Biomedical subjects

M J Glynias

Publications and source records attributed to M J Glynias.

8 recordsLinked to original sources

PubMed1997

Distinct multisite synergistic interactions determine substrate specificities of human chymase and rat chymase-1 for angiotensin II formation and degradation.

Human chymase and rat chymase-1 are mast cell serine proteases involved in angiotensin II (Ang II) formation and degradation, respectively. Previous studies indicate that both these enzymes have similar P1 and P2 preferences, which are the major determinants of specificity. Surprisingly, despite the occurrence of optimal P2 and P1 residues at the Phe8 downward arrow and Tyr4 downward arrow bonds (where downward arrow, indicates the scissile bond in peptide substrates) in Ang I (DRVYIHPFHL), human chymase cleaves the Phe8 downward arrow bond with an approximately 750-fold higher catalytic efficiency (kcat/Km) than the Tyr4 downward arrow bond in Ang II (DRVYIHPF), whereas rat chymase-1 cleaves the Tyr4 downward arrow bond with an approximately 20-fold higher catalytic efficiency than the Phe8 downward arrow bond. Differences in the acyl groups IHPF and DRVY at the Phe8 downward arrow and Tyr4 downward arrow bonds, respectively, are chiefly responsible for the preference of human chymase for the Phe8 downward arrow bond. We show that the IHPF sequence forms an optimal acyl group, primarily through synergistic interactions between neighboring acyl group residues. In contrast to human chymase, rat chymase-1 shows a preference for the Tyr4 downward arrow bond, mainly because of a catalytically productive interaction between the enzyme and the P'1 Ile5. The overall effect of this P'1 Ile interaction on catalytic efficiency, however, is influenced by the structure of the acyl group and that of the other leaving group residues. For human chymase, the P'1 Ile interaction is not productive. Thus, specificity for Ang II formation versus Ang II degradation by these chymases is produced through synergistic interactions between acyl or leaving group residues as well as between the acyl and leaving groups. These observations indicate that nonadditive interactions between the extended substrate binding site of human chymase or rat chymase-1 and the substrate are best explained if the entire binding site is taken as an entity rather than as a collection of distinct subsites.

Amino Acid Sequence↗

PubMed1996

Angiotensin II-forming activity in a reconstructed ancestral chymase.

The current model of serine protease diversity theorizes that the earliest protease molecules were simple digestive enzymes that gained complex regulatory functions and restricted substrate specificities through evolution. Among the chymase group of serine proteases are enzymes that convert angiotensin I to angiotensin II, as well as others that simply degrade angiotensins. An ancestral chymase reconstructed with the use of phylogenetic inference, total gene synthesis, and protein expression had efficient and specific angiotensin II-forming activity (turnover number, about 700 per second). Thus, angiotensin II-forming activity is the more primitive state for chymases, and the loss of such activity occurred later in the evolution of some of these serine proteases.

Amino Acid Sequence↗

PubMed1995

Evolutionary conservation of both the hydrophilic and hydrophobic nature of transmembrane residues.

An algorithm (HRG), developed to allow the pairwise comparisons of the aligned residues of several members of large gene families of polytopic integral membrane proteins is described. Using hydrophobicity scales, application of this algorithm allows the number and size of the membrane-spanning domains of bacteriorhodopsin, a polytopic protein whose structure has been partially determined, to be predicted with a high degree of accuracy (sensitivity 94%, specificity 82% for predicting the membrane embedded or extramembranous location of residues). As opposed to previously reported structure-prediction algorithms, delineation of putative transmembrane segments from connecting loops is also more clearly evident with the application of the HRG algorithm, even with proteins from widely divergent species. This indicates strong evolutionary pressure for the conservation of both the hydrophobic and hydrophilic character of residues in membrane-embedded regions of polytopic proteins, such as those of the G-protein-coupled receptor superfamily. These and other structural and functional implications evident from the application of the HRG algorithm are considered.

Algorithms↗

PubMed1992

Duck liver 'malic' enzyme. Expression in Escherichia coli and characterization of the wild-type enzyme and site-directed mutants.

