Purification and characterization of NADP-linked acetoacetyl-CoA reductase from Zoogloea ramigera I-16-M.

An NADP-linked acetoacetyl-CoA reductase was purified to electrophoretic homogeneity from Zoogloea ramigera I-16-M, a poly(3-hydroxybutyrate)-accumulating bacterium. The purified enzyme showed specific activity of 412 mumol acetoacetyl-CoA reduced per min per mg protein, which constituted an 880-fold purification compared to the crude extract, with a 32% yield. Electrophoretic analysis of the purified enzyme which had been cross-linked with dimethylsuberimidate showed that the native enzyme (Mr 92,000) is a tetramer of four identical subunits (Mr 25,500). Among the various D-(-)- and L-(+)-3-hydroxyacyl-CoAs tested, the purified enzyme oxidized only D-(-)-3-hydroxybutyryl-CoA and to a lesser extent D-(-)-3-hydroxyvaleryl-CoA in the presence of NADP+. The antiserum prepared against the purified enzyme completely inhibited poly(3-hydroxybutyrate) synthesis from acetyl-CoA by a crude extract of Z. ramigera I-16-M cells. These findings indicate that this enzyme plays an indispensable role as the supplier of D-(-)-3-hydroxybutyryl-CoA in poly(3-hydroxybutyrate) synthesis in this bacterium.

Formation of spirodilactone of 4-(2'-carboxyphenyl)-4,4-dihydroxybutyrate from 2-succinylbenzoate in cell-free extracts of Micrococcus luteus.

The enzyme mediated ATP- and, to a lesser extent, CoASH-dependent synthesis of the spirodilactone of 4-(2'-carboxyphenyl)-4,4-dihydroxybutyrate from 2-succinylbenzoate has been demonstrated in membrane-free extracts of Micrococcus luteus and Escherichia coli. The suggestion is made that the spirodilactone is the product of an aberrant reaction involving a compound that is normally an intermediate in the conversion of 2-succinylbenzoate to 1,4-dihydroxy-2-naphthoate.

The dependence of glucose formation from lactate on the adenosine triphosphate content in the isolated perfused rat liver.

Bilateral intercollicular lesions in the chick abolish or depress notonly calling, but also those phases of behavior when calling would have been occurring. These include: long bouts of excited feeding immediately after food is made available;examining and pecking moving targets and novel objects; persistent scanning, and inhibitionof other behaviour in a novel environment. Deaf birds behave precisely like controls,so that possible auditory deficits are not involved. During calling phases significantvisual stimuli are treated as if they were startling or conspicuous. Conversely,continuousexamination of a stimulus causes calling to diminish or disappear even though responsecontinues; a brief period when the stimulus is not seen causes calling to begin againwhen it is once more perceived. In addition to the increased effectiveness of relevantvisual stimuli, motor facilitation is usual in calling phases, as is inhibition ofirrelevant responses. Emotioanl behaviour in man and other mammals is composed tocalling phases in the chick

Hormonal control of ketogenesis. Biochemical considerations.

A two-site, bihormonal concept for the control of ketone body production is proposed. Thus, ketosis is viewed as the result of increased mobilization of free fatty acids from adipose tissue (site 1) to the liver (site 2), coupled with simultaneous enhancement of the liver's capacity to convert these substrates into acetoacetic and beta-hydroxybutyric acids. The former event is believed to be triggered by a fall in plasma insulin levels while the latter is considered to be effected primarily by the concomitant glucagon excess characteristic of the ketotic state. Although the precise mechanism whereby elevation of the circulating [glucagon]:[insulin] ratio stimulates hepatic ketogenic potential is not known, activation of the carnitine acyltransferase reaction, the first step in the oxidation of fatty acids, is an essential feature. Two prerequisites for this metabolic adaptation in liver appear to be an elevation in its carnitine content and depletion of its glycogen stores. Despite present limitations the model (evolved mainly from rat studies) provides a framework for the description of various types of clinical ketosis in biochemical terms and may be useful for future studies.

Studies on the relation of gamma-hydroxybutyric acid (GHB) to gamma-aminobutyric acid (GABA). Evidence that GABA is not the sole source for GHB in rat brain.

The effects of gamma-aminobutyric acid (GABA)-alpha-oxoglutarate aminotransferase (GABA-T) inhibitors, L-glutamic acid decarboxylase (GAD) inhibitors, and antipetit mal anticonvulsants on gamma-hydroxybutyric acid (GHB) and GABA were studied. Treatment with anticonvulsants and GABA-T inhibitors resulted in an increase in steady-state brain levels of both GHB and GABA. GAD inhibitors produced markedly decreased levels of brain GABA but no change in GHB concentrations. Studies of GHB derived exclusively from GABA showed that GABA-T inhibitors which produced an elevation of steady-state levels of GHB in brain also resulted in a decrease in GABA-derived GHB. Intracerebroventricular (i.c.v.) administration of GABA, putrescine, and 1,4-butanediol all produced significant elevations in brain GHB, but GABA-T inhibitors blocked this effect of GABA and putrescine. These data suggest that there may be another source for GHB in brain in addition to GABA and raise the possibility that 1,4-butanediol may be that source.

Differential effects of alanine on ketogenesis and triacylglycerol formation by isolated perfused livers from euthyroid and hyperthyroid rats.

