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L L David

Publications and source records attributed to L L David.

At least 37 records · Page 2Linked to original sources
PubMed1997

Activation of calpain in lens: a review and proposed mechanism.

The purpose of these experiments was to develop a hypothesis to explain activation of m-calpain in cataractogenesis observed in rodents. The in vitro model used to study m-calpain activation was to correlate breakdown of the 'reporter' protein alpha-crystallin with the appearance of activated m-calpain using protein sequencing and casein zymography. Incubation of alpha-crystallins with m-calpain and Ca2+ caused proteolysis of alpha-crystallins and accumulation of new polypeptides. E64 and calpain inhibitor I each inhibited proteolysis of alpha-crystallins. The N-terminus of the 80 kDa subunit of m-calpain was blocked at time 0 (pro calpain). After incubation with Ca2+, the remaining 80 kDa subunit of m-calpain gave a N-terminal sequence of KDREAAEGLG, indicating loss of nine amino acid from the N-terminus (autolysed calpain). The new 43 kDa m-calpain fragment also gave a N-terminal sequence of KDREAAEGLG, indicating the same loss of the first nine amino acids on the N-terminus as well as a major loss of the C-terminal half of the subunit (degraded calpain). In contrast, the N-terminus of the 80 kDa subunit of m-calpain remained blocked when E64 was present (unautolysed form). Moreover, the Ca2+ concentration required for proteolysis decreased when calpain was pre-incubated with Ca2+, although proteolysis of alpha-crystallin required a higher Ca2+ concentration than proteolysis of casein. These data suggested that the sequence of events for m-calpain activation were unautolysed, autolysed and finally degraded calpain. Unautolysed and/or autolysed calpains may be proteolytically active against alpha-crystallin.

Amino Acid Sequence↗
PubMed1997

Occurrence of Prevotella nigrescens and Prevotella intermedia in infections of endodontic origin.

The occurrence of Prevotella intermedia and Prevotella nigrescens in endodontic infections was studied using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of whole cell protein to distinguish between the species. Previous studies have shown an association between black-pigmented bacteria (BPB) and endodontic infections and that Prevotella intermedia (previously known as Bacteroides intermedius) was the most commonly isolated BPB. Recently, however, strains identified as P. intermedia were shown to in fact be composed of two separate species, P. intermedia and P. nigrescens. Fifty-six strains of BPB isolated from endodontic infections and previously identified as P. intermedia were used in this study. Following SDS-PAGE, P. nigrescens showed a unique 18.6 kDa band that was used to differentiate P. nigrescens from P. intermedia. Of the 56 strains of BPB, 41 (73.2%) were identified as P. nigrescens and 15 (26.8%) as P. intermedia. This study confirms that P. nigrescens, and not P. intermedia, is the BPB most often isolated from infections of endodontic origin.

Amino Acid Sequence↗
PubMed1996

The sequence of human betaB1-crystallin cDNA allows mass spectrometric detection of betaB1 protein missing portions of its N-terminal extension.

The sequence of human betaB1-crystallin cDNA encoded a protein of 251 amino acids in length. Mass spectrometric analysis of intact betaB1 from young human lens confirmed the deduced amino acid sequence. Lenses of human donors newborn to 27 years of age also contained partially degraded forms of betaB1 missing 15, 33, 34, 35, 36, 39, 40, and 41 amino acid residues from their N-terminal extensions. The similarity of the cleavage site between residues 15 and 16 in human betaB1 to the cleavage occurring in bovine betaB1 suggested that lenses of both species may contain a similar proteolytic activity. The remaining cleavage sites occurring in human betaB1 did not closely match those occurring in other species, possibly due to the widely divergent amino acid sequence of the N-terminal extension of betaB1 amoung species. Results from animal models suggest that cleavage of the N-terminal extension of betaB1-crystallin could enhance protein insolubilization and cataract in lens. However, the presence of partially degraded betaB1-crystallins in both water-soluble and water-insoluble fractions of lenses of young donors suggested that the rate that proteolyzed betaB1-crystallins become water-insoluble is relatively slow in humans.

