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R W Gracy

Publications and source records attributed to R W Gracy.

At least 37 records · Page 2Linked to original sources
PubMed1992

Relationship between the catalytic center and the primary degradation site of triosephosphate isomerase: effects of active site modification and deamidation.

Covalent modification of the active site Glu165 of triosephosphate isomerase (TPI) (EC 5.3.1.1) with the substrate analogue 3-chloroacetol phosphate (CAP) induces conformational changes similar to those observed during catalysis. We have introduced CAP into the active sites of TPI from yeast, chicken, pig, and rabbit, and assessed the effect of this modification on the structural integrity of the protein. CAP binding accelerated the specific deamidation of Asn71 in mammalian TPI. Transverse urea gradient gel electrophoretic analysis showed that the CAP-TPI dimer dissociates more readily than the native dimer. Hybrids composed of one CAP-modified subunit and one native subunit exhibited intermediate stability. The deamidated enzyme was more susceptible to proteases and denaturing conditions. Subtilisin cleaved the rabbit enzyme primarily at the Thr139-Glu140 bond. The resulting peptides remained noncovalently attached, and the enzyme retained catalytic activity. The data provide further evidence of the interactions between the catalytic center and the subunit interface and that the specific deamidation destabilizes the enzyme initiating its degradation. The enhancement of deamidation upon binding of substrate and catalysis suggest that molecular wear and tear may be involved in regulating proteolytic turnover of the enzyme.

Amides↗
PubMed1992

Effects of active site modification and reversible dissociation on the secondary structure of triosephosphate isomerase.

Binding of ligands to the catalytic center of mammalian triosephosphate isomerase (TPI) induces a conformational change(s) that enhances the specific deamidation of Asn71 at the subunit interface. Deamidation initiates dissociation and degradation of the enzyme in vivo and in vitro. We have utilized circular dichroism spectroscopy to examine the conformational changes in the enzyme upon ligand binding and subunit dissociation/reassociation. Native TPI from rabbit, chicken, and yeast exhibit similar spectra at pH 7.5, but are substantially different at pH 9.5. Covalent reaction of the active site Glu 165 with the substrate analogue 3-chloroacetol phosphate results in a conformational change (decrease in beta-sheet) which is similar in TPI from all three species. Reversible dissociation of the dimeric enzyme in guanidine followed by dialysis, although permitting full recovery of catalytic activity, results in refolded dimers with decreased alpha-helix. These conformational changes induced by ligand binding, pH, or reversible dissociation explain, in part, the differences in the chemical and physical properties of the enzyme from the three species at alkaline pH, the increased lability of the dissociated/reassociated enzyme, and corroborate 31P NMR data on substrate-induced conformational changes. These studies also support the concept of molecular wear and tear whereby ligand binding at the catalytic center induces conformational changes that increase the probability of covalent modification and ultimate degradation of the protein.

Animals↗
PubMed1991

Probing the catalytic sites of triosephosphate isomerase by 31P-NMR with reversibly and irreversibly binding substrate analogues.

