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Dehydroascorbic acid is generated in plants and animal cells by oxidation of ascorbic acid. The reaction is believed to occur by the one-electron oxidation of ascorbic acid to semidehydroascorbate radical followed by disproportionation to dehydroascorbic acid and ascorbic acid. Semidehydroascorbic acid may recycle to ascorbic acid catalyzed by membrane-bound NADH-semidehydroscorbate reductase. However, disproportionation of the free radical occurs at a rapid rate, 10(5) M-1 s-1, accounting for measurable cellular levels of dehydroascorbate. Dehydroascorbate reductase, studied earlier and more extensively in plants, is now recognized as the intrinsic activity of thioltransferases (glutaredoxins) and protein disulfide isomerase in animal cells. These enzymes catalyze the glutathione-dependent two-electron regeneration of ascorbic acid. The importance of the latter route of ascorbic acid renewal was seen in studies of GSH-deficient rodents (Meister, A. (1992) Biochem. Pharmacol. 44, 1905-1915). GSH deficiency in newborn animals resulted in decreased tissue ascorbic acid and increased dehydroascorbate-to-ascorbate ratios. Administration of ascorbic acid daily to GSH-deficient animals decreased animal mortality and cell damage from oxygen stress. A cellular role is proposed for dehydroascorbate in the oxidation of nascent protein dithiols to disulfides catalyzed in the endoplasmic reticulum compartment by protein disulfide isomerase.

Glycerol-3-phosphate dehydrogenase from pig brain mitochondria was stimulated 2.2-fold by the addition of 50 microm l-ascorbic acid. Enzyme activity, dependent upon the presence of l-ascorbic acid, was inhibited by lauryl gallate, propyl gallate, protocatechuic acid ethyl ester, and salicylhydroxamic acid. Homogeneous pig brain mitochondrial glycerol-3-phosphate dehydrogenase was activated by either 150 microm L-ascorbic acid (56%) or 300 microm iron (Fe(2+) or Fe(3+) (62%)) and 2.6-fold by the addition of both L-ascorbic acid and iron. The addition of L-ascorbic acid and iron resulted in a significant increase of k(cat) from 21.1 to 64.1 s(-1), without significantly increasing the K(m) of L-glycerol-3-phosphate (10.0-14.5 mm). The activation of pure glycerol-3-phosphate dehydrogenase by either L-ascorbic acid or iron or its combination could be totally inhibited by 200 microm propyl gallate. The metabolism of [5-(3)H]glucose and the glucose-stimulated insulin secretion from rat insulinoma cells, INS-1, were effectively inhibited by 500 microm or 1 mm propyl gallate and to a lesser extent by 5 mm aminooxyacetate, a potent malate-aspartate shuttle inhibitor. The combined data support the conclusion that l-ascorbic acid is a physiological activator of mitochondrial glycerol-3-phosphate dehydrogenase, that the enzyme is potently inhibited by agents that specifically inhibit certain classes of di-iron metalloenzymes, and that the enzyme is chiefly responsible for the proximal signal events in INS-1 cell glucose-stimulated insulin release.

Rat liver mitochondria were examined for their ability to reduce dehydroascorbic acid to ascorbic acid in an alpha-lipoic acid dependent or independent manner. The alpha-lipoic acid dependent reduction was stimulated by factors that increased the NADH dependent reduction of alpha-lipoic acid to dihydrolipoic acid in coupled reactions. Optimal conditions for dehydroascorbic acid reduction to ascorbic acid were achieved in the presence of pyruvate, alpha-lipoic acid, and ATP. Electron transport inhibitors, rotenone and antimycin A, further enhanced the dehydroascorbic acid reduction. The reactions were strongly inhibited by 1 mM iodoacetamide or sodium arsenite. Mitoplasts were qualitatively similar to intact mitochondria in dehydroascorbate reduction activity. Pyruvate dehydrogenase and alpha-ketoglutarate dehydrogenase reduced dehydroascorbic acid to ascorbic acid in an alpha-lipoic acid, coenzyme A, and pyruvate or alpha-ketoglutarate dependent fashion. Dehydroascorbic acid was also catalytically reduced to ascorbic acid by purified lipoamide dehydrogenase in an alpha-lipoic acid (K0.5 = 1.4 +/- 0.8 mM) and lipoamide (K0.5 = 0.9 +/- 0.3 mM) dependent manner.

