GTP hydrolysis by guinea pig liver transglutaminase

Homogeneous guinea pig liver transglutaminase was purified from a commercially available enzyme preparation by affinity chromatography on GTP-agarose. The purified transglutaminase exhibited a single band of apparent Mr = 80,000 on sodium dodecyl sulfate polyacrylamide gel and Western blotting and had enzyme activity of both transglutaminase and GTPase. The guinea pig liver transglutaminase has an apparent Km value of 4.4 microM for GTPase activity. GTPase activity was inhibited by guanine nucleotides in order GTP-gamma-S greater than GDP, but not by GMP. These results demonstrate that purified guinea pig liver transglutaminase catalyzes GTP hydrolysis.     

Guinea pig liver transglutaminase: A modified purification procedure affording enzyme with superior activity in greater yield.

Tissue transglutaminase purified from guinea pig livers has a very broad substrate specificity in comparison with other members of the transglutaminase family and therefore is useful for substrate analogue kinetic studies. Modifications made in our laboratory to the standard purification protocol (J. E. Folk and S. I. Chung, 1985, Methods Enzymol. 113, 358-364) have yielded a 28% increase in specific activity and 55% increase in overall yield, while reducing the number of steps to the purification. Herein we report some of the highest yields and specific activities for guinea pig liver transglutaminase found in the literature, as well as the use of lyophilization as a solution to the long-standing problem of enzyme stability during storage.     

Identification of a guanosine triphosphate-binding site on guinea pig liver transglutaminase. Role of GTP and calcium ions in modulating activity

Guanosine 5'-triphosphate (GTP) was found to inhibit guinea pig liver transglutaminase activity as measured by [3H]putrescine incorporation into casein. GDP and GTP-gamma-S also inhibited enzyme activity (GTP-gamma-S greater than GTP greater than GDP). Kinetic studies showed that GTP acted as a reversible, noncompetitive inhibitor and that CaCl2 partially reversed GTP inhibition. GTP also inhibited rat liver and adult bovine aortic endothelial cell transglutaminase, but did not inhibit Factor XIIIa activity. Guanosine monophosphate (GMP), cyclic GMP, and polyguanylic acid did not inhibit enzyme activity. Guinea pig liver transglutaminase adsorbed well to GTP-agarose affinity columns, but not to CTP-agarose columns, and the binding was inhibited by the presence of calcium ions. Specific binding of GTP to transglutaminase was demonstrated by photoaffinity labeling with 8-azidoguanosine 5'-[gamma-32P] triphosphate, which was inhibited by the presence of GTP or CaCl2. GTP inhibited trypsin proteolysis of guinea pig liver transglutaminase without affecting the trypsin proteolysis of chromogenic substrates. Proteolytic protection was reversed by the addition of calcium. This study demonstrates that GTP binds to transglutaminase and that both GTP and calcium ions function in concert to regulate transglutaminase structure and function.     

Studies on the effect of experimental inflammation on sialyltransferase in the mouse and guinea pig

The effect of inflammation on sialyltransferase was studied in the mouse and guinea pig. There was a three-fold increase in mouse liver sialyltransferase activity reaching a maximum at 72 hr after inflammation; serum levels were increased by five-fold at 72 hr after inflammation. The response of guinea pig sialyltransferase was slower and of lower magnitude compared with the response of the mouse enzyme; liver and serum sialyltransferase increased by about 50% reaching a maximum at 96 hr after inflammation. The specificity of the enzyme that responded to inflammation in the mouse and guinea pig was found to be Gal beta 1----4GlcNAc alpha 2----6 sialyltransferase, the same enzyme activity that was shown to be an acute phase reactant in earlier studies in the rat (Kaplan et al., 1983).     

Rabbit liver transglutaminase: physical, chemical, and catalytic properties.

