The molecular forms of gamma-glutamyl transferase in bile and serum of icteric rats

Calmodulin coupled to Sepharose has provided a rapid and sensitive means of isolating a cyclic nucleotide phosphodiesterase activity which is stimulated by the calmodulin-Ca2+ complex, from rat parotid gland. Initial experiments established that phosphodiesterase activity sensitive to calmodulin and Ca2+ could not be demonstrated in crude extracts of rat parotid gland or after partial purification of rat parotid phosphodiesterase over DEAE-cellulose. However, it was possible to readily demonstrate the presence of a cyclic nucleotide phosphodiesterase activity regulated by calmodulin if the extracts were first purified by batch ion-exchange chromatography over DEAE-cellulose followed by affinity chromatography with calmodulin coupled to Sepharose. The batch ion-exchange chromatography step removed the major portion of free parotid calmodulin which could compete with calmodulin-coupled Sepharose for the proteins regulated by calmodulin. Thus, by employing an initial chromatography step over DEAE-cellulose to separate phosphodiesterase activity from calmodulin, it was possible to increase the recovery of calmodulin-sensitive phosphodiesterase after affinity chromatrography with calmodulin coupled to Sepharose. This approach should be useful for demonstrating the presence of and for purifying other parotid proteins regulated by calmodulin.     

Effect of intestinal gamma-glutamyl transferase inhibitor on the amount of gamma-glutamyl metabolites in mouse.

Experimental mice fed a balanced rodent chow, called LSM fodder, had markedly lower gamma-glutamyl transferase activity in the epithelium of intestinal villi then control mice fed wheat. After oral administration of gamma-14C-glutamyglycine, oxidized 14C-glutathione or gamma-glutamyl-p-amino-benzoate the amounts of gamma-glutamyl substrates and their metabolites in intestines, livers and kidneys of experimental mice were significantly lower than those in control mice. L-serine simultaneously administered with gamma-14C-glutamylglycine reduced the radioactivity of gamma-glutamyl substances in organs of the control mice. No differences in organ radioactivity of experimental and control mice were observed when some uniformly labeled with 14C amino acids were given. The obtained results are not in aggreement with hypothesis on a role of gamma-glutamyl transferase in amino acid transport.     

Hematologic changes following chronic alcoholism

Changes in all three blood cell systems could be covered in 50 patients treated in hospital for a high consumption of alcohol for years and even decades. There is no symptom which would be pathognomonic for alcoholism in itself. Macrocytosis and macrovoluminity of erythrocytes, hyper-sideraemia and thrombocytopenia were findings frequently encountered and easily to be identified. Megaloblasts, vacuolization, and an increase of sideroblasts could be observed in the bone-marrow. The prompt reversibility of these changes mentioned by simply abstaining from alcohol has a considerable diagnostic utility. The impact of liver damage partly produced by an accompanying spleen enlargement could only be ensured for thrombocyte depression. The increase of methaemoglobin which is unequivocal but without any clinical importance can also be reversed by alcohol deprivation. From a haematological point of view an alcoholic is endangered by a deficient immunological system. Haemorrhagic diatheses due to thrombocytopenia, thromboembolic complications during rebound-thrombocytosis and severe haemolyses can rarely be found.     

A sensitive fluorimetric assay for gamma-glutamyl transferase.

A rapid, sensitive single-step assay for γ-glutamyl transferase is presented using l-γ-glutamyl-7-amino-4-methyl-coumarin as the donor substrate. Kinetic parameters and the relative activities with different acceptors were determined using this substrate. The results are compared with those obtained with an alternative substrate, l-γ-glutamyl-2-naphthyl-amide.     

Developmental studies on gamma-glutamyl transferase in rat intestinal mucosa.

The activity of gamma-GT in rat intestinal mucosa has been studied during normal development. No significant differences in enzymatic activity have been recorded between 21-day-old fetuses, neonates tested at 3, 10, 20 and 30 days and older animals (65 days). Only in the period immediately prior to birth did the liver gamma-GT display activity levels similar to those of intestinal gamma-GT. In neonates and in adult rats, the intestinal gamma-GT activity was much higher as compared to the enzymatic activity in the liver, possibly revealing a species feature in rats. The results of the studies show that the rat is particularly suitable for experimental studies on intestinal gamma-GT in pathological conditions.     

Histochemical quantitation of gamma-glutamyl transferase in human leukemia cell lines.

The enzyme gamma-glutamyl transferase (gamma-GT) is involved in many biochemical systems, including the signal transduction of hematopoietic growth factors. Standard colorimetric gamma-GT assays require larger cell numbers than may be obtainable in many cases, such as with highly purified stem-cell populations. To study gamma-GT expression in limited populations, we used a histochemical stain to analyze gamma-GT semiquantitatively in cells of hematopoietic origin. Several human leukemic cell lines, including one with inducible increases in gamma-GT, were stained for gamma-GT and graded 0 through 4+ for the amount of positive granules. The gamma-GT activity demonstrated by this stain was found to be directly proportional to the gamma-GT activity obtained with a colorimetric assay and could be used to calculate approximate gamma-GT activity. This stain therefore provides a useful method for determining gamma-GT activity when limited cell numbers are available.     

