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R W Gracy

Publications and source records attributed to R W Gracy.

At least 55 records · Page 3Linked to original sources
PubMed1989

Studies on the structure of lecithin:cholesterol acyltransferase (LACT)--comparisons of the active site region and secondary structure of the human and the porcine enzymes.

1. Chemical modification of essential serine, histidine and cysteine residues of porcine LCAT were accompanied by loss of enzymatic activity. 2. Modification of cysteine with DTNB inactivated the enzyme which could not be reactivated by KCN suggesting direct involvement of the cysteine residue(s) in catalysis. 3. About half of the primary structure of the porcine enzyme was determined. 4. Respective regions of the human and porcine LCAT are highly homologous; especially, the amino-terminus and the region surrounding the DFP-labeled serine residues. 5. The observed primary structure differences represent amino acid substitutions that are projected to induce significant changes in secondary structure.

Amino Acid Sequence↗
PubMed1989

Experimental parameters affecting the ultraviolet-induced tryptophan modification in the lens.

Experimental parameters affecting photolysis of tryptophan in the lens were evaluated, including: (a) the use of previously-frozen vs. freshly excised lenses, (b) the use of lenses in aqueous buffered solutions vs. in air, (c) the species source of the lens, and (d) the effects of oxygen depletion. Lenses were subjected to monochromatic ultraviolet radiation followed by monitoring of the tryptophan fluorescence spectra while exciting at two or more wavelengths. Previously frozen lenses showed faster UV-induced degradation of tryptophan, particularly in aqueous media. A rapid leakage of fluorescent components (including amino acids and peptides) from intact lenses was observed. Evidence for the artifactual production of free radicals during photometric monitoring of samples in solution was obtained. Species dependent differences were also observed.

Amino Acids↗
PubMed1989

Increased carbonyl content of proteins in synovial fluid from patients with rheumatoid arthritis.

The carbonyl content of proteins in the synovial fluid (SF) of patients with rheumatoid arthritis was significantly (p less than or equal to 0.10) elevated over levels in the SF of patients with osteoarthritis (OA). Other indicators of oxidative damage including catalse, ceruloplasmin, ferritin and superoxide dismutase also showed statistically significant differences (p less than or equal to 0.05) compared to patients with OA.

Arthritis, Rheumatoid↗
PubMed1988

Isolation and sequencing of a cDNA clone encoding lysosomal membrane glycoprotein mouse LAMP-1. Sequence similarity to proteins bearing onco-differentiation antigens.

We have isolated and sequenced a cDNA clone encoding the mouse LAMP-1 (mLAMP-1) major lysosomal membrane glycoprotein. The deduced protein sequence, which included the NH2-terminal portion of the mLAMP-1 molecule, consisted of 382 amino acids (Mr 41,509). The predicted structure of this protein included an NH2-terminal intralumenal domain consisting of two homology units of approximately 160 residues each separated by a proline-rich hinge region. Each homology unit contained four cysteine residues with two intercysteine intervals of 36-38 residues and one of 68 or 76 residues. The molecule also contained 20 asparagine-linked glycosylation sites within residues 1-287, a membrane-spanning region from residues 347 to 370, and a carboxyl-terminal cytoplasmic domain of 12 residues. The biochemical properties and amino acid sequence of mLAMP-1 were highly similar to those of two other molecules that have been studied as cell surface onco-differentiation antigens: a highly sialylated polylactosaminoglycan-containing glycoprotein isolated from human chronic myelogenous leukemia cells (Viitala, J., Carlsson, S. R., Siebert, P. D., and Fukuda, M. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, in press) and the mouse gp130 (P2B) glycoprotein, in which an increase in beta 1-6 branching of asparagine-linked oligosaccharides has been correlated with metastatic potential in certain tumor cells (Dennis, J.W., Laferte, S., Waghorne, C., Breitman, M.L., and Kerbel, R.S. (1987) Science 236, 582-585).

Amino Acid Sequence↗
PubMed1988

Molecular basis for the isozymes of bovine glucose-6-phosphate isomerase.

Glucose-6-phosphate isomerase exists as multiple, catalytically active isozymes which can be resolved by polyacrylamide gel electrophoresis, isoelectric focusing, and ion-exchange chromatography. GPI from bovine heart was purified to homogeneity and each of the isozymes resolved. Four of the five isozymes were characterized with regard to their physical, chemical, and catalytic properties in order to establish their possible physiological significance and to ascertain their molecular basis. The isozymes exhibited identical native (118,000) and subunit (59,000) molecular weights but had different apparent pI values of 7.2, 7.0, 6.8, and 6.6. Kinetic constants, such as turnover number, Km and Ki values, were identical for all isozymes in either reaction direction. Structural analyses showed that the amino termini were blocked and the carboxyl terminal sequences were -Glu-Ala-Ser-Gly for all four isozymes. The most basic isozyme was more stable than the more acidic isozymes at pH extremes, at high ionic strength, in the presence of denaturants, or upon exposure to proteases. When the most basic isozyme was incubated in vitro under mild alkaline conditions, there was a spontaneous generation of the more acidic isozymes with electrophoretic properties identical to those found in vivo. The simultaneous release of ammonia along with the spontaneous shift to more acidic isozymes indicates deamidation as the molecular basis for the formation of the acidic isozymes both in vivo and in vitro. The change in the peptide fragmentation patterns following cleavage by hydroxylamine further suggests that deamidation of specific Asn-Gly bonds accounts for the structural basis of the isozymes.