We have explored the degree of independence of the two catalytic centers, interactions between the catalytic centers and the subunit-subunit contact sites, and different conformations of triosephosphate isomerase (TPI), by simultaneously employing irreversibly (covalent) and reversibly binding substrate analogues and monitoring their 31P-NMR resonances. 3-Chloroacetol phosphate (CAP) was bound to the active site by reaction with Glu165. The resulting, inactive (CAP-TPI)2 complex exhibited two distinct 31P-NMR resonances which were independent of pH and represent two conformational forms of the enzyme. Dissociation in guanidine hydrochloride followed by redimerization resulted in a single conformation. This was observed with the enzyme from chicken, rabbit and yeast. The inactive (CAP-TPI)2 dimer was mixed with native TPI, and dissociated/reassociated to form heterodimers (CAP-TPI)(TPI) in which one subunit contained the CAP label and the other subunit was unmodified. This hybrid migrated intermediate between the native and CAP-modified enzyme on nondenaturing PAGE. The heterodimer exhibited 50% the activity of the native dimer, but kinetic properties were otherwise indistinguishable. The reversibly binding transition state analogue, 2-phosphoglycolate (PGA), was used to probe the remaining vacant active site of the heterodimer. Bound PGA exhibited a pH-independent 31P-NMR resonance which was readily distinguishable from resonances of CAP-TPI and free PGA. No differences were observed in the binding of PGA to the vacant subunit of the heterodimer or the native dimer, further pointing to the independent nature of the two catalytic centers. However, the (CAP-TPI)(TPI) heterodimer was more susceptible to subunit dissociation in guanidine hydrochloride than the native dimer. Thus, it appears that the two active sites function completely independently of each other, but that the binding of CAP at the active center loosens the subunit-subunit contact. In addition, the two forms of the enzyme-inhibitor complex trapped by reaction with CAP may represent conformations with the hinged lid or flexible loop (residues 166-176) in the open and closed positions.

Affinity Labels↗
PubMed1991

Characterization of the transcription unit and two processed pseudogenes of chimpanzee triosephosphate isomerase (TPI).

Three members of the chimpanzee TPI (encoding triosephosphate isomerase) gene family, the transcription unit and two processed pseudogenes, have been characterized by genomic blotting and nucleotide sequence analysis. The bona fide TPI gene spans 3.5 kb with seven exons and six introns, and is the first hominoid TPI gene to be completely sequenced. The chimpanzee gene exhibits a very high degree of sequence identity with human and rhesus TPI genes. For example, the polypeptides of 248 amino acids (aa) encoded by the chimpanzee and human TPI genes are identical, but the codons for five of these aa differ in the third codon wobble position. No alternative splice sites could be identified in the intervening sequences of the gene and, thus, the molecular basis for the synthesis of the proliferation-specific TPI isozyme observed in hominoids remains elusive. An Alu member occurs upstream from one of the processed pseudogenes, and short sequences with significant identity to the primate LINE-1 element flank the region encompassing the Alu member and TPI pseudogene. A solitary endogenous retroviral long terminal repeat occurs within the structural region of the other processed pseudogene. The ages of the processed pseudogenes are estimated to be 2.6 and 10.4 million years, implying that one was inserted into the genome before and one after the divergence of the chimpanzee and human lineages.

Amino Acid Sequence↗
PubMed1991

Effects of amiprilose hydrochloride on the components of human skin equivalents.

Amiprilose hydrochloride has been shown to inhibit the proliferation of a number of hyperproliferative cell types including psoriatic skin cells. In the present study, the effects of amiprilose hydrochloride on human tissue equivalents were examined by incubating a) dermal equivalents, b) skin equivalents in the process of epidermalization, and c) mature skin equivalents, with varying concentrations of the drug. In all three models amiprilose hydrochloride concentrations of 0.1% (wt/vol) and lower were not toxic to fibroblasts and keratinocytes and did not interfere with the differentiation of the skin equivalent and the developing skin equivalent. When tested in dermal equivalents, concentrations of amiprilose hydrochloride between 0.1 and 0.5% resulted in changes in fibroblast morphology with development of large intracellular vacuoles, and concentrations greater than 5% were toxic. In mature skin equivalents, in addition to changes in fibroblast morphology, amiprilose hydrochloride in concentrations of 1 to 10% affected the epidermis. When 0.5% amiprilose hydrochloride was present in the developing skin equivalent during differentiation, the epidermal keratinocytes were also affected. Thus the morphology of basal keratinocytes was modified, the differentiation was incomplete, and the dermal-epidermal attachment was compromised. These studies suggest the possibility of an extracellular mechanism of action of amiprilose hydrochloride and delineate acceptable dosage ranges for the potential drug.

Cell Differentiation↗
PubMed1991

Cellular models and tissue equivalent systems for evaluating the structures and significance of age-modified proteins.

