John F. Van Pilsum

Simplified assay for transamidinase activities of rat kidney homogenates

Abstract A simplified assay for transamidinase activities of rat kidney homogenates has been developed. Arginine and glycine are used as the substrates and the ornithine formed is measured by means of a ninhydrin color reaction.

Transamidinase activities, in vitro, of tissues from various mammals and from rats fed protein-free, creatine-supplemented and normal diets☆

Spleen, skeletal muscle, heart muscle, lung, brain, and testes from various mammals have been found to have measurable amounts of transamidinase activities, in vitro. These activities, expressed per unit weight of tissue, were only a fraction of the activities found in kidney and pancreas. However, in the rat, for example, the sum of the calculated total tissue activities of spleen, muscle, lung, brain, and testes was similar to the sum of the calculated total tissue activities of kidney and pancreas. Rats were fed normal, creatine-supplemented, and protein-free diets, and it was found that kidney and pancreas were the only tissues from the rats fed the latter two diets that had below normal activities. The possibility that there may be a significant synthesis of guanidinoacetic acid, in vivo, in tissues other than kidney and pancreas is discussed.

PHYLOGENY AND THE DISTRIBUTION OF CREATINE IN INVERTEBRATES

1. Fifteen species of invertebrates from seven phyla were examined for the presence of creatine, guanidinoacetic acid, and transamidinase.2. None of the animals which possessed creatine had detectable levels of transamidinase. All animals which possessed guanidinoacetic acid also possessed transamidinase activity. A third group of invertebrates was formed of those species in which neither of the guanidine compounds nor transamidinase could be demonstrated.3. Attempts using various procedures to obtain synthesis of creatine from appropriate precursors in those organisms in which it was found were uniformly negative.4. Accumulation of creatine from very dilute solution in the ambient medium was demonstrated for all of the invertebrates in which creatine was found.5. In Glycera and Saccoglossus, both of which possess creatine, creatine-C14 obtained from the ambient medium persists as creatine for at least 96 hours.6. Other organisms which do not normally possess creatine also showed the ability to accumulate...

Assay and Some Properties of Kidney Transamidinase.

Summary and Conclusions 1. An assay of transaminidase activity in mammalian kidneys was developed. 2. The enzyme was inhibited by p-chloromercuribenzoic acid. The inhibition could be reversed with reduced glutathione, cysteine, or dimercaprol. These results suggest that transaminidase is a sulfhy-dryl enzyme. 3. Kidneys from a mercury-poisoned rat showed reduced transaminidase activity. The activity could not be restored by addition of dimercaprol to the reaction flask. 4. Canavanine was found to be 3 times as effective as arginine as an amidine donor in the transamidinase system. 5. Kidney transaminidase activity in weanling rats fed a protein-free diet for 12 days was 24% of normal. The activity was restored to 80% of normal by feeding the rats a complete diet for 15 days following the feeding of a protein-free diet for 12 days. Thus, kidney transaminidase activity appears to be dependent on exogenous protein sources.

The purification and characterization of human kidney l-arginine:Glycine amidinotransferase

Human kidney L-arginine:glycine amidinotransferase (transamidinase) has been purified to a homogeneous state as defined by native and sodium dodecyl sulfate gel electrophoresis and by ultracentrifugation (sedimentation equilibrium) experiments. The four steps in the isolation procedure were chromatography with DEAE-cellulose, gel filtration with Sephadex G-150, chromatography with phenyl Sepharose, and high-pressure liquid chromatography with hydroxylapatite. The final product represented a 90-fold purification of the enzyme. Human kidney transamidinase is a dimer with a molecular mass of 89,000 Da and subunit masses of 44,000 Da. The Km for arginine and glycine were both 2.5 mM and the Vmax was 0.5 mumol ornithine/min/mg protein. The ultraviolet absorption spectrum, specific activity, and isoelectric points were determined for human kidney transamidinase. Multiple forms of the enzyme were obtained by isoelectric focusing. Human kidney transamidinase cross-reacted with polyclonal antibodies raised to rat kidney transamidinase. All of the properties of human kidney transamidinase that we have examined were similar to those of rat kidney transamidinase. A close evolutionary relationship between the rat and human kidney transamidinase is suggested.

