. The accumulation of the oxidized amino acids probably results from oxidation of Phe residues of the non-water soluble lens proteins. We found the presence of high-molecular weight protein aggregates in cataractous total homogenates, and a decrease of protein concentration in the water-soluble phase of cataractous lenses. The oxidation of lens proteins and the oxidative modification of Phe residues in key positions may lead to an altered interaction between protein and water molecules and thus contribute to lens opacification.     

Peroxide-metabolizing systems of the crystalline lens

The ability of transparent and cataractous human, rabbit and mice lenses to metabolize hydrogen peroxide in the surrounding medium was evaluated. Using a chemiluminescence method in a system of luminol-horseradish peroxidase and a photometric technique, the temperature-dependent kinetics of H₂O₂ decomposition by lenses were measured. The ability of opaque human lenses to catalyze the decomposition of 10^?4 M H₂O₂ was significantly decreased. However, this was reserved by the addition of GSH to the incubation medium. Incubation of the mice lenses with the initial concentration H₂O₂ 10^?4 M led to partial depletion of GSH in normal and cataractous lenses. Human cataractous lenses showed decreased activities of glutathione reductase, glutathione peroxidase (catalyzing reduction of organic hydroperoxides including hydroperoxides of lipids), superoxide dismutase, but no signs of depletion in activities of catalase or glutathione peroxidase (utilizing H₂O₂). The findings indicated an impairment in peroxide metabolism of the mature cataractous lenses compared to normal lenses to be resulted from a deficiency of GSH. An oxidative stress induced by accumulation of lipid peroxidation products in the lens membranes during cataract progression could be considered as a primary cause of GSH deficiency and disturbance of the redox balance in the lens.