A cDNA for duck liver 'malic' enzyme (EC 1.1.1.40) was subcloned into pUC-8, and the active enzyme was expressed in Escherichia coli TG-2 cells as a fusion protein including a 15-residue N-terminal leader from beta-galactosidase coded by the lacZ' gene. C99S and R70Q mutants of the enzyme were generated by the M13 mismatch technique. The recombinant enzymes were purified to near homogeneity by a simple two-step procedure and characterized relative to the enzyme isolated from duck liver. The natural duck enzyme has a subunit molecular mass of approx. 65 kDa, and the following kinetic parameters for oxidative decarboxylation of L-malate at pH 7.0: Km NADP+ (4.6 microM); Km L-malate (73 microM); kcat (160 s-1); Ka (2.4 microM) and Ka' (270 microM), dissociation constants of Mn2+ at 'tight' (activating) and 'weak' metal sites; and substrate inhibition (51% of kcat. at 8 mM-L-malate). Properties of the E. coli-derived recombinant wild-type enzyme are indistinguishable from those of the natural duck enzyme. Kinetic parameters of the R70Q mutant are relatively unaltered, indicating that Arg-70 is not required for the reaction. The C99S mutant has unchanged Km for NADP+ and parameters for the 'weak' sites (i.e. inhibition by L-malate, Ka'); however, kcat. decreased 3-fold and Km for L-malate and Ka each increased 4-fold, resulting in a catalytic efficiency [kcat./(Km NADP+ x Km L-malate x Ka)] equal to 3.7% of the natural duck enzyme. These results suggest that the positioning of Cys-99 in the sequence is important for proper binding of L-malate and bivalent metal ions.

Amino Acid Sequence↗

PubMed1987

GTP-binding domain: three consensus sequence elements with distinct spacing.

A sequence comparison of nine functionally different GTP-binding protein families has yielded further information on the general characterization of the conservation and importance of amino acid sequences in the GTP-binding domain, including a consensus sequence composed of three consensus elements GXXXXGK, DXXG, and NKXD with consensus spacings of either 40-80 or approximately equal to 130-170 amino acid residues between the first and second elements and approximately 40-80 amino acid residues between the second and third sequence elements; the sequence NKXW in place of NKXD in the sequence element responsible for base specificity allows the use of ITP as well as GTP; dGTP can be used with essentially the same efficiency as GTP; signal transducing proteins and enzymes have been identified in the nine families; and family conservations allow the identification of the most probable consensus sequence element when more than one is present. Employing these features we have screened the protein sequence data base of the Protein Identification Resource and have identified only known GTP-binding proteins with the exception of protein 2C from foot-and-mouth disease virus as matching the consensus sequence. Based on this finding we predict that foot-and-mouth disease virus protein 2C binds GTP and, by analogy, that protein 2C from several related viruses (polio, rhino, encephalomyocarditis, and cowpea mosaic) will bind a nucleotide as part of its biologic activity.

Amino Acid Sequence↗

PubMed1984

A cloned cDNA for duck malic enzyme detects abnormally large malic enzyme mRNAs in a strain of mice (Mod-1n) that does not express malic enzyme protein.

Sensitive immunochemical assays were used to measure the mass and rate of synthesis of malic enzyme protein in wild-type and Mod-1n mutant mice fed a high carbohydrate/low fat diet supplemented with thyroid hormone. Malic enzyme activity in the fed, wild-type mice was 100-fold higher than in starved, wild-type mice. Neither activity, mass, nor synthesis of malic enzyme could be detected in fed, mutant mice. However, glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase responded to these dietary manipulations with normal or supranormal increases in activities, respectively, in mutant mice. A cDNA clone containing an almost complete copy of the mRNA for malic enzyme from duck liver was used to analyze poly(A+) RNA from C57BL/6J-DBA/2J hybrid mice that had been fasted and refed a high carbohydrate/low fat diet supplemented with thyroid hormone. The 32P-cDNA probe hybridized to two RNAs of 2250 and 2950 nucleotides. The same two RNAs were detected in RNA from starved mice except at much lower concentrations. A similar analysis of RNA from Mod-1n mice fed the high carbohydrate-thyroid diet also revealed two hybridizing RNAs but each was 700-800 nucleotides longer than its counterpart in wild-type mice. The abundance of malic enzyme mRNA in the fed, mutant mice was about the same as that in fed, wild-type mice. The mutant malic enzyme mRNAs also were present in RNA from starved mice but at much lower concentrations. These results suggest that the mutation responsible for the Mod-1n phenotype is in the structural gene for malic enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1984

Regulation of the activity and synthesis of malic enzyme in 3T3-L1 cells.