The turnover of hepatoma cell gamma-glutamyl transferase

Glucagon and insulin both stimulated the ³²P-labelling of ribosomal protein S6 in rat hepatocytes that had been incubated with ³²P_i. Glucagon selectively enhanced the labelling of the tryptic peptide phosphorylated by cyclic AMP-dependent protein kinase, demonstrating that 6 S is a physiological substrate for this enzyme. Insulin stimulated the phosphorylation of distinct tryptic peptides, at least one of which appears to be very close in the primary structure to the sites phosphorylated by cyclic AMP-dependent protein kinase.     

In situ determination of the molecular weight of hepatic gamma-glutamyl transferase and gamma-glutamyl hydrolase activities

The molecular weight of gamma-glutamyl transferase from normal rat liver and hepatoma tissue was determined by radiation-inactivation and found to be approx 100000 in each case. The molecular weight previously reported for the subunit containing the gamma-glutamyl binding site (22000) is considerably less than that of the holoenzyme, suggesting that in situ the large subunit is implicated in both transferase and hydrolase activities.     

Induction of gamma-glutamyl transferase by dexamethasone in cultured fetal rat hepatocytes

Hepatocytes isolated as a relatively pure population from normal fetal rats were maintained in primary monolayer culture for 4-10 days. Hepatocytes exhibited a small increase in basal gamma-glutamyl transferase (GGT) activity over time. Exposure to dexamethasone (10(-6) mol/l) elicited a rise in GGT activity after a lag of 24 h. The presence of the steroid was necessary to maintain induction, and its removal resulted in reversal of induction. The maximal response was 2- to 3-fold, 72 h after exposure to the steroid. After this maximal response, a gradual decay in enzyme activity occurred, despite the continuous presence of the hormone. Actinomycin D or cycloheximide given prior to/or simultaneously with the steroid prevented the induction, thus suggesting that both RNA and protein biosynthesis are necessary for induction to occur.     

Purification and properties of gamma-glutamyl transferase from normal rat liver

gamma-Glutamyl transferase ((5-glutamyl)-peptide: amino-acid 5-glutamyltransferase, ED 2.3.2.2) has been partially purified from both whole rat liver (600-fold) and from isolated biliary tract (1200-fold). The most highly purified fraction gave two protein bands on polyacrylamide gel electrophoresis, the major band alone having enzyme activity. The enzyme purified from biliary tract appears identical to that from whole liver preparation according to molecular weight, kinetic parameters and the effects of various inhibitors. Three liver cell-types; parenchymal, Kupffer and biliary tract were isolated by perfusion of the rat liver in situ with collagenase, followed by selective cell isolation. Approx. 80-90% of the total recovered enzyme activity was found in the biliary tract. Nearly 50% of the apparent enzyme activity in the parenchymal cell was attributable to a nonspecific hydrolase.     

Interleukin-33 overexpression is associated with gamma-glutamyl transferase in biliary atresia

Interleukin-33 (IL-33) plays a crucial role in inflammation. However, it is not clear whether IL-33 levels are of clinical significance for patients with biliary atresia (BA). The purpose of this study was to determine correlations between serum IL-33 levels and the clinicopathologic features of BA. Serum samples were collected from 18 BA infants, 12 nonicteric choledochal cyst (CC) infants with normal liver function, and 10 healthy controls (HCs). Serum IL-33 levels were measured with an enzyme-linked immunosorbent assay (ELISA). Routine liver function tests were performed on the serum samples. qRT-PCR and Western blot analysis were used to detect IL-33 expression in BA liver biopsy tissues. Hepatic lobule localization of IL-33 expression in the hepatic lobule was conducted by immunohistochemistry (IHC). IL-33 levels in serum collected from BA infants were significantly elevated in comparison with CC and HC patients. Furthermore, the elevated serum levels of IL-33 in BA infants were correlated with gamma-glutamyl transferase (GGT) levels. The expression of IL-33 mRNA and protein levels were up-regulated in BA liver biopsy tissues in comparison with CC patients. IHC analysis revealed increased positive immunostaining for IL-33 in BA liver tissues as compared to that in CC tissues. These results suggest that IL-33 may play an important role in the pathogenesis of BA. In addition, the correlation of serum IL-33 levels with GGT levels may provide a novel marker for the diagnosis of BA.     

Phosphorylation and inhibition of gamma-glutamyl transferase activity by cAMP-dependent protein kinase

It was shown that preparations of bovine kidney gamma-glutamyl transferase with different degree of purity are phosphorylated by cAMP-dependent protein kinase. Phosphorylation is accompanied by a simultaneous decrease of both transferase and hydrolase activities of the enzyme. Hence, gamma-glutamyltransferase may serve as a substrate and target of regulation by cAMP-dependent protein kinase.     

Acute cardiomyopathy with rhabdomyolysis in chronic alcoholism.

Of five chronic alcoholics with acute skeletal muscle necrosis (rhabdomyolysis) three developed acute heart failure with disturbances of rhythm and conduction. Symptoms came on abruptly after a period of intensified drinking. Myocardial infarction, thiamine deficiency, and cobalt intoxication were excluded. Probably the whole spectrum of muscle disease in chronic alcoholism may be commoner than has been suspected.