Glycerol metabolism in the neonatal rat

1. The possible role of glycerol as a precursor in neonatal gluconeogenesis in the rat was investigated by recording the activities of glycerol kinase and l-glycerol 3-phosphate dehydrogenase in the liver, kidney and other tissues around birth and during the neonatal period. 2. Blood glycerol concentrations in the neonatal rat are high. 3. There is a marked increase after birth in the ability of both liver and kidney slices to convert glycerol into glucose plus glycogen that correlates with the increase in glycerol kinase activity. 4. High hepatic and renal l-glycerol 3-phosphate dehydrogenase activities are also found in the neonatal period. 5. The marked capacity for neonatal gluconeogenesis from glycerol thus demonstrated and the role of glycerol kinase in its control are discussed.     

The behavior of hepatic phosphorylase b kinase, phosphorylase a and b after administration of glucagon to patients with glycogen storage disease type VIa

In the patients with glycogen storage disease (GSD) type VIa and different serum glucose response to glucagon, the activities of hepatic phosphorylase b kinase, phosphorylase a and b were estimated before and after the intravenous administration of glucagon. 3 min after the administration of glucagon an increase in the activities of phosphorylase b kinase and phosphorylase a was found in liver tissue of all patients except one. These enzymatic activities, however, did not exceed the values of these enzymes in the control liver biopsies without glucagon loading. After the intravenous administration of glucagon an unsuspected increase of phosphorylase b activity was observed in the control liver tissues and in patients with GSD type VIa, except one. In vitro investigations revealed that an increase of hepatic phosphorylase b activity occurs during its conversion to phosphorylase a. We suppose that this phosphorylase b represents a partially phosphorylated form of this enzyme (an intermediate form) that is due to the action of the active phosphorylase b kinase. The correlations between the activities of phosphorylase b kinase, phosphorylase a and an intermediate form of phosphorylase b and hepatic glycogen degradation after administration of glucagon has been discussed.     

1-Thioglycerol: inhibitor of glycerol kinase activity in vitro and in situ

The infantile form of glycerol kinase (GK) deficiency (McKusick No. 30703) (1) is characterized by adrenal cortical insufficiency, adrenal hypoplasia and developmental delay. The underlying biochemical mechanism(s) responsible for the observed clinical presentations are undetermined. Pursuant to our examination of the molecular pathogenesis of this enzyme deficiency, we have endeavored to develop a model for this disorder. 1-thioglycerol (1-TG) was investigated as a potential GK inhibitor in adrenal gland, an organ consistently affected, and in cultured fibroblasts, available from affected individuals. In 105,000 g bovine adrenal supernatant the Ki for 1-TG was 1.9 mM. In human fibroblast 105,000 g supernatant, the Ki for 1-TG was 3.4 mM. In both tissues the inhibition was purely competitive with respect to glycerol. Using incorporation of [14C(U)]-glycerol into protein as an index of GK activity in situ in human skin fibroblasts, GK deficient fibroblasts incorporate less than 10% of that observed in normal fibroblasts. Addition of 1-TG to normal fibroblasts resulted in inhibited incorporation rates. The specificity of these effects in situ was examined. Our findings indicate that 1-TG may be a suitable inhibitor of GK activity for the development of a model for glycerol kinase deficiency.     

X-chromosome inactivation and enzymatic activities in diploid and triploid fibroblasts

Diploid and triploid rabbit embryos obtained by artificial fertilization were cultured. No Barr's body in XXY and only one Barr's body in XXX triploid fibroblasts was observed. Six enzymatic activities were also determined. Two X-chromosome-bound enzymatic activities, glucose-6-phosphate dehydrogenase and phosphoglycerate kinase, were significantly increased in triploid fibroblasts, while another X-chromosome-bound activity (hypoxanthine phosphoribosyl transferase) was not modified. Among the autosome-bound activities studied, pyruvate kinase and adenine phosphoribosyl transferase were not modified, whereas in the triploid cells the 6-phosphogluconate dehydrogenase activity was decreased. The relationship between these modifications of enzymatic activities in triploids and the X-chromosome inactivation is discussed.     