Malic enzyme activity in differentiated 3T3-L1 cells was about 20-fold greater than activity in undifferentiated cells. A new steady-state level was achieved about 8 days after initiating differentiation of confluent cultures with a 2-day exposure to dexamethasone, isobutylmethylxanthine, and insulin. This increase in enzyme activity resulted from an increase in the mass of malic enzyme as detected by immunotitration of enzyme activity with goat antiserum directed against purified rat liver malic enzyme. Malic enzyme synthesis was undetectable in undifferentiated cells and increased to about 0.2% of soluble protein in differentiated cells, suggesting that the increase in enzyme mass was due primarily to an increase in enzyme synthesis. Thyroid hormone, a potent stimulator of malic enzyme activity in hepatocytes in culture and in liver and adipose tissue in intact animals, decreased or increased malic enzyme activity in differentiating 3T3-L1 cells by about 40% when it was removed or added to the medium, respectively. Insulin, another physiologically important regulator of malic enzyme activity in vivo, had no effect on the initial rate of accumulation of malic enzyme activity in the differentiating cells and caused a 30 to 40% decrease in the final level of enzyme activity in the fully differentiated cells. Cyclic AMP, a potent inhibitor of malic enzyme synthesis in hepatocytes in culture, inhibited this process in 3T3-L1 cells by 30%. Malic enzyme is like several other enzymes in that the large increase in its concentration which accompanies differentiation of 3T3-L1 cells is due to increased synthesis of enzyme protein. However, the hormonal modulation of malic enzyme characteristic of liver and adipose tissue in intact animals does not appear to occur in differentiated 3T3-L1 cells, suggesting that differentiated 3T3-L1 cells may not be an appropriate model system in which to study the hormonal modulation of malic enzyme that occurs in liver and adipose tissue of intact animals.

1-Methyl-3-isobutylxanthine↗

PubMed1983

Molecular cloning of cDNA sequences for avian malic enzyme. Nutritional and hormonal regulation of malic enzyme mRNA levels in avian liver cells in vivo and in culture.

A double-stranded cDNA library constructed from the total poly(A+) RNA of goose uropygial gland was screened for recombinants containing sequences complementary to malic enzyme mRNA. Replicate arrays of 1400 colonies were hybridized independently with 32P-labeled cDNAs copied from two populations of hepatic RNA derived from tissues which differed by about 35-fold with respect to the relative synthesis of malic enzyme. Forty-eight of the colonies which gave differential signals were further screened by hybrid-selected translation. DNA from one of these contained an insert of 970 base pairs and selected an mRNA which directed the synthesis of malic enzyme in a cell-free system. The malic enzyme sequences were subcloned into the single-stranded bacteriophage M13mp8. The subclones were used to prepare 32P-labeled single-stranded hybridization probe. Northern analysis indicated that malic enzyme mRNA from both goose and chicken is about 2100 bases in length. Hepatic malic enzyme mRNA concentration is stimulated 30- to 50-fold or more when neonatal chicks or goslings, respectively, are fed for 24 h. When added to chick embryo hepatocytes in culture, triiodothyronine stimulated malic enzyme mRNA accumulation by more than 100-fold. Glucagon inhibited the thyroid hormone-stimulated accumulation of malic enzyme mRNA by 99%. In all instances, malic enzyme mRNA concentration was closely correlated with the relative rate of malic enzyme synthesis. These results suggest that nutritional and hormonal regulation of malic enzyme synthesis occurs at the pretranslational level.

Animals↗