The accumulation of modified proteins in aging is well documented in many aging models. For example, the deamidated isoforms of triosephosphate isomerase accumulate in: (a) old erythrocytes, (b) fibroblasts from old donors, (c) fibroblasts aged in vitro, (d) premature-aging syndromes and (e) old cells in the eye lens. However, a fundamental remaining question is: 'Do such modified proteins interfere with cellular function?' It has been difficult to assess this question at the molecular level using whole-organism models and equally frustrating to evaluate the physiological significance of such changes using classical cellular models. Tissue equivalent systems (TES) provide an opportunity for examining the molecular basis and physiological consequences of modified proteins during aging. TES are composed of differentiating and proliferating heterogeneous cell types with symbiotic cell-cell and cell-matrix interactions. They closely resemble, both morphologically and functionally, the tissues from which they were derived. Aging studies utilizing TES can provide information on modifications of protein structures, isozyme patterns, enzymes of the cellular environmental protection system and metabolic parameters which may regulate protein synthesis and degradation.

Adenosine Triphosphate↗
PubMed1990

Phosphorus-31 nuclear magnetic resonance spectroscopy reveals two conformational forms of chloroacetol phosphate-bound triosephosphate isomerase.

Chloroacetol phosphate covalently reacts with Glu-165 in the catalytic center of triosephosphate isomerase. Reaction of the enzyme with the substrate analogue results in two 31P resonances at 6.8 and 5.5 ppm. Dissociation with guanidinium chloride results in a single resonance at 4.5 ppm. Reassociation and redimerization of the triosephosphate isomerase-chloroacetol phosphate complex restores only the resonance at 5.5 ppm. The two 31P resonances appear to represent different conformations of the enzyme which are trapped upon reaction with the affinity label.

Affinity Labels↗
PubMed1990

Isoforms of chicken triosephosphate isomerase are due to specific oxidation of cysteine126.

The electrophoretic isoforms of mammalian triosephosphate isomerase (TPI; EC 5.3.1.1) are due to deamidation at two Asn-Gly sites (Asn15 and Asn71). Deamidation of these two asparagines in the subunit-subunit interface of the isologous dimer appears to destabilize the dimer and initiate degradation of the protein. Chicken TPI contains a lysine substitution for Asn71, thus precluding this deamidation site. Nevertheless, the chicken enzyme exhibits three electrophoretic isoforms. This multiplicity is not the result of deamidation of the remaining Asn15 site, but due to a specific site which is highly susceptible to oxidation. The three isoforms of chicken TPI can be reduced to a single form in the presence of high concentrations of reducing agents (e.g., greater than 15 mM dithiothreitol or greater than 50 mM 2-mercaptoethanol) and are also generated when oxidizing agents, such as oxidized glutathione, are present. The oxidized isoforms exhibit lowered catalytic activity and are more susceptible to denaturation and proteolytic degradation than the native enzyme. Structural analysis of the isoforms by chemical cleavage at the cysteine peptide bonds with 2-nitro-5-thiocyanobenzoic acid and subsequently at the methionines with CNBr followed by peptide sequencing reveals that Cys126 is the site of the modification. Since the oxidized isoforms of chicken TPI accumulate in vivo during aging analogous to the deamidated isoforms from mammals, it appears that TPI is the first example of a protein which has evolved two specific types of weak links which may initiate turnover of the protein.

Amino Acid Sequence↗
PubMed1990

Isolation and characterization of human glucose-6-phosphate isomerase isoforms containing two different size subunits.