Some properties of hepatic glycerol kinase and their relation to the control of glycerol utilization

1. Glycerol kinase (EC 2.7.1.30) is shown to catalyse a non-equilibrium reaction in rat liver; and, as it is the first enzyme in the pathway metabolizing glycerol, its properties may be pertinent to the metabolic regulation of glycerol uptake and utilization by this tissue. 2. The properties of hepatic glycerol kinase were studied by using a radiochemical technique to measure the enzyme activity. When the concentration of ATP is low the activity of glycerol kinase is inhibited by high concentrations of glycerol; but when the concentration of ATP is high there is no inhibition and the double-reciprocal plot is linear, providing a K(m) for glycerol of 3.16x10(-6)m. Glycerol kinase is activated by high ATP concentrations provided that the concentration of the second substrate (glycerol) is high; at low concentrations of glycerol ATP does not activate the enzyme so that the double-reciprocal plot is linear, providing a K(m) for ATP of 5.8x10(-5)m. It is suggested that these kinetics may be explained by a model similar to that described by Ferdinand (1966) for phosphofructokinase. 3. Hepatic glycerol kinase is inhibited by ADP and AMP, and raising the Mg(2+) concentration increases the inhibition by these two compounds; this suggests that ADP-Mg(2+) and AMP-Mg(2+) complexes are the inhibitory species. The physiological significance of these inhibitions may be to prevent phosphorylation of glycerol when the hepatic ATP concentration is low. It is suggested that this inhibition may provide an approach to the problem of measurement of rates of lipolysis by glycerol release in tissues that contain glycerol kinase (e.g. liver, kidney, muscle, adipose tissue). 4. Hepatic glycerol kinase is inhibited by l-3-glycerophosphate competitively with respect to glycerol. The physiological significance of this inhibition may be that factors that change the intracellular concentration of l-3-glycerophosphate could change glycerol uptake by the tissue. Thus it is suggested that thyroxine treatment or feeding rats on a diet high in glycerol, which increase the activity of glycerophosphate oxidase in liver and kidney cortex respectively, lead to an increased glycerol uptake through a decrease in the concentration of glycerophosphate in these tissues. It is known that ethanol administration decreases glycerol uptake by liver, and this can be explained by the increased concentration of l-3-glycerophosphate causing inhibition of glycerol kinase.     

GLYCEROL KINASE AND DIHYDROXYACETONE KINASE IN RAT BRAIN

—The enzymatic phosphorylation of glycerol and dihydroxyacetone by ATP to sn-glycerol-3-phosphate and dihydroxyacetone phosphate respectively in various subcellular fractions of rat brain was studied. A sensitive radiochemical assay was used where the labelled phosphorylated products were separated from the radioactive substrates by high voltage paper electrophoresis and the radioactivity in these compounds determined. Using this assay the glycerol kinase (EC 2.7.1.30) activity was found to be associated with the mitochondrial fraction of the brain. Under optimum conditions 2.45 nmol of glycerol was phosphorylated/min per mg of protein. The K_m for glycerol was 70 μm at pH 7. This mitochondrial enzyme, like other glycerol kinases from different sources, also phosphorylated dihydroxyacetone. Under optimum conditions 1.7 nmol of dihydroxyacetone phosphate was formed/min per mg of mitochondrial protein. The K_m for dihydroxyacetone was 0.6 mm . Glycerol kinase activity was also present in the cytoplasm of brain. However, the specific activity of this enzyme in cytosol is about 15% of the mitochondrial glycerol kinase. Compared to glycerol, dihydroxyacetone was phosphorylated by ATP in cytoplasm at a much higher rate. The pH optimum for this soluble dihydroxyacetone kinase was much lower (pH 6.5) than that of the soluble or mitochondrial glycerol kinase (pH 10.0). Using ammonium sulfate, brain cytoplasm was fractionated to yield a fraction in which the dihydroxyacetone kinase was enriched 2–3 fold with no glycerol kinase activity. Under optimum conditions 1.0 nmol of dihydroxyacetone was phosphorylated/min per mg protein. The K_m for dihydroxyacetone was 60 μm . This cytosol fraction was also found to phosphorylate d -glyceraldehyde and l -glyceraldehyde at a rate of 30–40% to that of the dihydroxyacetone phosphorylation. The properties and the possible metabolic role of these enzymes in brain are discussed.     

