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O P Srivastava

Publications and source records attributed to O P Srivastava.

At least 19 recordsLinked to original sources
PubMed2006

Truncation of motifs III and IV in human lens betaA3-crystallin destabilizes the structure.

The purpose of our study was to determine the effects of specific truncations on the structural properties of human betaA3-crystallin. The following eight deletion mutants of betaA3-crystallin were generated: (i) N-terminal extension (NTE) 21 amino acids (betaA3[21] mutant), (ii) NTE 22 amino acids (betaA3[22] mutant), (iii) NTE (betaA3[N] mutant), (iv) NTE plus motif I (betaA3[N+I] mutant), (v) NTE plus motifs I and II (betaA3[N+I+II] mutant), (vi) NTE plus motifs I and II and connecting peptide (betaA3[N+I+II+CP] mutant), (vii) motifs III and IV (betaA3[III+IV] mutant), and (viii) motif IV (betaA3 [IV] mutant). The DNA sequencing and MALDI-TOF mass spectrometric methods confirmed desired specific deletions, and the purified mutant proteins exhibited a single band during SDS-PAGE analysis. When ANS bound, all the mutant proteins exhibited fluorescence quenching and a red shift, suggesting that the truncations caused changes in the exposed hydrophobic patches. The CD spectra showed that deletion of either NTE or the N-terminal domain (motifs I and II) had a relatively weaker effect on the structural stability than deletion of the C-terminal domain (motifs III and IV). Intrinsic Trp fluorescence spectral studies suggested changes in the microenvironment of the mutant proteins following truncations. HPLC multiangle light scattering analyses showed that truncation led to higher-order aggregation compared to that in the wild-type protein. Equilibrium unfolding and refolding of WT betaA3 with urea were best fit to a three-state model with transition midpoints at 2.2 and 3.1 M urea. However, the two transition midpoints of betaA3[21] and betaA3[22] and betaA3[N] mutants were similar to those of the wild type, suggesting that these truncations had a minimal effect on structural stabilization. Further, the mutant proteins containing the N-terminal domain (i.e., betaA3[III+IV] and betaA3[IV] mutants) exhibited higher transition midpoints compared to the transition midpoints of the mutant protein with the C-terminal domain (i.e., betaA3[N+I+II+CP] mutant). The results suggested that the N-terminal domain is relatively more stable than the C-terminal domain in betaA3-crystallin.

Base Sequence↗
PubMed2003

BetaB2-crystallin undergoes extensive truncation during aging in human lenses.

Based on the present literature, it is unclear whether betaB2-crystallin undergoes age-related truncation in human lenses. To answer this question, the purpose of this study was to determine in vivo truncation of betaB2-crystallin in human lenses during aging by examining its fragments in the beta(H)-crystallin fraction. The WS-protein fraction was isolated from lenses of desired ages and separated by a size-exclusion Agarose A 1.5m column to recover alpha-, beta(H)-, beta(L)-, and gamma-crystallin fractions. The beta(H)-crystallin fractions, isolated from lenses of 24- and 70-year-old donors, were utilized for two-dimensional (2D) gel electrophoresis (isoelectric focusing in the first dimension followed by SDS-PAGE in the second dimension). The partial N-terminal sequences of the desired fragments (Molecular weights [M(r)]<18-19kDa) from a 2D-gel of WS-proteins from lenses of a 70-year-old donor were determined. More than 37 crystallin fragments with M(r) between 4 and 19kDa were observed on a 2D-gel. Nine fragments in beta(H)-crystallin fraction were from betaB2-crystallin but additional single fragments of alphaA-, gammas-, betaA4, and of either gammaB-, gammaC- or gammaD-crystallins were also observed. Seven cleavage sites in the betaB2-crystallin were identified, which included two sites at Q(7)-A(8) and A(8)-G(9) bonds in the N-terminal extension, two sites at E(46)-K(47) and G(49)-S(50) bonds in the motif 1, one site at S(94) -S(95) in the motif 2, and two sites at N(115)-F(116) and Q(135)-Y(136) in motif 3. No fragments with cleavage in the motif 4 and C-terminal extension of betaB2-crystallin were seen. Apparently, three betaB2-crystallin fragments with only N-terminal cleavage and five with both N- and C-terminal cleavages were observed. Additional fragments with cleavage sites at Q(54)-Y(55) in alphaA-crystallin, at E(112)-N(113) in betaA4-crystallin, at G(4)-T(5) in gammas-crystallin, at M(69)-G(70) in either gammaB-, gammaC- or gammaD-crystallins (three have identical sequences at the cleaved bond), and at G(1)-K(2) in gammaB or gammaC (both have identical sequences at the cleavage site) were observed.Conclusions. The results showed that betaB2-crystallin undergoes age-related truncation producing fragments with M(r) between 4 and 19kDa that existed in the beta(H)-crystallin oligomer. The beta(H)-crystallin fraction also contained single fragments of alpha-, betaA4-, gammas-, and other gamma-crystallins.