Amino Acid Sequence↗
PubMed1996

Crystallins from rat lens are especially susceptible to calpain-induced light scattering compared to other species.

PURPOSE: To compare the susceptibility of crystallins from various animal species to formation of light scattering elements after proteolysis by calpain II enzyme (EC 3.4.22.17). METHODS: Lens, total soluble proteins from: 12-day and 4-week old rat, fetal and adult bovine, 16-day embryonic and 10-week chicken, and young human cortex and nucleus were proteolyzed by either endogenous lens calpain or addition of purified calpain II for 24 h followed by incubation for up to 11 days. Absorbance of light at 405 nm estimated light scattering by crystallins; SDS-PAGE and 2D-electrophoresis assessed proteolysis on the crystallins. RESULTS: Most rapid light scattering occurred with total soluble proteins from young rat lens, either after adding purified calpain or by activating endogenous lens calpain with calcium. (Only rat lens showed activation of endogenous calpain II.) beta-crystallin polypeptides from rat, bovine, human, and to a more limited extent, chick lens were partially proteolyzed by addition of purified calpain II. In spite of this proteolysis, total soluble proteins from chicken, bovine, and human lenses showed no obvious light scattering by action of calpain. Crystallins from older rat lens showed approximately 50% of the light scattering displayed by crystallins from younger rats after 3 days, but only when purified calpain was added. CONCLUSIONS: Our results indicate an unusually high susceptibility of crystallin polypeptides from young rat lens to formation of light scattering elements after limited proteolysis. Thus, young rat lens provides a unique opportunity to investigate how properties of crystallins influence the development of light scattering found in cataract.

Animals↗
PubMed1995

Precipitation of crystallins from young rat lens by endogenous calpain.

The purpose of these experiments was to study the mechanism for precipitation of lens crystallins in cataract. An in vitro model was developed to activate the endogenous protease calpain II in the soluble proteins from young rat lens by addition of calcium in the presence of 120 mM KCl. Light-scattering, insoluble proteins were produced approximately 4-6 days after calpain II activation. Results showed that proteolysis was caused by activation of lens calpain II, proteolysis preceded precipitation by several days, and alpha-crystallin acted as a molecular chaperone against precipitation of crystallins caused by proteolysis. These data supported our hypothesis that calpain-induced proteolysis of the N-terminal arms of beta-crystallin polypeptides leads to a loss of normal oligomerization of beta-crystallin polypeptides and formation of abnormal insoluble aggregates, possibly stabilized by hydrophobic interactions.

Amino Acid Sequence↗
PubMed1995

Crystallin degradation and insolubilization in regions of young rat lens with calcium ionophore cataract.

PURPOSE: To determine if the susceptibility of rat lenses to cataract formation in culture changes with increasing age and to investigate the regional differences in crystallin degradation and insolubilization during the formation of cataracts in cultured lenses. METHODS: Lenses from 4-week-old (young group) and 12-week-old (adult group) rats were divided into four subgroups: noncultured control, cultured control, cultured in calcium ionophore A23187, and cultured in ionophore plus calpain inhibitor E64. Lenses were cultured for 7 days, and the cortex and nucleus were homogenized and separated into water-soluble and water-insoluble fractions. Two-dimensional electrophoresis and N-terminal sequencing were then performed. RESULTS: Young lenses treated with ionophore produced thin cortical and dense nuclear opacities. Adult lenses treated with ionophore also developed thin cortical opacity, but no nuclear opacity was observed, even though a large increase in the concentration of insoluble protein occurred. Two-dimensional electrophoresis and sequencing suggested that calpain caused protein degradation in the cortex region. However, unlike nuclear opacity, the formation of opacity in the cortex was not inhibited by E64 in young or adult lenses. CONCLUSIONS: Calpain was activated, and crystallins were proteolyzed in the cortex of ionophore-treated lenses. However, cortical opacity was not the result of proteolysis by calpain. Maturation also decreased the susceptibility of rat lens nucleus to calcium ionophore cataract.