Galactosialidosis: simultaneous deficiency of esterase, carboxy-terminal deamidase and acid carboxypeptidase activities

Esterase and deamidase activities at pH 7.0 and carboxypeptidase activity at pH 5.7 were markedly low or deficient in seven galactosialidosis fibroblast strains with deficient activity of "protective protein" for lysosomal beta-galactosidase and neuraminidase. No simultaneous deficiency of these three enzyme activities was observed in other lysosomal disease fibroblasts examined in this study. This result strongly suggests that "protective protein" is identical with a multifunctional protein with esterase/deamidase/carboxypeptidase activities and its mutation in galactosialidosis results in deficiency of these three enzyme activities.     

Role of glycerol 3-phosphate dehydrogenase in glyceride metabolism. Effect of diet on enzyme activities in chicken liver.

1. The metabolic role of hepatic NAD-linked glycerol 3-phosphate dehydrogenase (EC 1.1.1.8) was investigated vis-a-vis glyceride synthesis, glyceride degradation and the maintainence of the NAD redox state. 2. Five-week-old chickens were placed on five dietary regimes: a control group, a group on an increased-carbohydrate-lowered-fat diet, a group on a high-fat-lowered-carbohydrate diet, a starved group and a starved-refed group. In each group the specific activity (mumol/min per g wet wt. of tissue) of hepatic glycerol 3-phosphate dehydrogenase was compared with the activities of the beta-oxoacyl-(acyl-carrier protein) reductase component of fatty acid synthetase, glycerol kinase (EC 2.7.1.30) and lactate dehydrogenase (EC 1.1.1.27). 3. During starvation, the activities of glycerol 3-phosphate dehydrogenase, glycerol kinase and lactate dehydrogenase rose significantly. After re-feeding these activities returned to near normal. All three activities rose slightly on the high-fat diet. Lactate dehydrogenase activity rose slightly, whereas those of the other two enzymes fell slightly on the increased-carbohydrate-lowered-fat diet. 4. The activity of the beta-oxoacyl-(acyl-carrier protein) reductase component of fatty acid synthetase, a lipid-synthesizing enzyme, contrasted strikingly with the other three enzyme activities. Its activity was slightly elevated on the increased-carbohydrate diet and significantly diminished on the high-fat diet and during starvation. 5. The changes in activity of the chicken liver isoenzyme of glycerol 3-phosphate dehydrogenase in response to dietary stresses suggest that the enzyme has an important metabolic role other than or in addition to glyceride biosynthesis.     

Diacylglycerol metabolism in neonatal rat liver: characterization of cytosolic diacylglycerol lipase activity and its activation by monoalkylglycerols.

Diacylglycerol lipase (glycerol ester hydrolase, EC 3.1.1.3) activities were investigated in subcellular fractions from neonatal and adult rat liver in order to determine whether one or more different lipases might provide the substrate for the developmentally expressed, activity monoacylglycerol acyltransferase. The assay for diacylglycerol lipase examined the hydrolysis of sn-1-stearoyl,2- [14C]oleoylglycerol to labeled monoacylglycerol and fatty acid. Highest specific activities were found in lysosomes (pH 4.8) and cytosol and microsomes (pH 8). The specific activity from plasma membrane from adult liver was 5.8-fold higher than the corresponding activity in the neonate. In other fractions, however, no developmental differences were observed in activity or distribution. In both lysosomes and cytosol, 75 to 90% of the labeled product was monoacylglycerol, suggesting that these fractions contained relatively little monoacylglycerol lipase activity. In contrast, 80% of the labeled product from microsomes was fatty acid, suggesting the presence of monoacylglycerol lipase in this fraction. Analysis of the reaction products strongly suggested that the lysosomal and cytosolic diacylglycerol lipase activities hydrolyzed the acyl-group at the sn-1 position. The effects of serum and NaCl on diacylglycerol lipase from each of the subcellular fractions differed from those effects routinely observed on lipoprotein lipase and hepatic lipase, suggesting that the hepatic diacylglycerol lipase activities were not second functions of these triacylglycerol lipases. Cytosolic diacylglycerol lipase activity from neonatal liver and adult liver was characterized. The apparent Km for 1-stearoyl,2-oleoylglycerol was 115 microM. There was no preference for a diacylglycerol with arachidonate in the sn-2 position. Bovine serum albumin stimulated the activity, whereas dithiothreitol, N-ethylmaleimide, and ATP inhibited the activity. Both sn-1(3)- and 2-monooleylglycerol ethers stimulated cytosolic diacylglycerol lipase activity 2-3-fold. The corresponding amide analogs stimulated 28 to 85%, monooleoylglycerol itself had little effect, and 1-alkyl- or 1-acyl-lysophosphatidylcholine inhibited the activity. These data provide the first characterization of hepatic subcellular lipase activities from neonatal and adult rat liver and suggest that independent diacylglycerol and monoacylglycerol lipase activities are present in microsomal membranes and that the microsomal and cytosolic diacylglycerol lipase activities may describe an ambipathic enzyme. The data also suggest possible cellular regulation by monoalkylglycerols.     