A family of small molecular weight proteins with thiol-disulfide exchange activity have been discovered, widely distributed from E. coli to mammalian systems, called thioltransferases or glutaredoxins. There are no substantiated reports of thioltransferases-glutaredoxins in plants; however, partially purified dehydroascorbate reductase from peas had thiol-disulfide exchange catalytic activity using glutathione as reductant and S-sulfocysteine as thiosulfate cosubstrate (unpublished data). Thus, this class of proteins is universally distributed. Based on mutagenesis studies, a sequence of Cys-Pro-Tyr(Phe)-Cys- followed by Arg-Lys- or Lys alone is critical for both the thiol-disulfide exchange reaction and the dehydroascorbate reductase activity. The dithiol-disulfide loop represented by this structure is unique since the cystine closer to the N-terminus has a highly acidic thiol pKa (3.8 as determined for the pig liver enzyme) that contributes to the protein's high S- nucleophilicity. Compared with the microbial enzyme, the mammalian thioltransferases (glutaredoxins) are extended at both N and C termini by 10-12 amino acid residues, including a second pair of cysteines toward the C-terminus with no known special function. Yeast thioltransferase is more like mammalian enzymes in length (106 amino acids) but more like E. coli glutaredoxin in being unblocked at the N-terminus and having only one set of cysteines; that is, at the active center. The three mammalian enzymes, for which sequences are available, are blocked at the N-terminus by an acetyl group linked to alanine with no known special function other than possibly to impart greater cellular turnover stability. A report of carbohydrate (8.6%) content in rat liver thioltransferase has not been verified by more sensitive methods of carbohydrate analysis, nor has carbohydrate been identified in samples of purified glutaredoxin from any source. Thiol transferase and glutaredoxin are two names for the same protein based on similarity of amino acid sequence, immunochemical cross-reactivity, and other enzyme properties. The inability of thioltransferase from some mammalian sources to act as an electron carrier in ribonucleotide reductase systems, whether homologous or heterologous in origin, remains to be explained in future studies.

By using site-directed mutagenesis techniques, the essential amino acids at the catalytic center of porcine thioltransferase (glutaredoxin) were determined. Seven oligonucleotides were designed, synthesized, and used to construct mutants, ETT-C22S, ETT-C25S, ETT-C25A, ETT-R26V, ETT-K27Q, ETT-R26V: K27Q, and ETT-C78S:C82S, by altering their codons in pig liver thioltransferase cDNA/M13mp18 clones. Each of the thioltransferases was purified to homogeneity and its dithiol-disulfide exchange, and dehydroascorbate reductase activities were compared with those of the wild-type (ETT). Evidence was obtained that Cys22 was essential for catalytic activity, and the extremely low pKa value of its sulfhydryl group was facilitated primarily by Arg26. The role of Lys27 at the active center was different from that of Arg26 and may be important in stabilizing the E.S intermediate by electrostatic forces. The second pair of cysteines, Cys78 and Cys82, nearer the C terminus, were not directly involved in the active center, but may play a role in defining the native protein structure. The replacement of the original Cys with a Ser at position 25 increased rather than decreased the enzyme activity, suggesting that the proposed intramolecular disulfide bond between Cys22 and Cys25 is not necessary for the catalytic mechanism of the Ser25 mutant, but does not rule out such a mechanism for the wild-type enzyme.