Adult↗
PubMed2000

Acquisition of P-selectin binding activity by en bloc transfer of sulfo Le(x) trisaccharide to the cell surface: comparison to a sialyl Le(x) tetrasaccharide transferred on the cell surface.

Sialyl Le(x), NeuNAcalpha2 --> 3Galbeta1 --> 4(Fucalpha1 --> 3)GlcNAcbeta --> R, is known to be a ligand for E-selectin in various assays. The sulfated counterpart of sialyl Le(x), sulfo Le(x), (Sulfo --> 3) Galbeta1 --> 4 (Fucalpha1 --> 3) GlcNAcbeta --> R, was also shown to be a ligand for E-selectin in solid-phase assays employing immobilized oligosaccharides. In order to determine whether sulfo Le(x) structure on the cell surface also works as E-selectin or P-selectin ligand, a novel approach for in vitro transfer of oligosaccharides (S. Tsuboi, Y. Isogai, N. Hada, J. K. King, O. Hindsgaul, and M. Fukuda (1996) J. Biol. Chem. 271, 27213-27216) was utilized. A synthetic GDP-fucose harboring sialyl Le(x) or sulfo Le(x) oligosaccharide was enzymatically transferred to Chinese hamster ovary (CHO) cells with a milk fucosyltransferase. The resultant cells, CHO-sialyl Le(x) and CHO-sulfo Le(x) were tested for adhesion to E-selectin. IgG or P-selectin. IgG chimeric protein coated on plates. The results indicate that CHO-sialyl Le(x) adhered efficiently to E-selectin, while adhesion of CHO-sulfo Le(x) was very poor despite the fact that near equal number of the ligands had been attached to the cell surface. In contrast, CHO-sulfo Le(x) adhered efficiently to P-selectin, while CHO-sialyl Le(x) adhered modestly to P-selectin. These results demonstrate that sialyl Le(x) and sulfo Le(x) structures on the cell surface differ substantially in their ability to adhere to E- and P-selectin.

Animals↗
PubMed1999

Characterization of a sodium deoxycholate-activatable proteinase activity associated with betaA3/A1-crystallin of human lenses.

A human lens proteinase was purified by a five-step procedure that included two consecutive size-exclusion agarose A 1.5 m chromatographies, a preparative non-denaturing gel-electrophoretic separation, HPLC on a size-exclusion column (TSK G-3000 PW(XL)) followed by preparative isoelectric focusing. A 2300-fold purified enzyme showed a major band of 22 kDa during SDS-PAGE, a pH optimum of 7.8, pI between 4.5 and 5.0, a loss of activity above 45 degrees C and a serine type nature. The partial N-terminal sequence of the enzyme, i.e. P-M-P-G-S-L-G-P-W, matched with the sequence of human lens betaA3/A1-crystallin starting at residue No. 23. Based on the Western blot results of the enzyme with five different site-specific polyclonal antibodies raised against betaA3/A1-crystallin, it was concluded that the 22 kDa crystallin enzyme had a cleaved N-terminus but an intact C-terminus. The betaA3/A1-crystallin, isolated from human lenses, also exhibited proteinase activity following detergent activation and size-exclusion chromatography. The mouse recombinant betaA3/A1-crystallin proteinase was purified by the above five-step procedure, from a homogenate of Sf-9 cells transfected with baculovirus containing the full length coding sequence of betaA3/A1-crystallin. The mouse 22 kDa species also exhibited proteinase activity and immunoreactivity with anti-betaA3/A1-C-terminal antibody. Together, the data suggest that a truncated species of betaA3/A1-crystallin exhibits proteinase activity.