Aging↗
PubMed1994

Cataract and the acceleration of calpain-induced beta-crystallin insolubilization occurring during normal maturation of rat lens.

PURPOSE: To determine if limited proteolysis of beta-crystallins is associated with insolubilization of proteins in rats lens during maturation and to test if the protease, calpain II, is involved. METHODS: Soluble and insoluble lens proteins from 4-day-old to 4-month-old rat lens cortexes and nuclei were separated by two-dimensional electrophoresis. The insoluble proteins from 4-month-old nuclei were electroblotted and the NH2 termini of proteins sequenced. Cleavage sites appearing at 4 months of age were compared to cleavage sites produced by purified calpain II and to cleavage sites appearing in cataracts induced by selenite in vivo or in lenses cultured with calcium ionophore A23187 or diamide. RESULTS: In solubilization of more than 50% of proteins occurred in the nucleus of the transparent rat lens by 4 months of age. The insoluble protein that formed contained an abundance of partially degraded beta-crystallin polypeptides missing portions of their NH2 terminal extensions. In contrast, these truncated beta-crystallins were largely absent from both the cortex and soluble fraction of the nucleus. The cleavage sites in the insoluble beta-crystallins appearing during maturation in the lens nucleus were similar to cleavage sites produced by purified calpain II and also similar to cleavage sites appearing in the insoluble protein of cataractous lenses. CONCLUSIONS: These results suggest that proteolysis of beta-crystallins by the protease calpain II contributes to protein insolubilization during lens maturation and that acceleration of this insolubilization process is associated with cataract formation in rodent lenses.

Amino Acid Sequence↗
PubMed1993

alpha-Crystallin chaperone activity is reduced by calpain II in vitro and in selenite cataract.

This study reports the first demonstration of a marked reduction in alpha-crystallin chaperone activity in an experimental model of cataract, and the study implicates activation of the cysteine protease calpain II (EC 3.4.22.17) as the in vivo protease responsible for decreased chaperone activity. Chaperone activity of normal alpha-crystallin from lenses of young rats was assayed by measuring attenuation of heat-induced aggregation and scattering of beta L-crystallin. alpha-Crystallin from the nucleus of lenses with selenite cataract showed specific selective proteolysis, and chaperone activity was diminished. Proteolysis of alpha-crystallin from selenite cataract lenses was mimicked by incubating normal alpha-crystallin with calpain II, and this also resulted in loss of chaperone activity. Two-dimensional gel electrophoresis and peptide mapping were used to identify four partially degraded alpha A- and alpha B-crystallin polypeptides following incubation of normal alpha-crystallin with calpain. Similar partially degraded alpha A and alpha B polypeptides were found in selenite cataract. Previous experiments indicated that alpha-crystallin chaperone activity decreases because of removal of the COOH terminus. Our experiments support this observation and suggest that calpain proteolysis of alpha-crystallin at the COOH terminus may result in a loss of chaperone activity in selenite cataract.

Amino Acid Sequence↗
PubMed1993

Beta-crystallins insolubilized by calpain II in vitro contain cleavage sites similar to beta-crystallins insolubilized during cataract.

Incubation of soluble proteins from rat lens with the protease calpain II caused the precipitation of beta-crystallin polypeptides. Two-dimensional electrophoresis and sequence analysis identified beta-crystallin polypeptides both before and after their precipitation by calpain II. beta-crystallin polypeptides precipitated by calpain were cleaved at their NH2-terminal extensions. These cleavage sites were similar to cleavage sites occurring in beta-crystallin polypeptides precipitated during formation of experimental cataract induced by an overdose of selenite. These data suggested that calpain II caused beta-crystallin insolubilization during cataract formation, and indicated that the process can be mimicked in vitro.