The sex difference in the regulation of liver regeneration after partial hepatectomy in the rat

The increases in the activity of hepatic thymidylate synthetase and thymidine kinase, which catalyzes the formation of thymidylate via the de novo and salvage pathways, respectively, were significantly suppressed 24 h after 70% partial hepatectomy in female rats administered either alpha- or beta-adrenoreceptor antagonists. The injection of beta-antagonist to male or ovariectomized female rats had no effect on the activities of these enzymes. Only alpha-adrenoceptor antagonist depressed these enzymatic activities of 24-h-regenerating liver in male and ovariectomized female rats. The decrease of the activities of thymidylate synthetase and thymidine kinase was accompanied by a concomitant reduction of DNA content in 24-h-regenerating liver. It is concluded that catecholamine regulates the female rat liver regeneration through both alpha- and beta-adrenergic pathways by the inductions of thymidylate synthase and thymidine kinase, while in adult male and ovariectomized female rats, only the alpha-mediated pathway is involved.     

Synthesis and hydrolysis of cholesteryl esters by isolated rat-liver lysosomes and cell-free extracts of human lung fibroblasts

The objective of this study was to examine and characterize the cholesteryl ester synthesizing [S] and hydrolyzing [H] properties of the acid cholesteryl ester hydrolase (acid cholesteryl ester hydrolase), both in isolated rat liver lysosomes and in cell-free extracts from cultured fibroblasts. For both liver lysosomes and fibroblasts extracts, the major synthesizing activity was found around pH 4 and did not require exogenous ATP. The rate of hydrolysis was measured at pH 4.5. Several different inhibitors were used in order to characterize the reactions. Ammonium chloride did not markedly affect the activity of acid cholesteryl ester hydrolase at pH 4 [S] or 4.5 [H], whereas chloroquine was a potent inhibitor of acid CEase in both liver lysosomes and fibroblast extracts. The [S] activity of the acid cholesteryl ester hydrolase in either material was not affected by the acylCoA:cholesterol acyltransferase inhibitor Compound 58-035 from Sandoz. Progesterone, on the other hand, which is an often used acylCoA:cholesterol acyltransferase inhibitor, markedly blocked both activities of the acid CEase. Our results indicate that the lysosomal compartment of both studied tissues, in addition to hydrolysis activity, also have a significant esterification activity. It appears that both activities are carried out by the same enzyme.     

Proportional activities of glycerol kinase and glycerol 3-phosphate dehydrogenase in rat hepatomas.

The activities of glycerol 3-phosphate dehydrogenase (EC 1.1.1.8), glycerol kinase (EC 2.7.1.30), lactate dehydrogenase (EC 1.1.1.27), "malic' enzyme (L-malate-NADP+ oxidoreductase; EC 1.1.1.40) and the beta-oxoacyl-(acyl-carrier protein) reductase component of the fatty acid synthetase complex were measured in nine hepatoma lines (8 in rats, 1 in mouse) and in the livers of host animals. With the single exception of Morris hepatoma 16, which had unusually high glycerol 3-phosphate dehydrogenase activity, the activities of glycerol 3-phosphate dehydrogenase and glycerol kinase were highly correlated in normal livers and hepatomas (r = 0.97; P less than 0.01). The activities of these two enzymes were not strongly correlated with the activities of any of the other three enzymes. The primary function of hepatic glycerol 3-phosphate dehydrogenase appears to be in gluconeogenesis from glycerol.     