Aged↗
PubMed1999

Age-related degradation of betaA3/A1-crystallin in human lenses.

The aim of this study was to determine age-related degradation of betaA3/A1-crystallin in human lenses. The betaA3/A1-crystallin fragments were identified by Western blot analysis using two site-specific anti-betaA3/A1-crystallin antibodies. The first antibody was raised against a N-terminal region (residues 35-66), and the second to the C-terminal (residues 203-214) region of the crystallin. During the analyses, either preparative SDS-PAGE-separated fragments from betaH-crystallin fraction or water-soluble (WS) protein fractions from lenses of different aged donors were used. In lenses from 27- to 30-year-old donors, four major crystallin fragments of about 5, 16, 17, and 18 kDa immunoreacted with the anti-betaA3/A1-N-terminal antibody, suggesting their intact N-terminus but cleaved C-terminus. A similar analysis with the anti-betaA3/A1-C-terminal antibody identified 15-, 18-, 19-, and 20-kDa species and also five species between 4 and 11 kDa that had intact C-terminus but cleaved N-terminus. In lenses from a 5-year-old donor only two crystallin species, a major 15-kDa and a minor 18-kDa species, showed an intact N-terminus and cleaved C-terminus, whereas, eight species with Mr's between 4 and 19 kDa exhibited intact C-terminus but cleaved N-terminus. Upon two-dimensional gel electrophoresis of a betaH-crystallin fraction from the lenses of a 70-year-old donor, a degradation profile almost similar to the crystallin mentioned above was observed. However, the existence of multiple spots with identical Mr's of truncated betaA3/A1-crystallin species on the 2D-gel suggests their existence as isoforms (identical size species with different charges) because of post-translational modifications. Five species of 4, 6, 11, 15, and 18 kDa showed an identical partial N-terminal sequence of N-F-Q-G, suggesting cleavage at the E39-N40 bond during their production. Together, the data suggest that the majority of age-related cleavages in betaA3/A1-crystallin occur at the N-terminal region, with a major cleavage site at the E39-N40 bond generating some of these fragments.

Aging↗
PubMed1999

cAMP-dependent phosphorylation of betaig-h3 protein in human corneal endothelial cells.

PURPOSE: To determine the identity of a major membrane/ extracellular matrix (ECM)-associated 66-kDa protein in human corneal endothelial cells and its phosphorylation in vivo. METHODS: A membrane/ECM-associated 66 kDa protein was purified from human corneal endothelial cells of 50-80 year-old donors by a three-step procedure that included preparative SDS-PAGE, preparative isoelectric focusing (IEF) and HPLC using a C-18 column. The phosphorylation of the 66-kDa protein was determined by both endogenous kinases and exogenous protein kinase A in the presence of endogenous or added cAMP, or added Walsh inhibitor (a specific inhibitor of protein kinase A). The phosphorylated proteins were analyzed by SDS-PAGE followed by autoradiography. The phosphoamino acids were identified following hydrolysis of the purified phosphorylated 66-kDa protein and thin layer chromatography with standard phosphoamino acids. RESULTS: Following purification, the 66-kDa protein showed a single band on SDS-PAGE, a single species on two dimensional (2D)-gel electrophoresis and a single peak during C-18 HPLC. The partial N-terminal sequence of the 66-kDa protein matched with that of the 68-kDa beta ig-h3 protein (minus the signal peptide) of lung adenocarcinoma cells. Furthermore, on Western blot analysis, the 66-kDa protein immunoreacted with anti-beta ig-h3 past signal peptide (residue nos.24-32) antibody but not with the anti-beta ig-3 signal peptide (residue nos.1-9) antibody. During the phosphorylation of endothelial proteins by endogenous kinases or added protein kinase A in the presence of endogenous or added cAMP, the 66 kDa protein was the major substrate with its Ser residues phosphorylated in both cytosolic- and membrane/ECM-fractions. CONCLUSIONS: The human corneal endothelial 66-kDa protein is identical to the 68-kDa beta ig-h3 protein (minus signal peptide) from lung adenocarcinoma cells. The corneal protein exists in a phosphorylated state in vivo with its Ser residues phosphorylated by a cAMP-dependent protein kinase A.