Amino Acid Sequence↗
PubMed1993

Sequence analysis of lens beta-crystallins suggests involvement of calpain in cataract formation.

Abnormal activation of the protease calpain in the lens may be a cause of cataracts. Cataracts were induced in 10-day-old rats by a single overdose of sodium selenite. The water-insoluble protein from the opaque lens nucleus was separated by two-dimensional electrophoresis, electroblotted onto membranes, and the NH2-terminal sequence of partially degraded beta-crystallin polypeptides determined. Selenite cataractous lenses contained four major structural proteins, beta B1, beta B3, beta A3/A1, and beta A4 crystallins, missing from 5 to 49 amino acids from their NH2 termini. Incubation of intact beta-crystallins with calpain II in vitro produced identical cleavage sites. This provided further evidence for the role of calpain in the production of light scattering insoluble protein in cataractous lenses and also suggested that a similar process may lead to lens protein insolubilization during aging.

Amino Acid Sequence↗
PubMed1993

Role of calpain in hydrogen peroxide induced cataract.

The purpose of these experiments was to examine the relationship between oxidation cataract and proteolysis in cultured rat lens. Hydrogen peroxide cataract showed insolubilization of protein, loss of 31 kDa beta B1-crystallin polypeptide, decreases in soluble calpain, and increases in insoluble calpain. This suggested that calpain may be activated in hydrogen peroxide treated lenses, since beta B1 is a known calpain substrate, and calpain undergoes autolysis and degradation when activated. Furthermore, the cysteine protease inhibitor E64 was partially effective in preventing development of H2O2-cataract. E64 also prevented the loss of the 31 kDa beta B1-crystallin polypeptide and decreased the loss of calpain in the lens. These results suggested that development of hydrogen peroxide induced cataract in rat lenses was associated with activation of calpain.

Animals↗
PubMed1992

Superior prevention of calcium ionophore cataract by E64d.

The purposes of this experiment were: (1), to compare effect of three E64 derivatives, E64, E64c and E64d in preventing nuclear opacity and proteolysis in calcium ionophore-induced cataract and (2), to measure the accumulation of E64 derivatives in the cultured lenses. In vitro E64 and E64c strongly inhibited purified calpain II from porcine heart, while E64d showed weaker inhibition than E64 and E64c. In cultured lenses, all three E64 derivatives reduced nuclear opacity by calcium ionophore A23187 in a concentration-dependent manner, and E64d, the ethyl-ester of E64c, was the most effective. When lenses were cultured in E64d for 2 h, the resulting concentration of E64 derivative in the lens was markedly higher than during culture in E64 or E64c. All three E64 derivatives prevented proteolysis of crystallins seen in A23187 cataract. The stronger effect of E64d against A23187 cataract was likely due to an earlier penetration into the lens, conversion to E64c and inhibition of activated calpain.

Animals↗
PubMed1992

Calpain II induced insolubilization of lens beta-crystallin polypeptides may induce cataract.

Addition of calpain II (EC 3.4.22.17) to soluble proteins from 10-day-old rat lens caused an increase in turbidity and production of water-insoluble protein. The insolubilization increased with higher concentrations of both lens protein and calpain II, it could be prevented by the cysteine protease inhibitor E-64; it required at least 0.5 mM Ca2+, it was limited to 6% of the soluble protein present and resulted from precipitation of proteolyzed beta-crystallin polypeptides. When compared by two-dimensional electrophoresis, the insoluble beta-crystallin polypeptides produced by calpain II were similar to insoluble beta-crystallin polypeptides found in cataractous lenses. Trypsin also caused insolubilization of beta-crystallin polypeptides, but these polypeptides were unlike polypeptides produced during cataract formation. These data suggested that the loss of solubility was due to a specific removal of N/or C-terminal extensions from beta-crystallin polypeptides by calpain II, and that a similar process may occur in vivo during cataract formation. It is hypothesized that the insoluble protein produced by calpain II causes cataract by increasing light scatter in the lens.