Purification and properties of phosphofructokinase 2/fructose 2,6-bisphosphatase from chicken liver and from pigeon muscle

Phosphofructokinase 2 and fructose 2,6-bisphosphatase extracted from either chicken liver or pigeon muscle co-purified up to homogeneity. The two homogeneous proteins were found to be dimers of relative molecular mass (Mr) close to 110,000 with subunits of Mr 54,000 for the chicken liver enzyme and 53,000 for the pigeon muscle enzyme. The latter also contained a minor constituent of Mr 54,000. Incubation of the chicken liver enzyme with the catalytic subunit of cyclic-AMP-dependent protein kinase in the presence of [gamma-32P]ATP resulted in the incorporation of about 0.8 mol phosphate/mol enzyme. Under similar conditions, the pigeon muscle enzyme was phosphorylated to an extent of only 0.05 mol phosphate/mol enzyme and all the incorporated phosphate was found in the minor 54,000-Mr constituent. The maximal activity of the native avian liver phosphofructokinase 2 was little affected by changes of pH between 6 and 10. Its phosphorylation by cyclic-AMP-dependent protein kinase resulted in a more than 90% inactivation at pH values below 7.5 and in no or little change in activity at pH 10. Intermediary values of inactivation were observed at pH values between 8 and 10. Muscle phosphofructokinase 2 had little activity at pH below 7 and was maximally active at pH 10. Its partial phosphorylation resulted in a further 25% decrease of its already low activity measured at pH 7.1 and in a negligible inactivation at pH 8.5. Phosphoenolpyruvate and citrate inhibited phosphofructokinase 2 from both origins non-competitively. The muscle enzyme and the phosphorylated liver enzyme displayed much more affinity for these inhibitors than the native liver enzyme. Fructose 2,6-bisphosphatase from both sources had about the same specific activity but only the chicken liver enzyme was activated about twofold upon incubation with ATP and cyclic-AMP-dependent protein kinase. All enzyme forms were inhibited by fructose 6-phosphate and this inhibition was released by inorganic phosphate and by glycerol 3-phosphate. Both liver and muscle fructose 2,6-bisphosphatases formed a 32P-labeled enzyme intermediate when incubated in the presence of fructose 2,6-[2-32P]bisphosphate.     

Rat liver dihydroxyacetone-phosphate acyltransferases and their contribution to glycerolipid synthesis

Differential and isopycnic centrifugation of rat liver homogenates showed that, besides its established localization in peroxisomes and endoplasmic reticulum, dihydroxyacetone-phosphate acyltransferase is also present in mitochondria. The three activities differed in a number of properties (pH optimum, palmitoyl-CoA and dihydroxyacetone-phosphate dependence, and sensitivity toward N-ethylmaleimide) and are therefore likely associated with three distinct proteins. Glycerol 3-phosphate (5 mM) did not inhibit peroxisomal dihydroxyacetone-phosphate acyltransferase but inhibited the extraperoxisomal activities virtually completely. Peroxisomal dihydroxyacetone-phosphate acyltransferase was located at the inner aspect of the peroxisomal membrane, but the enzyme was not latent. Purified microsomes, from which intact peroxisomes had been removed, were still contaminated with peroxisomal membranes as deduced from the presence of two dihydroxyacetone-phosphate acyltransferase activities: a glycerol 3-phosphate-resistant activity with properties similar to those of peroxisomal dihydroxyacetone-phosphate acyltransferase and a glycerol 3-phosphate-sensitive "true" microsomal dihydroxyacetone-phosphate acyltransferase. We propose that, assayed in the presence of 5mM glycerol 3-phosphate, dihydroxyacetone-phosphate acyltransferase can be used as a marker enzyme for peroxisomal membranes. Such a marker enzyme has not hitherto been available. The differential effect of 5 mM glycerol 3-phosphate on peroxisomal and extraperoxisomal dihydroxyacetone-phosphate acyltransferases enabled us to determine the relative contribution of these activities to overall dihydroxyacetone-phosphate acylation in whole liver homogenates. At near-physiological pH and at near-physiological concentrations of unbound palmitoyl-CoA and of dihydroxyacetone-phosphate plus glycerol 3-phosphate, peroxisomes contributed 50-75%. The remaining percentage was mostly accounted for by the microsomal enzyme. At near-physiological concentrations of glycerol 3-phosphate plus dihydroxyacetone-phosphate, glycerolphosphate acyltransferase contributed 93% and dihydroxyacetone-phosphate acyltransferase 7% to overall glycerolipid synthesis in homogenates. This suggests that the dihydroxyacetone-phosphate pathway is of minor quantitative importance in overall hepatic glycerolipid synthesis but that its main function lies in the synthesis of ether lipids, which have acyldihydroxyacetone-phosphate as obligatory precursor.(ABSTRACT TRUNCATED AT 400 WORDS)     

Adenine nucleotide metabolism in relation to purine enzymes in liver, erythrocytes and cultured fibroblasts.