Adenocarcinoma↗
PubMed1999

Characterization of the substrate specificity of alpha1,3galactosyltransferase utilizing modified N-acetyllactosamine disaccharides.

alpha1,3galactosyltransferase (alpha1,3GalT) catalyzes the synthesis of a range of glycoconjugates containing the Galalpha1,3Gal epitope which is recognized by the naturally occurring human antibody, anti-Gal. This enzyme may be a useful synthetic tool to produce a range of compounds to further investigate the binding site of anti-Gal and other proteins with a Galalpha1,3Gal binding site. Thus, the enzyme has been probed with a series of type 2 disaccharide-C8(Galbeta1-4GlcNAc-C8) analogs. The enzyme tolerated acceptors with modifications at C2 and C3 of the N-acetylglucosamine residue, producing a family of compounds with a nonreducing alpha1,3 linked galactose. Compounds that did not serve as acceptors were evaluated as inhibitors. Interestingly, the type 1 disaccharide-C8, Galbeta1-3GlcNAc-C8, was a good inhibitor of the enzyme (Ki = 270 microM vs. Km = 190 microM for Galbeta1-4GlcNAc-C8). A potential photoprobe, based on a modified type 2 disaccharide (octyl 3-amino-3-deoxy-3-N-(2-diazo-3, 3, 3-trifluoropropionyl-beta-D-galactopyranosyl-(1, 4)-2-acetamindo-2-deoxy-beta-D-glycopyranoside, (DTFP-LacNAc-C8)), was evaluated as an inhibitor of alpha1,3GalT. alpha1,3GalT bound DTFP-LacNAc-C8 with an affinity (Ki = 300 microM) similar to that displayed by the enzyme for LacNAc-C8. Additional studies were done to determine the enzyme's ability to transfer a range of sugars from UDP-sugar donors. The results of these experiments demonstrated that alpha1,3GalT has a strict specificity for UDP-Gal. Finally, inactivation studies with various amino acid modifiers were done to obtain information on the importance of different types of amino acids for alpha1,3GalT activity.

Amino Sugars↗
PubMed1998

Degradation of gamma D- and gamma s-crystallins in human lenses.

The aim of this study was to determine age-related degradation of gammaD-crystallin and the cleavage sites in the connecting peptide regions of the two domains of gammaD- and gammas-crystallins. The water-soluble (WS) proteins from lenses of donors of different ages and a purified gamma-crystallin fraction were analyzed for the fragments of gammaD-crystallin by the Western blot method. Four site-specific antibodies (Ab) raised to the four regions of human gammaD-crystallin, i.e., anti-gammaD-N-Ab to the N-terminal end (residue nos. 1-9), anti-gammaD-C-Ab to the C-terminal end (residue nos. 165-173), and two to the middle regions, anti-gammaD-M1 Ab (residue nos. 78-86) and anti-gammaD-M2 Ab (residue nos. 87-95), were used. The gamma-crystallin fragments were also separated by a preparative SDS-PAGE method prior to Western blot analysis. The two-dimensional gel electrophoretic method (first dimension of isoelectric focusing followed by the second dimension of SDS-PAGE) was used to separate crystallin fragments and desired fragments were analyzed for their partial N-terminal sequences. The Western blot results showed seven major gammaD-crystallin fragments of about 4, 5, 11, 14, 15, and 17 kDa with intact N-termini but cleaved C-termini. In contrast, only three fragments with Mrs of about 5, 9, and 11 kDa were observed with intact C-termini but cleaved N-termini. Similar analysis also identified fragments with Mrs of about 5, 9, 11, and 14 kDa that originated via cleavage in the middle region of the molecule. The partial N-terminal sequencing results of the 9- to 10-kDa fragments showed cleavage in the connecting peptide region, i. e., two cleavage sites at D73-S74 and G86-S87 in gammaD-crystallin whereas four such sites at R83-A84, A84-V85, H85-L86 and G90-G91 in gammas-crystallin. Together, the results suggest that the degradation in the gammaD-crystallin mostly occurs at the C-terminal region with repeated cleavage of certain sites during aging. In addition, the major fragments with Mr of 9-10 kDa were produced via cleavages within or close to the connecting peptide regions of gammaD- and gammas-crystallins at the two and four cleavage sites, respectively, as described above.