Amino Acids↗
PubMed1987

The catalytic center of lecithin:cholesterol acyltransferase: isolation and sequence of diisopropyl fluorophosphate-labeled peptides.

Lecithin:cholesterol acyltransferase (LCAT) was purified from hog plasma and subsequently reacted with [3H]-Diisopropyl fluorophosphate (DFP). The labeled enzyme was digested with pepsin and the peptides separated by high performance liquid chromatography (HPLC). Two radioactive peptides were isolated, subjected to automated amino acid sequencing and yielded the following data: A) Ile-Ser-Leu-Gly-Ala-Pro-Trp-Gly-Gly-Ser, and B) Tyr-Ile-Phe-Asp-x-Gly-Phe-Pro-Tyr-x-Asp-Pro-Val. Both of these sequences represent very highly conserved regions of the enzyme when compared to the sequence of human LCAT. Peptide (A) is considered to represent the catalytic center of LCAT based on comparisons with data reported in the literature.

Amino Acid Sequence↗
PubMed1986

In vitro deamidation of human triosephosphate isomerase.

The effects of pH, temperature, buffer ion, ionic strength, protein concentration, and substrate on the rates of specific, spontaneous deamidations of Asn-15 and Asn-71 of human triosephosphate isomerase were examined. Elevated temperature and pH facilitate the deamidations, and the deamidation rate is dependent on the specific buffer ions indicating a general base catalysis mechanism. The presence of substrate also enhances the rates of deamidation. The effect of substrate may be related to conformational changes in the catalytic center which are known to cause changes in the subunit-subunit contact sites where Asn-15 and Asn-71 are located. The enhanced deamidation in the presence of substrate may, in part, account for the more rapid rate of deamidation observed in vivo.

Asparagine↗
PubMed1986

Molecular basis of the isozymes of bovine glucose-6-phosphate isomerase.

Glucose-6-phosphate isomerase occurs in different bovine tissues as multiple, catalytically active isozymes which can be resolved by polyacrylamide gel electrophoresis (PAGE) and isoelectric focusing (IEF). Most differentiated tissues have five distinct forms with apparent pI values of 7.2, 7.0, 6.8, 6.6, and 6.4. Young, mitotically active, cells of the intestinal mucosa and the epithelium of the eye lens show only the two more basic isozymes, while old cells in the cortex and nucleus of the eye lens accumulate the more acidic isozymes. All of the isozymes exhibit equal separation based on charge-to-mass ratio (PAGE) and charge (IEF), thus indicating only charge changes. The isozyme patterns are unchanged in the presence of reducing agents or protease inhibitors. Each isozyme was purified to homogeneity and shown to exhibit identical subunit molecular weights (59,000) on SDS-gel electrophoresis. Each of the isolated isozymes, when subjected to PAGE or IEF, exhibited a single band, indicating that the isozymes are not generated as a result of electrophoresis. When the most basic isozyme was incubated in vitro under mild alkaline conditions, there was a spontaneous generation of the more acidic isozymes with properties identical to those found in vivo. The isozymes, thus, appear to be the result of spontaneous, postsynthetic modifications involving the addition of equal numbers of negative charges and are consistent with the deamidation of specific asparagine and/or glutamine residues.

Aging↗
PubMed1986

The redox state in lymphocytes from patients with rheumatoid arthritis.

Our study was designed to quantitate levels of NAD+, NADH, NADP(H), and ATP in peripheral blood lymphocytes with and without mitogenic stimulation from patients with rheumatoid arthritis (RA). No differences were found in patients with RA and healthy controls. Our data suggest the ability of the redox system of circulating peripheral lymphocytes to respond to mitogenic stimulation in patients with RA is not markedly impaired.

Adenosine Triphosphate↗
PubMed1985

The importance of disulfide bridges in human endopeptidase (enkephalinase) after proteolytic cleavage.

The neutral endopeptidase (NEP) is a membrane-bound enzyme, which is solubilized by treatment with the protease, papain. Papain did not affect the apparent catalytic activity or the molecular mass of the purified human enzyme in SDS-PAGE. When NEP was treated with a reducing agent after papain digestion, it dissociated into smaller, lower molecular mass fragments. Amino acid analysis and s-carboxymethylation of the half cystine residues indicated that NEP contains four S-S bridges. We concluded that, although covalent bonds appear to be cleaved in NEP by papain, its activity and structure are sustained by S-S bridges.