To evaluate the regulation of adenine nucleotide metabolism in relation to purine enzyme activities in rat liver, human erythrocytes and cultured human skin fibroblasts, rapid and sensitive assays for the purine enzymes, adenosine deaminase (EC 2.5.4.4), adenosine kinase (EC 2.7.1.20), hyposanthine phosphoribosyltransferase (EC 2.4.28), adenine phosphoribosyltransferase (EC 2.4.2.7) and 5'-nucleotidase (EC 3.1.3.5) were standardized for these tissues. Adenosine deaminase was assayed by measuring the formation of product, inosine (plus traces of hypoxanthine), isolated chromatographically with 95% recovery of inosine. The other enzymes were assayed by isolating the labelled product or substrate nucleotides as lanthanum salts. Fibroblast enzymes were assayed using thin-layer chromatographic procedures because the high levels of 5'-nucleotidase present in this tissue interferred with the formation of LaCl3 salts. The lanthanum and the thin-layer chromatographic methods agreed within 10%. Liver cell sap had the highest activities of all purine enzymes except for 5'-nucleotidase and adenosine deaminase which were highest in fibroblasts. Erythrocytes had lowest activities of all except for hypoxanthine phosphoribosyltransferase which was intermediate between the liver and fibroblasts. Erhthrocytes were devoid of 5'-nucleotidase activity. Hepatic adenosine kinase activity was thought to control the rate of loss of adenine nucleotides in the tissue. Erythrocytes had excellent purine salvage capacity, but due to the relatively low activity of adenosine deaminase, deamination might be rate limiting in the formation of guanine nucleotides. Fibroblasts, with high levels of 5'-nucleotidase, have the potential to catabolize adenine nucleotides beyond the control od adenosine kinase. The purine salvage capacity in the three tissues was erythrocyte greater than liver greater than fibroblasts. Based on purine enzyme activities, erythrocytes offer a unique system to study adenine salvage; fibroblasts to study adenine degradation; and liver to study both salvage and degradation.     

Hepatic alcohol dehydrogenase activity in chick hepatocytes towards the major alcohols present in commercial alcoholic beverages: comparison with activities in rat and human liver

We have compared hepatic alcohol dehydrogenase activities in chick, rat and human liver with the major alcohols in commercial alcoholic beverages. 1. Chick and rat hepatic alcohol dehydrogenase was greater when assayed at a physiological pH in buffer containing chloride ions, as compared with the activity in pyrophosphate buffer at alkaline pH. 2. In contrast to reports of instability of ADH to freezing, we found the enzyme from all three species stable to freezing in 0.25 M sucrose. 3. Rat liver enzymatic activity was unstable in the presence of substrate, where as that of chick and human was not. 4. For all three species, the Km of hepatic ADH for substrate decreased with increasing chain length of alcohols. In both chick and human samples, the Vmax values for the higher chain alcohols were similar to that with ethanol, while in rat samples, ADH activity was dramatically lower with the higher chain alcohols compared to ethanol.     