Adult↗
PubMed1998

Purification of gamma-crystallin from human lenses by acetone precipitation method.

PURPOSE: The aim of this study was to develop a new purification method for human lens gamma-crystallin by utilizing its unique property of remaining soluble during acetone precipitation of water soluble (WS) proteins. METHODS: The WS protein fractions from lenses of donors of different ages were precipitated with 50% acetone (v/v) and the supernatant and precipitated protein fractions were collected following centrifugation. Among lens crystallins, gamma-crystallin remained soluble (recovered in the supernatant following centrifugation) while other crystallins were precipitated. To determine the recovery of maximal levels of gamma-crystallin as soluble protein during acetone precipitation, the WS proteins were precipitated under different conditions, and both supernatant and precipitated fractions were quantified for proteins and analyzed by size-exclusion chromatographic and Western blot methods. Based on these results, a three-step purification procedure for gamma-crystallin was developed which consisted of acetone precipitation followed by preparative isoelectric focusing (IEF) and size-exclusion HPLC of the soluble fraction. RESULTS: During precipitation of WS proteins by 50% (v/v) acetone, only gamma-crystallin remained soluble. The identity of gamma-crystallin was based on its Mr of 20 kDa on SDS-PAGE, co-elution with lens homogenate gamma-crystallin during a size-exclusion Agarose chromatography, immunoreactivity with anti-gamma-crystallin antibody on a Western blot and an overlap of its partial N-terminal sequence with gammaC-crystallin. A three-step procedure, as described above, provided a highly purified preparation of gammaC-crystallin from the WS protein fraction. The three-step procedure was also utilized to recover a highly purified human lens recombinant gammaD-crystallin preparation from E. coli lysate. CONCLUSIONS: The unique property of human lens gamma-crystallin of remaining soluble during acetone precipitation can be utilized to purify this crystallin by a three-step procedure. This procedure is also applicable in the purification of recombinant gammaD-crystallin from E. coli lysate.

Acetone↗
PubMed1997

Field trial of graded care profile (GCP) scale: a new measure of care.

AIM: The graded care profile (GCP) scale was developed as a practical tool in response to the Children Act 1989 to provide a measure of care in four areas: physical, safety, love, and esteem, on a bipolar continuum. This field trial was to assess its user friendliness and inter-rater agreement. METHODS: 43 nursery children and 11 registered for neglect were each scored on this scale independently by two different raters (health visitor and nursery teacher or social worker). Their inter-rater agreement was assessed by weighted kappa and user friendliness by time taken for and completeness of scoring. RESULTS: An almost perfect level of agreement was achieved in physical care (kappa = 0.899; confidence interval (CI) = 0.850 to 0.948), safety (kappa = 0.894; CI = 0.854 to 0.933), esteem (kappa = 0.877; CI = 0.808 to 0.946), and a substantial level in love (kappa = 0.785; CI = 0.720 to 0.849). Mean time taken for scoring was 20 minutes (range 10 to 30); of 54 paired scales, area of safety was not scored only in three by one of the raters. CONCLUSIONS: This scale appeared user friendly and provided grading of care with high inter-rater agreement. Its use in practice could provide an opportunity for useful comparison with other means of assessment of care, studying outcomes of different care profiles, targeting intervention, and monitoring change.

Child Abuse↗
PubMed1996

Synthesis of alpha-D-Glcp-(1-->2)-alpha-D-Glcp-(1-->3) -alpha-D-Glcp-O-(CH2)8 COOCH3 for use in the assay of alpha-glucosidase I activity.

The chemical synthesis of alpha-D-Glcp-(1-->2)-alpha-D-Glcp p-(1-->3) -alpha-D-Glcp-O-(CH2)8 COOCH3 (9), a substrate specific for alpha-glucosidase I, is reported. This enzyme removes the terminal alpha-D-Glcp unit to produce alpha-D-Glcp-(1-->3)-alpha-D-Glcp-O-(CH2)8 COOCH3 (10). This is the first synthetic substrate described for glucosidase I that allows kinetic evaluation of substrates and inhibitors of this enzyme. Tetramethylrhodamine was coupled to 9 through an ethylenediamine linker to produce a brilliant red derivative. Addition of this fluorescent dye did not affect enzyme binding to the substrate, as determined by a comparison of the Km value (1.3 mM). The fluorescent label allows visual detection of 2-3 pmol of product by TLC.