Transglutaminase (R-glutaminyl-peptide:amine alpha-glutamyl-yltransferase [EC 2.3.2.13]) has been purified to apparent homogeneity from extracts of rabbit liver. The enzyme is a single polypeptide chain of approximately 80 000 molecular weight containing one catalytic site per molecule. That the isolated enzyme is the rabbit counterpart of the well-characterized guinea pig liver transglutaminase is evidenced by the similarities in their amino acid compositions and in their enzymic activities toward several substrates, together with the fact that the isolated rabbit enzyme is immunologically distinct from both rabbit plasma and rabbit platelet blood coagulation factor XIII. A striking difference between the catalytic activities of the rabbit and guinea pig enzymes is the low activity of rabbit transglutaminase for hydroxylamine incorporation into benzyloxycarbonyl-L-glutaminylglycine, a reaction for which the guinea pig enzyme shows a high reactivity. This finding reveals the cause of error in an earlier report (Tyler, H.M., and Laki, K. (1967) Biochemistry 6, 3259) that rabbit liver contains little, if any, of the enzyme. Preparation of, and analytical data on, several glutamine-containing peptide derivatives used in this study are reported here.     

Lipids associated with tissue transglutaminase

A substantial amount of lipids (cholesterol and its esters, mono-, di- and triacylglycerols, free fatty acids and the phospholipids phosphatidylethanolamine and phosphatidylinositol) was found associated with tissue transglutaminase purified to apparent homogeneity from guinea pig liver. Removal of lipids results in an increased tendency of the enzyme for self-association and a decreased stability. Lauric acid was detected following hydroxylamine treatment of the enzyme, suggesting the occurrence of a fatty acid-type, covalent, posttranslational modification of transglutaminase. The results provide support for the idea that part of tissue transglutaminase may be localized in the cell membrane.     

Cellular transglutaminase. Lung matrix-associated transglutaminase: characterization and activation with sulfhydryls

Transglutaminase in the rat lung is tightly associated with the insoluble matrix which is not extractable with detergent, 0.5 M NaCl, and 40% glycerol solutions. The insoluble matrix was found to be rich in heparin sulfate and poor in collagen, elastin, and DNA. The lung transglutaminase was found to be distinct from tissue transglutaminase (identifiable with the well-characterized guinea pig liver transglutaminase) in its retention volume in DEAE-Sephacel columns and its Kd value in gel-filtration columns. The enzyme was activated 6-8-fold with the sulfhydryl reagent dithiothreitol. This activation was accompanied with the dissociation of enzyme from the tightly bound insoluble matrix and resulted in changes of the molecular properties of the enzyme--increase in affinity for anion-exchanger and decrease in Stokes radius. Addition of 50 mM KSCN induced a 2-fold increase in SH-dependent activation of transglutaminase activity. These results suggest that sulfhydryl agents may play a role in the activation and compartmental translocation of the transglutaminase in the lung.     

Structural features of glutamine substrates for transglutaminases. Role of extended interactions in the specificity of human plasma factor XIIIa and of the guinea pig liver enzyme

Amino acid residues at several locations in close primary vicinity to a substrate glutamine residue have been recognized as important determinants for the specificities of human plasma factor XIIIa and guinea pig liver transglutaminase (Gorman, J. J., and Folk, J. E. (1981) J. Biol. Chem. 256, 2712-2715). The present studies measure the influence on transglutaminase specificity of some changes in amino acid side chains in a small synthetic glutamine peptide amide, Leu-Gly-Leu-Gly-Gln-Gly-Lys-Val-Leu-GlyNH2, which was designed to contain most of the known elements needed for enzyme recognition. The results are in agreement with previous findings and show that full catalytic activity of each enzyme may be retained upon replacement of the lysine residue by certain other amino acid residues. Evidence is provided that serine in place of glycine at one or more positions causes a significant increase in specificity with factor XIIIa, but not with liver enzyme. The effective substrate property for factor XIIIa seen with the model peptide amide is lost upon reversal of the sequence Val-Leu. This is not the case with the liver enzyme even though replacement of either of these amino acids by alanine causes a pronounced loss in activity with this enzyme. These differences and the effects of various other substitutions in the model peptide amide on the enzymes' specificities points up the relatively stringent structural requirements of factor XIIIa and the rather broad requirements for liver transglutaminase.     