Evidence for the role of pancreatic acinar cells in the production of ornithine and guanidinoacetic acid by L-Arginine:glycine amidinotransferase

l-Arginine:glycine amidinotransferase (transamidinase) occurs at high concentrations in the kidney and the pancreas of rats. The cellular localization of transamidinase was investigated in fetal, neonatal, and adult rat pancreatic tissue using three indicators of the presence of transamidinase: (1) immunofluorescence microscopy, (2) in vitro enzymatic activity measurements on homogenates of whole pancreas and on isolated acinar and islet tissue from adult rats, and (3) ornithine production from perfused adult rat pancreas. The cellular localization of transamidinase was determined in fetal, neonatal, and adult rat pancreas, using a polyclonal guinea pig antibody made against a highly purified preparation of kidney transamidinase. Immunoreactive transamidinase was detected only in the pancreatic acinar cells. The cellular distribution of the immunostaining was compatible with the presence of transamidinase in mitochondria. The transamidinase enzymatic activity of whole pancreatic homogenates was 13.4 ± 0.7 U/g wet weight (n = 11). In pancreata where islets had been isolated away from the acinar tissue, the transamidinase activity was similar to that of the whole pancreatic homogenates (16.8 ± 2 U/g wet weight). Any transamidinase activity present in isolated islets was below the sensitivity of the assay. Transamidinase activity in the isolated perfused pancreas was determined by measuring the amount of ornithine released into the perfusate. The transamidinase activity of the perfused pancreas was 16.4 ± 1.8 U/g pancreas and is an estimate of the physiological production capacity of the enzyme (270 ± 29 nmol ornithine/min/g pancreas). These results indicate that transamidinase is present at high concentrations in the pancreas. Within the pancreas, the enzyme was present only in acinar cells of fetal, neonatal, and adult rats. Transamidination products that may be provided to the liver by way of the portal vein are largely, if not exclusively, derived from the pancreatic acinar cells.

Effect of castration and steroid sex hormones on rat kidney transamidinase

Abstract The rat kidney enzyme activity of transamidinase has been found to be lower in the female compared with the male. Castration of the female increases the kidney enzyme activity, but the enzyme values remain unchanged after castration in the male. Enzyme levels were significantly lowered in intact male rats receiving daily injections of estradiol at 1 μg/day, estrone at 10μg/day, and estriol at 100μg/day. Intact female rats receiving 500 μg of testosterone per day had slightly increased levels of transamidinase. These changes in transamidinase were also observed in the castrated and hypophysectomized rats treated with steroid sex hormones. The relationship of exogenous hormones with food consumption and gain in body weight is discussed.

Studies on the uptake of creatine from sea water by the marine annelid, Glycera dibranchiata

Abstract 1. 1. A variety of compounds were tested for their effects on the uptake of creatine from sea water by the marine annelid, Glycera dibranchiata. 2. 2. Guanidino-carboxylic acids and 2-amino carboxylic acids were competitive and uncompetitive inhibitors, respectively, of the creatine uptake by Glycera. Some spatial and chemical requirements for inhibitors of the creatine uptake by Glycera are defined. 3. 3. The Km and Vmax values for the creatine uptake by Glycera were 0·037 mM and 5 × 10−3 μmoles/g worm per min, respectively. 4. 4. The creatine transport system in Glycera is compared with that reported for rat skeletal muscle.

S-Adenosylmethionine: Guanidinoacetate N-Methyltransferase Activities in Livers from Rats with Hormonal Deficiencies or Excesses

Summary S-Adenosylmethionine: guanidinoacetate N-methyltransferase activities were determined in livers from rats after removal of either adrenals, pituitaries, gonads, or thyroids and parathyroids and in livers from intact rats given large doses of either insulin, estradiol, testosterone, cortisol, thyroxine, or growth hormone. The only differences that were statistically significant (p < 0.01) were between the activities of livers from intact rats and the slightly lower (~20%) activities of livers from rats with their thyroid and parathyroid glands removed or from intact rats given large doses of either testosterone, thyroxine, or growth hormone. Further, liver GA-methyltransferase activities were virtually unaffected by replacement of either thyroxine to thyroidectomized rats, growth hormone to hypophysectomized rats, or testosterone or estradiol to castrated rats. Therefore, direct hormonal regulation of rat liver GA-methyltransferase is contraindicated. Thus, the second enzyme involved in creatine synthesis (GA-methyltransferase) differs greatly from the first enzyme (arginine: glycine transamidinase) in that transamidinase activities have previously been reported to be altered markedly by removal of certain endocrine glands or by injections of large doses of certain hormones into intact rats. The authors thank Neil Derechin for technical assistance.

On the proposed origin of creatinine from creatine phosphate

Abstract 1. 1. Solutions containing extracts of normal rabbit muscle, glucose 1-phosphate, creatine phosphate, and cysteine were incubated and analyzed for creatine and creatinine formation and creatine phosphate utilization. 2. 2. The muscle extract catalyzed the conversion of creatine phosphate to creatine without any detectable formation of creatinine.