Carbohydrate Conformation↗
PubMed1996

Characterization of three isoforms of a 9 kDa gamma D-crystallin fragment isolated from human lenses.

The purpose of this study was to determine whether a 9 kDa gamma D-crystallin fragment, on in vivo post-translational modifications, exists as isoforms in the water soluble protein fraction of human lenses. In this study, three isoforms of the 9 kDa polypeptide (named as 9 kDa I, II and III) were identified and purified. In addition, the possible modified amino acids and their locations in the three isoforms were identified. The purification of the three isoforms was achieved by four steps which included separation of a mixture of crystallin fragments from the intact crystallins by a Sephadex G-50 chromatography under denaturing conditions, followed by purification of the 9 kDa polypeptide isoforms by a non-denaturing gel electrophoresis, preparative SDS-PAGE and HPLC using a C-18 column. Each of the isoforms showed a single protein band and a single peak during SDS-PAGE and HPLC analyses respectively. The three isoforms on their partial N-terminal sequence analyses, exhibited sequence identical to gamma D-crystallin starting at residue no. 87 suggesting that the isoforms contained residues no. 87 to 173 of gamma D-crystallin. On comparison of the amino acid compositions of the isoforms with that of the identical 9 kDa gamma D-crystallin fragment, the isoforms showed relatively lower amino contents of Asp, Arg, Leu and Tyr residues suggesting modifications of these residues in the isoforms. To identify the specific regions at which these amino acid residues were modified, the Western blot analysis with six site-specific polyclonal antibodies to six regions of the 9 kDa gamma D-crystallin polypeptide was carried out. Of the six antibodies raised, one was to the N-terminal region (residue nos 87-95; named as anti-9 kDa N-Ab), second to the C-terminal region (residue nos 165-173; named as anti-9 kDa C-Ab) and four to the four different middle regions [named as anti-9 kDa M1 (nos 94-100)-Ab, M2 (nos 114-120)-Ab, M3 (nos 137-143)-Ab and M4 (nos 149-154)-Ab] of the polypeptide. The Western blot analysis suggested that the 9 kDa I and 9 kDa II isoforms had modified amino acid residues in the regions of residue nos 114-120 and 165-173 whereas the 9 kDa III isoform in the regions of residue nos 114-120, 137-143, 149-154 and 165-173. It was also determined whether the 9 kDa isoforms exhibit an age-related appearance in human lenses. Western blot analysis as above of the WS-proteins from lenses from donors of different ages was carried out. On comparison of these results with an identical Western blot analysis of the three purified 9 kDa isoforms, I, II and III, it was inferred that the 9 kDa isoform III appeared earlier than other isoforms during aging in human lenses.

Adolescent↗
PubMed1996

Levels of crystallin fragments and identification of their origin in water soluble high molecular weight (HMW) proteins of human lenses.

PURPOSE: The aims of this study were to determine in the human lens water soluble-high molecular weight (WS-HMW)-proteins: (a) the levels of degraded polypeptides (crystallin fragments), and (b) the in vivo cleavage sites in the parent crystallins to produce the major fragments. METHODS: The WS-HMW proteins (Mr > 15 x 10(6) daltons) were isolated as a void volume peak from homogenates of lenses of donors of different ages using Agarose A 15m gel-filtration chromatography. The degraded polypeptides (Mr < 18 kDa), present in the WS-HMW proteins, were separated by a preparative SDS-PAGE method and quantified as a percent of total WS-HMW proteins. In addition, the parent crystallins of the major polypeptides were identified by the Western blot method using antibodies raised either to the whole crystallin molecule or to desired regions at N- and C-terminals or middle of individual crystallins. The partial N-terminal sequences of purified individual polypeptides were determined to identity the cleavage sites in parent crystallins. RESULTS: The levels of degraded polypeptides as percent of the total WS-HMW proteins increased with aging, i.e. about 5% in lenses of 16 to 19 year-old-donors compared to 27% in the 60-80 year-old-donors. As many as thirteen polypeptide species with Mr's between 3 to 17 kDa were separated from WS-HMW proteins by a preparative SDS-PAGE method. The Western blot analyses showed that the polypeptides originated from alpha-, beta- and gamma-crystallins and the cleavage sites varied in different regions of crystallins as identified by partial N-terminal sequence analyses. CONCLUSIONS: The data showed an age-related increase in levels of degraded polypeptides in the WS-HMW proteins and the polypeptides were derived from alpha-, beta- and gamma-crystallins.

