r-Glutamyltranspeptidase (r-GT) from a rat hepatoma induced by 3'-methyl-4-dimethylaminoazobenzene (3'-Me DAB) was purified 833 fold. The purified enzyme had a specific activity of 15.0 U/mg protein with an overall yield of 3.8%. The molecular weight of native r-GT was estimated as about 350,000 daltons, whichs a multicomplex of a single polypetide having a M W of 59,000. Anti r-GT rabbit antiserum cross-reacted with kidney r-GT as well as liver r-GT. Tryptic digestion of r-GT followed by separation with Con A sepharose column chromatography resulted in two major glycopeptides. A tumor associated antigen was prepared by the conjugation of a tryptic glycopeptide of r-GT to keyhole limpets hemocyanin and an antibody against this antigen cross-reacted preferentially with r-GT in rat hepatoma tissue.
Animal ; Antigens, Neoplasm/isolation and purification ; Liver Neoplasms, Experimental/*enzymology/immunology ; Male ; Molecular Weight ; Rats ; Support, Non-U.S. Gov't ; gamma-Glutamyltransferase/immunology/*isolation and purification

The relevance of transglutaminases with neural function and several disorders has been emphasized recently. Especially, many polypeptides associated with neurodegenerative diseases are suggested to be putative transglutaminase substrates such as beta amyloid protein of Alzheimer's disease, microtubule-associated proteins and neurofilaments, etc. In addition, the CAG repeated gene products with probable polyglutamine tract, putative transglutaminase substrates, were identified in several neurodegenerative disorders. However, the identity of the brain transglutaminase has not been confirmed, because of enzymic stability and low activity. In the present experiment, we have isolated brain-specific transglutaminases, designated as TGase NI and TGase NII, which are different from other types of transglutaminases in respects of molecular weights (mw. 45 kDa, 29 kDa respectively), substrate affinity, elution profile on ion-exchange chromatography, sensitivity to proteases and ethanol, and immunological properties. The enzymes were localized specifically in the brain tissues but not in the liver tissue. And neural cells such as pheochromocytoma cell, glioma cell, primary neuronal and glial cells were shown to be enriched with TGase NI and TGase NII. The possible biological roles of the enzymes were discussed not only on the aspect of crosslinking activity but also of signal transducing capacity of the enzyme in the brain.
Animal ; Astrocytes/enzymology ; Blotting, Western ; Brain/enzymology* ; Calcium/metabolism ; Chromatography, Ion Exchange ; Endopeptidases/pharmacology ; Enzyme Stability ; Ethanol/pharmacology ; Glioma ; Immunoblotting ; Immunohistochemistry ; Male ; Molecular Weight ; Neurons/enzymology ; PC12 Cells ; Protein-Glutamine gamma-Glutamyltransferase/isolation & purification* ; Protein-Glutamine gamma-Glutamyltransferase/immunology ; Protein-Glutamine gamma-Glutamyltransferase/chemistry* ; Rats ; Rats, Sprague-Dawley ; Trypsin/pharmacology ; Tumor Cells, Cultured

We previously reported an identification of a 77-kDa GTP-binding protein that co-purified with the alpha 1-adrenoceptor following ternary complex formation. In the present paper, we report on the purification and characterization of this GTP-binding protein (termed G alpha h5) isolated from pig heart membranes. After solubilization of pig heart membranes with NaCl, G alpha h5 was purified by sequential chromatographies using DEAE-Cellulose, Q-Sepharose, and GTP-agarose columns. The protein displayed high-affinity GTP gamma S binding which is Mg(2+)-dependent and saturable. The relative order of affinity of nucleotide binding by G alpha h5 was GTP > GDP > ITP >> ATP > or = adenyl-5'-yl imidodiphosphate, which was similar to that observed for other heterotrimeric G-proteins involved in receptor signaling. Moreover, the G alpha h5 demonstrated transglutaminase (TGase) activity that was blocked either by EGTA or GTP gamma S. In support of these observations, the G alpha h5 was recognized by a specific antibody to G alpha h7 or TGase II, indicating a homology with G alpha h (TGase II) family. These results demonstrate that 77-kDa G alpha h5 from pig heart is an alpha 1-adrenoceptor-coupled G alpha h (TGase II) family which has species-specificity in molecular mass.
Animal ; Binding Sites ; Binding, Competitive ; Cross Reactions ; GTP-Binding Proteins/metabolism* ; GTP-Binding Proteins/isolation & purification* ; GTP-Binding Proteins/immunology ; Guanosine 5'-O-(3-Thiotriphosphate)/metabolism ; Molecular Weight ; Myocardium/chemistry* ; Protein-Glutamine gamma-Glutamyltransferase/metabolism ; Receptors, Adrenergic, alpha-1/metabolism ; Swine