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R W Gracy

Publications and source records attributed to R W Gracy.

At least 73 records · Page 4Linked to original sources
PubMed1982

Increased lability of triosephosphate isomerase in progeria and Werner's syndrome fibroblasts.

Triosephosphate isomerase was found to have an increased thermolabile component in skin fibroblasts from patients with progeria and Werner's syndrome when compared with normal fibroblasts. Mixtures of cell extracts from progeria or Werner's syndrome with normal fibroblasts gave intermediate levels of the heat-labile triosephosphate isomerase suggesting the absence of cytosolic destabilizing factors. The incorporation of the protease inhibitors 1-tosylamide-2-phenylethyl chloromethyl ketone, N-alpha-p-tosyl-L-lysine chloromethyl ketone, phenylmethyl-sulfonyl fluoride, and pepstatin A in cell extracts failed to affect the level of the labile form of triosephosphate isomerase. The labile component also accumulates in normal fibroblasts in late passage in tissue culture and appears to be identical to the deamidated form of the enzyme which accumulates in other aging cells.

Adolescent↗
PubMed1981

Chemical modification of critical catalytic residues of lysine, arginine, and tryptophan in human glucose phosphate isomerase.

Human glucose phosphate isomerase was subjected to a series of chemical modifications aimed at identifying residues essential for catalytic activity. A specific lysine was found to stoichiometrically react with pyridoxal 5'-phosphate forming a reversible Schiff base which could be reduced with NaBH4. The covalently modified enzyme was specifically cleaved with hydroxylamine at three labile Asn-Gly sequences yielding a series of peptides which were separated by sodium dodecyl sulfate-polyacrylamide electrophoresis. The modified lysine was located in the COOH-terminal peptide. A critical arginine residue/subunit was found to be stoichiometrically modified with either 2,3-butadione or cyclohexadione. At high concentrations of butadione, an irreversible nonspecific modification of essentially all arginines occurred. An essential tryptophan residue was found to be stoichiometrically modified with N-bromosuccinimide in a similar fashion. Each of the chemical modifications of these three residues followed pseudo-first order and rate saturation kinetics and the modifications were prevented by the presence of substrates or competitive inhibitors. Circular dichroic spectral studies and analytical gel filtration indicated that these modifications have no effect on the quarternary structure and little effect on the secondary and tertiary structures of the enzyme. However, the extensive modification of arginine with butadione caused a dissociation of the enzyme into monomers and significant changes in tertiary structure. These studies provide new insights into functional aspects of isomerization and also provide an effective method for evaluating structural consequences of chemical or genetic modification of the enzyme.

Affinity Labels↗
PubMed1981

Impaired glycolysis of human lymphocytes during aging.

When peripheral lymphocytes from young persons are stimulated in vitro with phytohemagglutinin, an increase in the levels of all the glycolytic enzymes occurs concomitantly with blastogenesis. The specific activities of enzymes increase approximately 200%, with a greater induction of the latter half of the pathway. The increases do not represent a general enzyme induction, since nonglycolytic enzymes such as glucose 6-phosphate dehydrogenase and isocitrate dehydrogenase do not increase during transformation. Human lymphocytes from a geriatric population were also subjected to mitogen stimulation under identical conditions. The initial levels of the enzymes were essentially identical in lymphocytes from young and old subjects as were cultured controls which received no mitogen. However, during mitogen stimulation the cells from the old subjects failed to increase the glycolytic enzymes. This inability to activate glycolysis may be related to the decline in cell-mediated immunity which is known to occur with advancing age.

Adult↗
PubMed1981

Molecular basis for the accumulation of acidic isozymes of triosephosphate isomerase on aging.

Triosephosphate isomerase exhibits acidic electrophoretic subforms in many tissues and these isozymes appear to increase during aging of erythrocytes and the eye lens. Incubation of the pure enzyme under mild alkaline conditions results in the generation of acidic forms which are identical to those found in vivo. Structural analysis of these isozymes from both in vivo and in vitro studies showed that they are the result of deamidation of two specific asparagines (Asn-15 and Asn-71). These labile asparagines are located in the subunit-subunit contact sites, and the deamidations introduce a total of four new negative charges in the contact site. The positions of the new aspartic acid residues are juxtaposed, thus creating charge-charge interactions which cause the dimeric enzyme to dissociate more readily. These studies (1) explain the molecular basis for the acidic isozymes observed in many tissues, (2) show that the deamination process is spontaneous and requires no intrinsic cell factors, (3) show that the deamination occurs in a sequential fashion with the deamidation of Asn-71 preceding the deamidation of Asn-15, and (4) suggest that proteolytic degradation processes may become altered during aging resulting in the accumulation of the deamidated intermediates of the normal catabolic process.

Aging↗
PubMed1980

Affinity labeling and characterization of the active site histidine of glucosephosphate isomerase. Sequence homology with triosephosphate isomerase.

N-Bromoacetylethanolamine phosphate was found to act as a specific affinity label for the active center of glucosephosphate isomerase. The inactivation process followed pseudo-first order kinetics, was irreversible and exhibited rate saturation kinetics with minimal half-lives of inactivation of 4.5 and 6.3 min for the enzyme isolated from human placenta and rabbit muscle, respectively. The pH dependence of the inactivation process closely paralleled the pH dependence of the overall catalytic process with pKa values at pH 6.4 and 9.0. The stoichiometry of labeling of either enzyme, as determined with N-bromo[14C2]acetylethanolamine phosphate, was 1 eq of the affinity label/subunit of enzyme. After acid hydrolysis and amino acid analysis of the radioactive affinity-labeled human enzyme, only radioactive 3-carboxymethyl histidine was found. In the case of the rabbit enzyme, the only radioactive derivative obtained was 1-carboxymethyl histidine. Active site tryptic peptides were isolated by solvent extraction, thin layer peptide fingerprinting, and ion exchange chromatography before and after removal of the phosphate from the active site peptide. Amino acid analysis of the labeled peptides from the two species were very similar. Using high sensitivty methods for sequence analysis, the primary structure of the active site was established as Val-Leu-His-Ala-Glu-Asn-Val-Asp (Gly,Thr,Ser) Glu-Ile (Thr-Gly-His-Lys-Glx)-Tyr-Phe. Apparent sequence homology between the catalytic center of glucosephosphate isomerase and triosephosphate isomerase suggest that the two enzymes may have evolved from a common ancestral gene.

Affinity Labels↗
PubMed1979

A deletion mutation in glucosephosphate isomerase (GPI Denton).

A new genetic variant form of glucosephosphate isomerase has been found in a family heterozygous for the mutant allele. The mutant enzyme, unlike other phenotypic variants, does not appear to be the result of a single amino acid replacement. The allozyme exhibits an isoelectric point of 5.7 and is thus much more acidic than the normal enzyme (pI = 9.3). The allozyme has been isolated from placenta and separated from the normal homodimer and heterodimer by isoelectric focusing. The enzyme exhibits normal Km and Ki values for the substrates and competitive inhibitors. The allozyme exhibits a normal pH optimum and thermal stability. However, the molecular specific activity of the variant enzyme as quantitated by radioimmunoassay is significantly lower than normal. Analytical gel filtration revealed that the molecular weight of the weight of the enzyme is significantly lower than the normal enzyme. These data thus suggest that the phenotype is unlike any previously reported and is due to a deletion mutation.

Alleles↗