Animals↗
PubMed1992

Association of calpain with insoluble pellet of rat lens.

Calpains are calcium-activated neutral proteases found in many tissues including the lens. The purpose of this research was to localize calpain in various biochemical fractions of the rat lens. Lenses were homogenized (with and without added calcium) and separated into water-soluble and -insoluble fractions, which were further extracted with urea, NaOH, and SDS. Of the total calpain 10% was insoluble. In the lens calpain was found to be both insoluble and associated with the membrane. Extraction of calpain from the insoluble fraction suggested calpain was loosely and tightly associated with the membrane. Calpain associated with membrane-rich fractions was obtained from discontinuous sucrose gradients, confirming the above. Calcium increased the amount of calpain associated with the insoluble fraction up to 30% of the total calpain. When the calcium was chelated, this calpain once again became soluble, and its specific activity was higher than water-soluble calpain. The translocation of calpain from the water-soluble fraction to insoluble fractions by calcium may be important because: (1) it may bring calpain into proximity with its substrates; and (2) it may activate calpain, since membrane phospholipids lower the protease's calcium requirement.

Animals↗
PubMed1992

Comparison of cell-permeable calpain inhibitors and E64 in reduction of cataract in cultured rat lenses.

E64, an inhibitor of calpain (EC 3.4.22.17) and other cysteine proteases, slows the rate of formation of cataract in cultured rat lenses. The purpose of this study was to determine (1) why E64, a charged compound with little cell permeability, was effective in reducing cataract in cultured lens and (2) whether uncharged more permeable protease inhibitors are more effective than E64 in preventing cataract. Results showed that E64 entered the lens, but only after the lens was treated with the calcium ionophore, A23187, or sodium selenite, both of which cause cataracts. Therefore, the uptake and subsequent effectiveness of E64 may be related to a generalized increase in membrane permeability during induction of cataract in culture. Three protease inhibitors, reported to have improved cell permeability, were compared with E64 for their ability to prevent cataracts in cultured lenses. cBz-ValPheH, calpain inhibitors I and II, are uncharged-aldehyde inhibitors of calpain. Calpain inhibitors I and II even at high concentrations were not effective at reducing lens opacity caused by calcium ionophore and were toxic to the lens. cBz-ValPheH, which is slightly toxic to the lens, was able to significantly reduce lens opacity induced by calcium ionophore. The presented data suggest that while E64 decreases cataract formation in cultured lens, the more cell permeable inhibitor, cBz-ValPheH, may have greater efficacy as an anticataract drug in vivo.

Amino Acid Sequence↗
PubMed1992

Hydration and elevated calcium alone do not produce xylose nuclear cataract: role of proteolysis by calpain.

The purpose of this experiment was to determine the contribution of calpain proteolytic enzyme (EC 3.4.22.17) in the formation of nuclear cataract during lens culture in xylose. Increased lens calcium was found to be required for formation of xylose nuclear cataract in our culture system. Inhibition of calpain by the cysteine protease inhibitor E64 was effective in slowing the formation of nuclear cataract, even though lens calcium and hydration were markedly elevated. These results showed that hydration and elevated calcium alone do not produce xylose nuclear cataract, and they indicated that calpain proteolysis may be necessary for xylose nuclear cataract in the rat lens.

Animals↗
PubMed1992

Review of selenite cataract.

Recent advances in understanding the mechanism of selenite cataract have resulted from locating the cleavage sites on proteolyzed beta-crystallins from the cataract, mimicking the insolubilization of crystallins found in the cataract in an in vitro system, studying cataract produced in lenses cultured in selenite, and permanently or temporarily reducing the rate formation of selenite cataract by use of various inhibitors. The present review discusses the selenite cataract as a useful model for understanding the role calcium-induced proteolysis in cataract formation.

Amino Acid Sequence↗