Previously undetected isoforms of human glucose-6-phosphate isomerase (GPI) have been isolated utilizing substrate-induced elution of the enzyme from spherical cross-linked phosphocellulose as an affinity ligand and subjected to a series of physical and chemical studies. The two major isoforms (1, 48%, pI 9.13; 2, 36%, pI 9.00) are homodimers of subunits of 63.2 kDa (Type-A) and are charge isomers, probably representing deamidation of specific Asn-Gly sequences as in other species. Isoform 3 (13%, pI 8.84) is a heterodimer composed of the Type-A subunit and a previously unreported larger subunit of 69.8 kDa (Type-B). Isoform 4 (3%, pI 8.62) is a BB-homodimer. Structural differences in the two types of subunits are also apparent from CNBr fragmentation patterns. Carbohydrate analyses show that, even though potential N- and O-linked glycosylation sites exist, the isoforms are not due to glycosylation. Recently recognized sequence similarities between GPI and the neurotropic lymphokine, neuroleukin (NLK) suggest that GPI and NLK are either derived from the same gene or represent modifications of the same protein. The possibility of NLK-GPI dimers exists, but the new isoforms identified in this study do not appear to represent hybrids of GPI subunits with mature NLK.

Amino Acids↗
PubMed1990

In vivo degradation of oxidized, regenerated cellulose.

Oxidized, regenerated cellulose (ORC) was surgically implanted on the uterine horns of rabbits, and its biodegradation was studied in vivo. Samples of peritoneal lavages, serum, and urine were collected during the degradation process and analyzed for carbohydrate components utilizing high-performance liquid chromatography with pulsed amperometric detection (h.p.l.c.-p.a.d.). Degradation was rapid, and oligomeric products were evident primarily in the peritoneal fluid from the implantation site, with no apparent accumulation in either the serum or the urine. The size distribution and the amount of the oligomeric products decreased after day one, and by day four peritoneal lavages were essentially free of oligomers. The structure of the products formed was consistent with the lability of the polymer in solution, and the kinetics of degradation paralleled the results of the previously reported in vitro studies. Rabbit peritoneal macrophages, when incubated with ORC in vitro were observed to readily ingest and hydrolyze the polymeric material. A mechanism of degradation consisting of chemical depolymerization, followed by enzymatic hydrolysis mediated by glycosidases endogenous to peritoneal macrophages, is proposed.

Animals↗
PubMed1990

Biodegradation of oxidized regenerated cellulose.

The in vitro solubilization and degradation of regenerated cellulose was studied under conditions which approximate those found in vivo, when the material is used as an adhesion barrier to assist normal wound repair. Factors affecting solubilization which were examined included the effects of serum or plasma, and the presence of hydrolytic enzymes. Products of the solubilization and degradation processes were examined by high performance liquid chromatography coupled with pulsed amperometric detection. The oxidized polymer readily undergoes chain shortening to give oligomers which, in the presence of plasma or serum, are further hydrolyzed to smaller fragments, including glucuronic acid and glucose. Proposed mechanisms of degradation are discussed.

Biodegradation, Environmental↗
PubMed1990

Inhibition of psoriatic cell proliferation in in vitro skin models by amiprilose hydrochloride.

Amiprilose hydrochloride, a 3-substituted glucose derivative, was found to inhibit the proliferation of human fibroblasts and keratinocytes originating from psoriatic lesions. Fibroblasts and keratinocytes were obtained from skin biopsies of normal donors, and from the biopsies of active/involved and uninvolved sites of psoriatic donors. The cells were cultured as monolayers or as components of tissue equivalent models. Keratinocytes and fibroblasts originating from biopsies of psoriatically involved areas were shown to proliferate at a significantly higher rate than those derived from uninvolved areas. The antiproliferative effect of amiprilose hydrochloride was not observed with normal keratinocytes or fibroblasts from the skin of healthy donors or from uninvolved areas of psoriatic donors. Amiprilose hydrochloride was not cytotoxic to any of these cells at levels below 0.1%. The combination of the low cytotoxicity and the selective antiproliferative effect indicates that this compound may be a useful antipsoriatic agent. The use of monolayer cultures and tissue equivalent models in this study illustrates the utility of such a progressive strategy in the evaluation of potential topical pharmaceuticals.

Cell Division↗
PubMed1990

The accumulation of oxidized isoforms of chicken triosephosphate isomerase during aging and development.