Posttranslational modification of ornithine decarboxylase by its product putrescine

Putrescine, spermidine, and spermine, as well as other primary amine substances, when added exogenously to growth-stimulated systems, inhibit ornithine decarboxylase (ODC) activity in a dose- and time-dependent manner. Evidence is presented to support a direct posttranslational modification of ODC by transglutaminase-mediated putrescine incorporation. Purified ODC serves as an acceptor protein for putrescine in the presence of transglutaminase purified from guinea pig liver. The transamidation of putrescine to ODC results in a linear decrease in activity. The Km for putrescine is 0.4 mM and the Ki for putrescine inhibition of ODC activity by transglutaminase is 0.4 mM. The kinetics are identical to those reported for physiological systems. In regenerating rat liver, protein conjugated putrescine parallels increased transglutaminase activity and the rapid disappearance of ODC activity at 8 h. These data strongly suggest that posttranslational modification of ODC by putrescine may be an important regulatory step in the trophic cascade.     

Variations in induction of drug-metabolizing enzymes by trans-Stilbene oxide in rodent species

trans-Stilbene oxide (400 mg/kg) produced a 500% increase in the microsomal in the microsomal epoxide hydratase activity in rat and mouse with little change in the soluble enzyme activity. However, in guinea pig, the soluble epoxide hydratase activity increased by about 33% with only a small increase (47.6%) in the microsomal enzyme activity. The soluble glutathione S-transferase activities were also induced in both rat and mouse, with little change in that of the guinea pig. Increasing dosage of trans-stilbene oxide from 400 mg/kg to 1000 mg/kg had little effect on the above enzyme activities. That the guinea pig was not relatively refractory to all inducing agents was shown by the fact phenobarbital (100 mg/kg) and 3-methylcholanthrene (25 mg/kg) produced relatively similar increases in the activities of aniline hydroxylase and P-aminopyrineP-demethylase in rat, mouse and guinea pig. However, these inducers produced only a 15–20% stimulation in the soluble glutathione S-transferase and microsomal epoxide hydratase activities in guinea pig, when compared to a 50–80% increase in rat and mouse, suggesting a general resistance to induction by the phase II enzymes in guinea liver. In all animal models, the inducer markedly increased th emicrosomal total phospholipid content, although the sphingomyelin content itself was decreased. In both rat and mouse, the microsomal cholesterol content was significantly decreased while that in guinea pig was unaffected. Possible factors responsible for the observed species differences are discussed.     

Hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase activity and cholesterol 7 alpha-hydroxylase activity in neonatal guinea pig.

Optimal assay conditions for hepatic HMG-CoA reducatase activity and cholesterol 7 alpha-hydroxylase activity in the guinea pig were determined. These two enzyme activities were studied in the liver of newborn guinea pigs during the first three postnatal weeks. Hepatic HMG-CoA reductase activity of neonatal guinea pigs was similar to that of adult animals. However, cholesterol 7 alpha-hydroxylase activity of newborns was about one-third of that in adult guinea pigs. This finding suggests that the system for bile acid synthesis in the neonatal guinea pigs is underdeveloped.     

A sensitive fluorometric assay for tissue transglutaminase

We have devised a highly sensitive fluorometric well plate assay for tissue transglutaminase that is suitable for multiple kinetic analyses/high-throughput screening of chemical inventories for inhibitors of this enzyme. The procedure measures the rate of fluorescence enhancement (lambda(exc) 260 nm, lambda(em) 538 nm) when 1-N-(carbobenzoxy-l-glutaminylglycyl)-5-N-(5'N'N'-dimethylaminonaphthalenesulfonyl)diamidopentane (glutaminyl substrate) is cross-linked to dansyl cadaverine (amine substrate). The assay procedure can be used to measure the activity of as little as 60 microU of purified guinea pig liver tissue transglutaminase (4.2 ng or 54 fmol of enzyme).     