Amino Acids↗
PubMed1985

Interrelationships among human aldo-keto reductases: immunochemical, kinetic and structural properties.

We have proposed earlier a three gene loci model to explain the expression of the aldo-keto reductases in human tissues. According to this model, aldose reductase is a monomer of alpha subunits, aldehyde reductase I is a dimer of alpha, beta subunits, and aldehyde reductase II is a monomer of delta subunits. Using immunoaffinity methods, we have isolated the subunits of aldehyde reductase I (alpha and beta) and characterized them by immunocompetition studies. It is observed that the two subunits of aldehyde reductase I are weakly held together in the holoenzyme and can be dissociated under high ionic conditions. Aldose reductase (alpha subunits) was generated from human placenta and liver aldehyde reductase I by ammonium sulfate (80% saturation). The kinetic, structural and immunological properties of the generated aldose reductase are similar to the aldose reductase obtained from the human erythrocytes and bovine lens. The main characteristic of the generated enzyme is the requirement of Li2SO4 (0.4 M) for the expression of maximum enzyme activity, and its Km for glucose is less than 50 mM, whereas the parent enzyme, aldehyde reductase I, is completely inhibited by 0.4 M Li2SO4 and its Km for glucose is more than 200 mM. The beta subunits of aldehyde reductase I did not have enzyme activity but cross-reacted with anti-aldehyde reductase I antiserum. The beta subunits hybridized with the alpha subunits of placenta aldehyde reductase I, and aldose reductase purified from human brain and bovine lens. The hybridized enzyme had the characteristic properties of placenta aldehyde reductase I.

Alcohol Oxidoreductases↗
PubMed1984

Solid-phase protein and peptide sequencing using either 4-N,N-dimethylaminoazobenzene 4'-isothiocyanate or phenylisothiocyanate.

Automated solid-phase sequencing using 4-N,N-dimethylaminoazobenzene 4'-isothiocyanate (DABITC) double coupling or regular phenylisothiocyanate (PITC) degradation procedures have been investigated. Employing sensitive high-performance liquid chromatography for the identification of amino acid thiohydantoin derivatives (PTH and DABTH), both methods were capable of sequencing immobilized peptides or proteins at the subnanomole levels. In the sequencing program using DABITC, alternate methanol and dichloroethane washes and automated conversion using methanolic HCl containing dithiothreitol were introduced to obtain clean thiazolinones and to ensure high recovery yields of the thiohydantoins. Using regular PITC degradation with a 59-min program, the background peaks of the side products could be reduced to enhance HPLC identification. Peptides or proteins attached to the glass beads or resins via the carboxyl terminii or epsilon-amino groups of lysyl residues could be readily sequenced up to 30 identifiable degradation cycles, where the sequencing is generally terminated due to the increased background components.

Amino Acid Sequence↗
PubMed1984

Primary structure of human triosephosphate isomerase.

Human placental triosephosphate isomerase was isolated by an improved procedure and recovered with the highest specific activity ever reported. Employing this purification procedure, sufficient amounts of the enzyme were obtained for detailed primary structural studies. For sequences analysis, the enzyme was reduced and carboxymethylated and subjected to tryptic and chymotryptic digestions. The peptide mixtures were separated by high-performance liquid chromatography using octyl or alkylphenyl reverse-phase columns and trifluoroacetic acid/acetonitrile gradient elution systems. Sequence analyses of the intact enzyme, tryptic, chymotryptic, and cyanogen bromide peptides were accomplished using high-sensitivity solid-phase sequencing procedures with either 4-N,N-dimethylaminoazobenzene-4'-isothiocyanate or phenylisothiocyanate. The primary structure of human triosephosphate isomerase is constructed from the alignment of the tryptic peptides with the analysis of the overlapping chymotryptic peptides. The enzyme is a dimeric molecule consisting of two identical polypeptide chains with 248 amino acid residues and a calculated subunit molecular mass of 26,750 daltons. A comparison of the amino acid sequences from the human placental enzyme and from other species such as rabbit, chicken, and coelacanth muscles showed relatively high sequence homology, indicating that the evolution of the enzyme is very conservative. The amino acids of the active-site pocket and the subunit-subunit contact sites exhibit few changes.

Amino Acid Sequence↗
PubMed1983

Changes in NAD levels in human lymphocytes and fibroblasts during aging and in premature aging syndromes.

NAD levels markedly increase upon mitogen stimulation of lymphocytes from young subjects. In contrast, lymphocytes from old subjects do not increase NAD levels upon stimulation. A survey of 35 individuals aged 18-79 years revealed a significant age-dependent decrease in the NAD response to mitogen stimulation. No significant differences were noted in lymphocytes from age-matched individuals with Down's syndrome or diabetes mellitus. On the other hand, cultured skin fibroblasts showed elevated NAD levels with age. However, this effect appears to be due to increased size of the cells since the NAD/protein ratio is unchanged. Skin fibroblasts from patients with progeria exhibit much higher levels of NAD and protein per cell than age-matched controls.

Adolescent↗