Triosephosphate isomerase (TPI) from mammals undergoes two specific deamidations (Asn-15 and Asn-71) which destabilize the isologous dimer and lead to the degradation of the protein. In aging cells and tissues, the deamidated isoforms accumulate apparently due to age-related changes in protein turnover. Chicken TPI lacks one of these sites (i.e., Asn 71----Lys), but also exhibits unstable isoforms. These isoforms are the result of the specific oxidations which occur both in vitro and in vivo. Electrophoretic analyses of various tissues from chicken show that the most oxidized isoform, which is present in adult tissues, is only present in small quantities in tissues of the newborn chick. Moreover, embryonic tissues contain almost exclusively the fully reduced form of TPI. Thus, it appears that oxidation rather than deamidation constitutes the first step in the degradation of avian TPI. TPI may be the first example of a protein which has evolved two different types of modifications (deamidation and oxidation) which trigger its degradation. The accumulation of both deamidated and oxidized isoforms in different species may provide clues to the underlying basis for the accumulation of modified proteins in aging.

Aging↗
PubMed1990

Rapid isolation of triosephosphate isomerase utilizing high-performance liquid chromatography.

A new method for the isolation of homogeneous triosephosphate isomerase (TPI, EC 5.3.1.1) has been developed. The method utilizes high-performance liquid chromatography on DEAE 5PW and Hydrophase-polyethyleneimine columns, which results in the rapid isolation and essentially quantitative recovery of the enzyme. The procedure is superior to previous methods with respect to specificity, recovery, and time. In addition, this rapid process minimizes the potential for postsynthetic modifications of the protein. Milligram quantities of TPI can be isolated from 100 g of tissue.

Animals↗
PubMed1990

Effects of aging and xerosis on the amino acid composition of human skin.

Amino acid compositions of skin samples from young and old subjects and from age-matched donors with dry skin syndrome (xerosis) were examined. The amino acid contents of the free amino acid (FAA) fraction, soluble hydrolysate (SH) fraction, and whole cell hydrolysate (WCH) were determined. The greatest differences were observed between the FAA compositions of the young and old normal subjects. Xerosis did not appear to affect the amino acid compositions of samples from young subjects as much as old subjects. Overall, the effect of aging on the amino acid contents was more pronounced than the effect of xerosis. The amino acid composition of the FAA showed a high degree of similarity to filaggrin, whereas the WCH showed a similarity to keratin.

Aging↗
PubMed1989

Simultaneous assay for plasmin and DNase using radiolabeled human fibroblasts on microcarriers.

A critical step in tissue and wound repair is the removal of eschar--accumulation of denatured cellular and extracellular macromolecules. Enzymatic debridement using a combination of plasmin (fibrinolysin) and DNase has been successfully utilized on a variety of types of wounds. Monitoring the activity of these enzymes by measuring the rate of fibrinolysis, or by viscometric changes due to DNA hydrolysis, is exceedingly cumbersome, time consuming, and, at best, only semiquantitative. Although spectrophotometric assays using synthetic substrates offer several advantages, they do not allow extrapolation of the data to the more complex natural substrates encountered in vivo. We have, therefore, developed an in vitro radioisotopic assay for the simultaneous and quantitative measurement of the hydrolytic activity of both plasmin and DNase. Double labeled ([3H]thymidine, [14C]leucine) human dermal fibroblasts grown on microcarrier beads are utilized as sources of nucleic acid and protein substrates. The assay meets all the criteria of analytical validity, is sensitive and rapid, and is amenable to adaptation for analysis of other hydrolytic enzymes. The method offers a direct evaluation of the enzymatic debridement of wounds using actual human cellular substrates. Moreover, the microcarriers provide a greatly increased surface area for cell attachment and growth, are amenable to rapid separation from the cells by simple mechanical methods, and are ideally suited to analytical manipulations.

Carbon Radioisotopes↗