We examined the effects of infusion of alanine on hepatic concentration of glycero-3-phosphate (glycero-3-P) and output of triacylglycerol (TG) by isolated perfused livers from triiodothyronine (T3)-treated rats. It was expected that because of its gluconeogenic and antiketogenic properties, alanine might stimulate accumulation of glycero-3-P, which in turn might result in enhanced TG production by the hyperthyroid livers. The hepatic concentration of glycero-3-P is lower in livers from T3-treated rats than in euthyroid rats. Infusion of 1.83 and 4.23 mmol alanine/4 h did not alter the hepatic concentration of glycero-3-P and output of triacylglycerol by livers from T3-treated rats. However in these livers, the two concentrations of infused alanine increased the output of glucose and decreased the output of ketone bodies. In livers from euthyroid animals, the infusion of 1.83 mmol alanine/4 h had no effect, whereas 4.23 mmol/4 h decreased hepatic concentration of glycero-3-P. These concentrations of alanine did not alter the output of ketone bodies or TG, but progressively decreased the output of glucose by the euthyroid livers. Our data suggested that the decreased availability of glycero-3-P in livers from T3-treated rats is rate-limiting for TG production and correlated with the diminished output of TG, whereas in livers from euthyroid rats, the glycero-3-P concentration is sufficient to maintain maximal synthesis and secretion of TG. Furthermore, the effects of alanine on the output of ketone bodies or glucose appear to be independent of its effects on hepatic glycero-3-P.

The formation of 2-hydroxybutyric acid in experimental animals.

1. The formation of 2-hydroxybutyric acid (2-HB) has been studied by animal experiments. 2. 2-HB was excreted in the urine together with lactic acid following intravenous administration of huge amounts of 3-hydroxybutyric acid (sodium salt) to a dog. 3. Rats made diabetic by an injection of streptozotocin were found to excrete large quantities of 2HB in the urine (up to 300 mumol/24 h) together with the development of ketoacidosis. 4. The use of 14-C-labelled precursors clearly showed that the amino acids methionine, threonine and homoserine can be converted to 2-HB. 5. 2-Aminobutyric acid is also converted to 2-HB, with 2-oxobutyric acid as an intermediate metabolite. The ratio between the urinary concentrations of 2-HB and 2-oxobutyric acid was increased by the ingestion of ethanol. 6. In normal rats neither a prolonged fasting period nor loading with large doses of methionine, threonine and homoserine resulted in the excretion of 2-HB. 7. The mechanisms behind the formation of 2-HB are discussed, and it is concluded that an increased NADH2/NAD ratio in the cytoplasma is the most important factor.

A new antitumor antibiotic, FR900840. I. Discovery, identification, isolation and characterization.

A new antitumor antibiotic, FR900840, was isolated from a culture broth of Streptomyces strain No. 8727 as a pale yellowish prism and the molecular formula was determined to be C7H11N3O5. The antibiotic exhibited prominent antitumor effects on human tumor cell lines both in vitro and in vivo.

Relative utilization of fatty acids for synthesis of ketone bodies and complex lipids in the liver of developing rats.

The regulation of hepatic ketogenesis, as related to the metabolism of fatty acids through oxidative and synthetic pathways, was studied in developing rats. [1-14C] palmitate was used as a substrate to determine the proportions of free fatty acids utilized for the production of ketone bodies, CO2 and complex lipids. Similar developmental patterns of hepatic ketogenesis were obtained by measuring the production of either [14C] acetoacetate from exogenous [1-14C] palmitate or the sum of unlabeled acetoacetate and beta-hydroxybutyrate from endogenous fatty acids. The production of total ketone bodies was low during the late fetal stage and at birth, but increased rapidly to a miximum value within 24 hr after brith. The maximal ketogenic capacity appeared to be maintained for the first 10 days of life. 14CO2 production from [1-14C] palmitate increased by two- to fourfold during the suckling period, from its initial low rate seen at birth. The capacity for synthesis of total complex lipids was low at birth and had increased by day 3 to a maximal value, which was comparable to that of adult fed rats. The high lipogenic capacity lasted throughout the remaining suckling period. When ketogenesis was inhibited by 4-pentenoic acid, the rate of synthesis of complex lipids did not increase despite an increase in unutilized fatty acids. During the mid-suckling period, approximately equal amounts of [1-14C] palmitate were utilized for the synthesis of ketone plus CO2 and for complex lipid synthesis. By contrast, in adult fed rats, the incorporation of fatty acids into complex lipids was four times higher than that of ketone plus CO2. These observations suggest that stimulated hepatic ketogenesis in suckling rats results from the rapid oxidation of fatty acids and consequent increased production of acetyl CoA, but not from impaired capacity for synthesis of complex lipids.

Alcohol metabolism at high alcohol concentrations.

The incorporation rate of tritium from (R,S)ethanol-1-3H, (R)ethanol-1-3H and (S)ethanol-1-3H in lactate and beta-hydroxybutyrate is investigated by means of a GLC method. Preliminary results show no decrease in incorporation of 3H from (R)ethanol during the period of labelling (15 min). These results indicate that either a shift in the isotope effect occurs at high concentrations of ethanol, or the results may indicate that ethanol does not exhibit a substrate inhibitory effect on alcohol dehydrogenase in the liver cells. Furthermore, the fraction of acetaldehyde metabolized in the cytosol is determined.