Ontogeny of microsomal activities of triacylglycerol synthesis in guinea pig liver

Because the onset of triacylglycerol-rich lipoprotein synthesis occurs in guinea pig liver during fetal life, we investigated the microsomal enzyme activities of triacylglycerol synthesis in fetal and postnatal guinea pig liver. Hepatic monoacylglycerol acyltransferase specific and total microsomal activities peaked by the 50th day of gestation and declined rapidly after birth to levels that were virtually unmeasurable in the adult. Peak fetal specific activity was more than 75-fold higher than observed in the adult. The specific activities of fatty acid CoA ligase and lysophosphatidic acid acyltransferase increased 2- to 3-fold before birth; lysophosphatidic acid acyltransferase increased a further 2.6-fold during the first week of life. Specific activities of phosphatidic acid phosphatase, microsomal glycerophosphate acyltransferase, and diacylglycerol acyltransferase varied minimally over the time course investigated. These data demonstrate that selective changes occur in guinea pig hepatic microsomal activities of triacylglycerol synthesis before birth. Because of an approximate 11-fold increase in hepatic microsomal protein between birth and the adult, however, major increases in total microsomal activity of all the triacylglycerol synthetic activities occurred after birth. The pattern of monoacylglycerol acyltransferase specific and total microsomal activities differs from that of the rat in occurring primarily during the last third of gestation instead of during the suckling period. This pattern provides evidence that hepatic monoacylglycerol acyltransferase activity probably does not function to acylate 2-monoacylglycerols derived from partial hydrolysis of diet-derived triacylglycerol.     

In vitro modification of betaine-homocysteine S-methyltransferase by tissue-type transglutaminase

Transglutaminases catalyze the cross-linking and amine incorporation of proteins, and are implicated in various biological phenomena. To elucidate the physiological roles of transglutaminase at the molecular level, we need to identify its physiological protein substrates and clarify the relationship between transglutaminase modification of protein substrates and biological responses. Here we examined whether betaine-homocysteine S-methyltransferase (BHMT: EC 2.1.1.5) can be a substrate of tissue-type transglutaminase by in vitro experiments using porcine liver BHMT and guinea pig liver transglutarninase. Guinea pig liver transglutaminase incorporated 5-(biotinamido) pentylamine and [3H] histamine into BHMT in a time-dependent manner. Putrescine and spermidine also seemed to be incorporated into BHMT by transglutaminase. In the absence of the primary amines, BHMT subunits were cross-linked intra- and intermolecularly. BHMT activity was decreased significantly through the cross-linking by transglutaminase. Histamine incorporation slightly reduced the BHMT activity. Peptide fragments of BHMT containing the glutamine residues reactive for transglutaminase reaction were isolated through biotin labelling, proteinase digestion, biotin-avidin a affinity separation, and reverse phase HPLC. The results of amino acid sequence analyses of these peptides and sequence homology alignment with other mammalian liver BHMT subunits showed that these reactive glutamine residues were located in the region near the carboxyl terminal of porcine BHMT subunit. These results suggested that the liver BHMT can be modified by tissue-type transglutaminase and its activity is regulated repressively by the modification, especially by the cross-linking. This regulatory reaction might be involved in the regulation of homocysteine metabolism in the liver.     

Colorimetric assay for cellular transglutaminase

A colorimetric assay for cellular transglutaminase using 5-(biotinamido)pentylamine and polyvinylidine difluoride membranes for crude cellular preparations and purified enzyme has been developed. The biotinpentylamine substrate was incorporated into N,N-dimethylcasein by transglutaminase, the biotinylated products were adsorbed onto the membrane disks and conjugated with streptavidin-beta-galactosidase, and the absorbance resulting from the formation of p-nitrophenol from hydrolysis of p-nitrophenyl-beta-D-galactopyranoside was measured at 405 nm. The validity of the assay was established by showing a good correlation, gamma = 0.922, between the colorimetric procedure and the commonly used radiometric filter paper method for the enzyme. The procedure offers a rapid, sensitive, and nonisotopic method for the estimation of cellular transglutaminase activity in as low as 20 ng of purified guinea pig liver transglutaminase and 10 micrograms of crude fibroblast cytosol protein.     

