Purification and some kinetic properties of rat liver glucosamine synthetase

1. Glucosamine synthetase (l-glutamine-d-fructose 6-phosphate aminotransferase, EC 2.6.1.16) was purified about 300-fold from rat liver by two techniques. One procedure utilized the protective action of fructose 6-phosphate and gave a relatively stable preparation, the other yielded an unstable enzyme (half-life of about 20h), free of contaminant activities, on which kinetic experiments were performed. Although the properties of the two preparations showed slight differences, the unstabilized form could be converted into the stabilized form. 2. During preparation the enzyme retained its sensitivity to the feedback inhibitor, UDP-N-acetylglucosamine. 3. The reversibility of the enzyme-catalysed reaction could not be demonstrated. There was no apparent requirement for a cofactor. 4. The pH optimum was at 7.5, at which pH the reaction obeyed a Ping Pong Bi Bi rate equation. At pH values outside the range 6.9-7.6 and at temperatures below 29 degrees C the velocity was described by an ordered Bi Bi rate equation. 5. The molecular weight of the enzyme, determined by two procedures, was 360000-400000. 6. The aminotransferase was unable to utilize ammonia as a substrate.     

Effect of porphyrinogenic agents on protein synthesis and bilirubin formation by the isolated perfused rat liver

We established an isolated rat liver perfusion system for the study of heme catabolism. The liver of rats fasted for 48 h is perfused with an erythrocyte-free medium. Ultrastructural analysis shows integrity of all subcellular organelles with the exception of minor alterations in the rough endoplasmic reticulum. The perfused liver synthesis serum proteins at a constant rate for 5 h. Albumin is secreted at a mean rate of 17 ± 2 mg/h per 100 g liver, hemopexin at 5.0 ± 0.7. haptoglobin at 3.2 ± 0.6 and transferrin at 5.1 ± 0.8 mg/h per 100 g liver. The mean ratio of ATP : ADP is 3.5 ± 0.1, and that of lactate : pyruvate 27 ± 6. The rate of conversion of heme into bilirubin is comparable to that reported for in vivo studies.A minimal effect on protein synthesi is observed after administration of the porphyrinogenic agents, allylisopropylacetamide (AIA) and 3,5-diethoxycabonyl-1,4 dihydrocollidine (DDC). Pretreatment of the rats with the iron chelator, Desferal, causes a 3–4-fold increase in hemopoxin but not in albumin and transferrin synthesi. A striking 2–3-fold enhancement of bile bilirubin production follows treatment with DDC and Desferal, but not with AIA. The amount of bilirubin formed from heme added to the perfusate is reduced by AIA and DDC and enhanced by Desferal treatment. It is proposed that unavailability of iron in a certain hepatic tissue pool causes protoporphyrin IX accumulation which may serve as an alternate source for bilirubin production.     

Studies on the in vitro effects of insulin on glycogen synthesis and ultrastructure in isolated rat liver hepatocytes

Rat liver hepatocytes were isolated by collagenase $\text{in vitro}$ perfusion technique and effect of insulin on glycogen synthesis and ultra-structure was studied. Addition of insulin stimulated glycogen synthesis and maintained better cellular structure. Synthesis of glycogen was linear in isolated hepatocytes when incubated with various concentrations of glucose (0–800 mg%) reaching initial levels. Concanavaline A inhibited epinephrine stimulated glycogenolysis but had no effect on glucagon stimulated glycogenolysis. These studies indicate that insulin is required for glycogen synthesis and for maintaining hepatocytes ultrastructure. Furthermore, isolated hepatocytes retain various receptors and that different hormones utilize different receptor sites.     

Effect of some antioxidants and caffeine on xanthine dehydrogenase activity in rat liver in vitro

The regulation of xanthine dehydrogenase activity by reductors-antioxidants (ascorbic acid, glutatione-SH, gentathione dithiothreitol, cysteine and hydrocortisone) and caffeine was investigated in the purified enzymatic preparation in vitro. It was shown, that reductors-antioxidants were incompetitor inhibitors of xanthine dehydrogenase and caffeine was a competitor-inhibitor of it. The mechanisms of inhibition of xanthine dehydrogenase activity by these agents were discussed.     

Regulation of protein synthesis in intact rat liver by calcium mobilizing agents

1. Exposure of intact perfused rat liver to EGTA, vasopressin or phenylephrine resulted in a rapid decrease in polysome formation. Pretreatment with phentolamine, an alpha-adrenergic antagonist, blocked the effect of phenylephrine. 2. Hormonal inhibitions of leucine incorporation into protein in isolated hepatocytes and of polysome formation in perfused liver were reversed in the presence of supraphysiologic extracellular Ca2+ concentrations. 3. The beta-adrenergic agonist isoproterenol exerted minimal effects on polysome content. 4. It is proposed that intracellular Ca2+ stores sensitive to hormonal modulation are necessary for maintenance of protein synthesis in hepatocytes.     

Effect of insulin and glucagon on aldolase turnover in rat liver

The effects of exogenous and endogenous insulin and glucagon on aldolase turnover in rat liver and blood were studied. Some effects of these hormones on the biosynthesis and degradation of hepatic aldolase were specified. The rate of the "de novo" synthesis of aldolase was investigated in hepatocyte mitochondria and in blood plasma. The exogenous and endogenous hormones were shown to produce different effects on the biosynthesis and spontaneous degradation of rat liver aldolase.