Adolescent↗
PubMed1994

A spectrophotometric assay for glucosidase I.

A spectrophotometric assay for glucosidase I using the synthetic trisaccharide alpha-D-Glc 1-->2 alpha-D-Glc 1-->3 alpha-D-Glc-O(CH2)8COOCH3 is reported. The terminal glucose is released from the substrate by the enzyme and quantitated using glucose oxidase, peroxidase, and o-dianisidine. The trisaccharide is specific for glucosidase I and provides all the necessary structural features for correct interaction in the enzyme active site. The utility of the assay for monitoring enzyme activity during isolation and for use in kinetic and inhibition studies (i.e., with 1-deoxynorjirimycin) is demonstrated.

1-Deoxynojirimycin↗
PubMed1994

Covalent modification at the C-terminal end of a 9 kDa gamma D-crystallin fragment in human lenses.

The presence of a 9-kDa gamma D-crystallin fragment among water-soluble (WS) and water-insoluble (WI) proteins of human lenses was investigated using individual site specific antibodies to the N- and C-terminal regions of the molecule. The polyclonal antibodies were raised against nonapeptides corresponding to the N- and C-terminal ends and are referred to as anti-9-kDa-N and anti-9-kDa-C antibodies respectively. On Western blot analysis of WS and WI proteins from lenses of donors of different ages, the WS9-kDa species showed immunoreactivity to both the anti-9-kDa-N and anti-9-kDa-C antibodies whereas WI 9 kDa species showed immunoreactivity to only the anti-9-kDa-N antibody. This suggested that possible modification had occurred at the C-terminal region of the WI 9-kDa polypeptide. The 9-kDa species of WS, water-soluble-high-molecular-weight (WS-HMW), water-insoluble-urea-soluble (WI-US) and water-insoluble-urea-insoluble (WI-UI) protein fractions was purified by preparative SDS-PAGE separation followed by HPLC on a C-18 column. Two forms of 9-kDa species were isolated from the WS proteins; one associated with the gamma-crystallin, immunoreactive to both the antibodies, and the other associated with high-molecular-weight protein, immunoreactive to only the anti-9-kDa-N antibody. In contrast, only one form of the 9-kDa species, immunoreactive to the anti-9-kDa-N antibody could be detected in the WI-US and WI-UI protein fractions.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗
PubMed1994

Identification of origin of two polypeptides of 4 and 5 kD isolated from human lenses.

PURPOSE: To purify crystallin fragments (degraded polypeptides molecular weight < 18 kD) and identify their parent crystallins. METHODS: The purification of polypeptides with apparent molecular weights of 4 and 5 kD was carried out using three sequential steps: Sephadex G-50 chromatography under denaturing conditions, preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and high-performance liquid chromatography using a C-18 column. The parent crystallins of the two polypeptides were identified by the Western blotting method using polyclonal antibodies raised against individual 4 and 5 kD polypeptides and by comparing N-terminal amino acid sequences of the polypeptides with crystallins. RESULTS: Two polypeptides of 4 and 5 kD were purified by the three sequential steps as described from water-soluble proteins of lenses from 60-80-year-old donors. Both purified polypeptides showed a single major peak during high-performance liquid chromatography on a C-18 column and also a single band during sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The Western blot analyses showed maximum immunoreactivity of the anti-4 kD polypeptide antibody to a 22 kD species of beta-crystallin, whereas the anti-5 kD polypeptide antibody showed maximum reactivity to only the alpha B crystallin. These results were further confirmed during comparison of the N-terminal amino acid sequences of the two polypeptides with crystallins. Such comparison showed that the 4 kD polypeptide originated from beta A 3/A1 crystallin after cleavage at His187-His188 bond. Further, the 5 kD polypeptide was a fragment of alpha B crystallin that originated after cleavage at Val145-Asn146 bond. CONCLUSION: These results showed that specific bonds of beta A3/A1 and alpha B crystallins are posttranslationally cleaved in vivo to produce 4 kD and 5 kD polypeptides, respectively.

Aged↗