Amino acid sequence of guinea pig liver transglutaminase from its cDNA sequence

Transglutaminases (EC 2.3.2.13) catalyze the formation of epsilon-(gamma-glutamyl)lysine cross-links and the substitution of a variety of primary amines for the gamma-carboxamide groups of protein-bound glutaminyl residues. These enzymes are involved in many biological phenomena. In this paper, the complete amino acid sequence of guinea pig liver transglutaminase, a typical tissue-type nonzymogenic transglutaminase, was predicted by the cloning and sequence analysis of DNA complementary to its mRNA. The cDNA clones carrying the sequences for the 5'- and 3'-end regions of mRNA were obtained by use of the sequence of the partial-length cDNA of guinea pig liver transglutaminase [Ikura, K., Nasu, T., Yokota, H., Sasaki, R., & Chiba, H. (1987) Agric. Biol. Chem. 51, 957-961]. A total of 3695 bases were identified from sequence data of four overlapping cDNA clones. Northern blot analysis of guinea pig liver poly(A+) RNA showed a single species of mRNA with 3.7-3.8 kilobases, indicating that almost all of the mRNA sequence was analyzed. The composite cDNA sequence contained 68 bases of a 5'-untranslated region, 2073 bases of an open reading frame that encoded 691 amino acids, a stop codon (TAA), 1544 bases of a 3'-noncoding region, and a part of a poly(A) tail (7 bases). The molecular weight of guinea pig liver transglutaminase was calculated to be 76,620 from the amino acid sequence deduced, excluding the initiator Met. This enzyme contained no carbohydrate [Folk, J. E., & Chung, S. I. (1973) Adv. Enzymol. Relat. Areas Mol. Biol. 38, 109-191], but six potential Asn-linked glycosylation sites were found in the sequence deduced.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fibrin membrane endowed with biological function. IV. Formation of cross-links between fibrinogen (or fibrin) and ribonuclease by transglutaminase.

Transglutaminase from guinea pig liver catalyzed the formation of cross-links between fibrinogen (or fibrin) and ribonuclease. Using transglutaminase, immoblized ribonuclease was prepared by two separate methods: (1) fibrinogen-ribonuclease conjugates formed by transglutaminase were treated with thrombin to make fibrin membrane bound covalently to the enzyme; (2) fibrin polymer formed from fibrinogen with thrombin was covalently bound to ribonuclease by transglutaminase to make fibrin-ribonuclease conjugates.     

Co-overexpression of folding modulators improves the solubility of the recombinant guinea pig liver transglutaminase expressed in Escherichia coli

Transglutaminases (EC 2.3.2.13) catalyze the formation of epsilon-(gamma-glutamyl)lysine cross-links and the substitution of primary amines for the gamma-carboxamide groups of protein bound glutamine residues, and are involved in many biological phenomena. Transglutaminase reactions are also applicable in applied enzymology. Here, we established an expression system of recombinant mammalian tissue-type transglutaminase with high productivity. Overexpression of guinea pig liver transglutaminase in Escherichia coli, using a plasmid pET21-d, mostly resulted in the accumulation of insoluble and inactive enzyme protein. By the expression culture at lower temperatures (25 and 18 degrees C), however, a fraction of the soluble and active enzyme protein slightly increased. Co-overexpression of a molecular chaperone system (DnaK-DnaJ-GrpE) and/or a folding catalyst (trigger factor) improved the solubility of the recombinant enzyme produced in E. coli cells. The specific activity, the affinity to the amine substrate, and the sensitivity to the calcium activation and GTP inhibition of the purified soluble recombinant enzyme were lower than those of the natural liver enzyme. These results indicated that co-overexpression of folding modulators tested improved the solubility of the overproduced recombinant mammalian tissue-type transglutaminase, but the catalytic properties of the soluble recombinant enzyme were not exactly the same as those of the natural enzyme.     

Expression and rapid purification of highly active hexahistidine-tagged guinea pig liver transglutaminase

Tissue transglutaminase has been identified as a contributor to a wide variety of diseases, including cataract formation and Celiac disease. Guinea pig tissue transglutaminase has a very broad substrate specificity and therefore is useful for kinetic studies using substrate analogues. Here, we report the expression in Escherichia coli of a hexahistidine-tagged guinea pig liver tissue transglutaminase (His(6)-tTGase) allowing rapid purification by immobilized-metal affinity chromatography. Using this procedure we have obtained the highest reported specific activity (17 U/mg) combined with a high yield (22 mg/L of culture) for recombinant TGase using a single-step purification protocol. Using two independent spectrophotometric assays, we determined that the K(m) value of the recombinant enzyme with the substrate Cbz-Gln-Gly is in the same range as values reported in the literature for the native enzyme. We have thus developed a rapid and reproducible protocol for the preparation of high quality tissue TGase.