Glycerol kinase activities in muscles from vertebrates and invertebrates

1. Glycerol kinase (EC 2.7.1.30) activity was measured in crude extracts of skeletal muscles by a radiochemical method. The properties of the enzyme from a number of different muscles are very similar to those of the enzyme from rat liver. Glycerol kinase from locust flight muscle was inhibited competitively by l-3-glycerophosphate with a K(i) of 4.0x10(-4)m. 2. The activity of glycerol kinase was measured in a variety of muscles from vertebrates and invertebrates in an attempt to explain the large variation in the activity of this enzyme in different muscles. 3. In vertebrates glycerol kinase activities were generally higher in red muscle than in white muscle; the highest activities (approx. 0.2mumole/min./g. fresh wt.) were found in the red breast muscle of some birds (e.g. pigeon, duck, blue tit) whereas the activities in the white breast muscle of the pheasant and domestic fowl were very low (approx. 0.02mumole/min./g.). 4. On the basis of glycerol kinase activities, muscles from insects can be classified into three groups: muscles that have a low enzyme activity, i.e. <0.3mumole/min./g. (leg muscles of all insects studied and the flight muscles of cockroaches and the tsetse fly); muscles that have an intermediate enzyme activity, i.e. 0.3-1.5mumoles/min./g. (e.g. locusts, cockchafers, moths, water-bugs); and muscles that have a high enzyme activity, i.e. >1.5mumoles/min./g. (e.g. bees, wasps, some blowflies). 5. The function of glycerol kinase in vertebrate and insect muscles that possess a low or intermediate activity is considered to be the removal of glycerol that is produced from lipolysis of triglyceride or diglyceride by the muscle. Therefore in these muscles the activity of glycerol kinase is related to the metabolism of fat, which is used to support sustained muscular activity. A possible regulatory role of glycerol kinase in the initiation of triglyceride or diglyceride lipolysis is discussed. 6. The function of glycerol kinase in the insect muscles that possess a high activity of the enzyme is considered to be related to the high rates of glycolysis that these muscles can perform. The oxidation of extramitochondrial NADH, and therefore the maintenance of glycolysis, is dependent on the functioning of the glycerophosphate cycle; if at any stage of flight (e.g. at the start) the rate of mitochondrial oxidation of l-3-glycerophosphate was less than the activity of the extramitochondrial glycerophosphate dehydrogenase, this compound would accumulate, inhibit the latter enzyme and inhibit glycolysis. It is suggested that such excessive accumulation of l-3-glycerophosphate is prevented by hydrolysis of this compound to glycerol; the latter would have to be removed from the muscle when the accumulation of l-3-glycerophosphate had stopped, and this would explain the presence of glycerol kinase in these muscles and its inhibition by l-3-glycerophosphate.     

Enzymes of glycerol and glyceraldehyde metabolism in mouse liver: effects of caloric restriction and age on activities

The influence of caloric restriction on hepatic glyceraldehyde- and glycerol-metabolizing enzyme activities of young and old mice were studied. Glycerol kinase and cytoplasmic glycerol-3-phosphate dehydrogenase activities were increased in both young and old CR (calorie-restricted) mice when compared with controls, whereas triokinase increased only in old CR mice. Aldehyde dehydrogenase and aldehyde reductase activities in both young and old CR mice were unchanged by caloric restriction. Mitochondrial glycerol-3-phosphate dehydrogenase showed a trend towards an increased activity in old CR mice, whereas a trend towards a decreased activity in alcohol dehydrogenase was observed in both young and old CR mice. Serum glycerol levels decreased in young and old CR mice. Therefore increases in glycerol kinase and glycerol-3-phosphate dehydrogenase were associated with a decrease in fasting blood glycerol levels in CR animals. A prominent role for triokinase in glyceraldehyde metabolism with CR was also observed. The results indicate that long-term caloric restriction induces sustained increases in the capacity for gluconeogenesis from glycerol.     

Changes during Development in the Activities and Intracellular Locations of Glycerol Kinase and Hexokinase in Rat Brain and Liver

The changes in activities and intracellular locations of glycerol kinase in rat brain and liver during development were compared. Glycerol kinase activity was consistently much higher in the liver than in the brain from just before the birth to the adult stage. Most of the activity was bound to mitochondria in the brain, but the intracellular distribution of the activity in the liver changed during development, the amount of activity bound to mitochondria being high just after birth and then decreasing gradually. These changes of glycerol kinase during development were compared with those of hexokinase, and the significance of the changes in the two enzymes is discussed in relation to dietary changes during development.     

Effect of hydrazine exposure on hepatic triacylglycerol biosynthesis.

Acute hydrazine exposure elevated rat liver triacylglycerol content and produced a rapid rise in triacylglycerol production from sn-[1,3-14C]glycerol 3-phosphate by liver homogenate and microsomal fractions. Hydrazine treatment also increased the incorporation of [1,3-14C]glycerol into hepatic triacylglycerol by the intact animal. Homogenates of hepatocyte monolayers exposed to hydrazine in vitro also exhibited an increased capacity to form triacylglycerol from sn-[1,3-14C]glycerol 3-phosphate. Hydrazine-dependent increases in hepatic triacylglycerol production measured in vitro correlated well with an increase in microsomal phosphatidate phosphohydrolase (EC 3.1.3.4) activity. Therefore, the fatty liver associated with hydrazine exposure may be explained in part by a rise in the enzymatic capacity of hepatic triacylglycerol biosynthesis.