Homogeneous native and recombinant porcine liver thioltransferase (glutaredoxin), bovine thymus and human placenta thioltransferase (glutaredoxin) were examined for dehydroascorbate reductase activity (EC 1.8.5.1) involving the direct catalytic reduction of dehydroascorbic acid (DHA) by glutathione. Each enzyme had substantial activity with apparent Km and Vmax for dehydroascorbate between 0.2 and 2.2 mM and 6-27 nmol min-1, respectively, and for gluathione between 1.6 and 8.7 mM and 11-30 nmol min-1, respectively. In the presence of purified bovine liver thioredoxin reductase, homogeneous bovine liver thioredoxin failed to reduce DHA to ascorbic acid as measured by NADPH oxidation. Highly purified bovine liver protein disulfide isomerase (PDI) reacted directly with DHA and GSH to catalyze the reduction of DHA to ascorbic acid. The apparent Km for DHA was 1.0 mM and the Vmax was 8 nmol min-1, and for GSH were 3.9 mM and 14 nmol min-1, respectively. These results suggest that thioltransferase and PDI contribute to the regeneration of oxidized ascorbic acid in mammalian cells, and based on their cellular location, thioltransferase is proposed to be the major cytoplasmic activity, whereas interaction of DHA with microsomal membrane PDI may catalyze regeneration of ascorbic acid and initiate oxidation of intralumenal protein thiols to disulfides.

We report the first high-level expression of a mammalian thioltransferase (glutaredoxin) in Escherichia coli. A NcoI site (CCATGG) was introduced into the cDNA encoding pig liver thioltransferase (glutaredoxin) by site-directed mutagenesis, in which the first G of the original sequence, GCATGG, was replaced by a C. The altered cDNA was cloned into an expression vector, plasmid pKK233-2, between the unique NcoI and HindIII sites and expressed in E. coli JM105 at a high level (8% of total soluble protein) after 6 h of isopropyl-beta-D-thiogalactopyranoside induction. The soluble and unfused product was measured by the thiol-transferase thiol-disulfide exchange assay and immunoblotting analysis. The recombinant enzyme was purified to a single band as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing. The amino acid composition of the expressed enzyme agreed with that of the known sequence of pig liver thioltransferase (glutaredoxin). N-terminal sequence analysis revealed that unlike the native pig liver protein which is N-acetylated, the recombinant enzyme was unblocked at the N terminus (alanine). Various kinetic properties of the recombinant enzyme with regard to the exchange reaction were identical with those of the native enzyme.

Phosphate uptake across the plasma membrane of rat hepatocytes was characterized in primary cultures of rat hepatocytes. Phosphate uptake by cultured rat hepatocytes consisted of a Na(+)-dependent and Na(+)-independent component. A major portion of the uptake was Na(+)-dependent, which was saturable and exhibited Michaelis-Menten kinetics. Kinetic analysis showed that the apparent Km value for phosphate at 137 mM Na+ was 0.25 mM and Vmax was 13.3 pmol/microgram protein for 30 min. Hill coefficient was 1.76. This study is the first to demonstrate the presence of a phosphate carrier on the plasma membrane of hepatocytes.

Nuclear envelopes were prepared from purified rat liver nuclei by lysis with heparin, digestion with deoxyribonuclease I (DNase I), or sonication. The envelopes were fractionated by centrifugation on sucrose density gradients and analyzed for protein kinase activity using endogenous and exogenous protein substrates and [gamma-32 P]ATP. The protein kinase activity toward endogenous proteins was markedly affected by the method used to isolate the envelopes, with sonication producing a preparation with very low activity. At least 12 phosphoproteins in nuclear envelopes isolated by the heparin or DNase I method were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. A 32P-labeled material migrating with an apparent Mr = 3000 was extracted with chloroform:methanol:HCl and was identified as a mixture of phospholipids. Total 32P incorporation into nuclear envelopes peaked at 5 min of incubation, followed by a decrease in labeled products. This decrease was due to both phosphoprotein phosphatase activity and degradation of the lipid products. The highest protein kinase activity toward endogenous proteins was expressed with [gamma-32P]ATP in the presence of MgCl2; however, some phosphorylation also occurred with MnCl2, CoCl2, NiCl2, and [gamma-32P]GTP in the presence of MgCl2. Nuclear envelope protein phosphorylation was unaffected by cyclic nucleotides and calmodulin, slightly inhibited by CaCl2, MnCl2, CoCl2, disulfides, and sulfhydryl alkylating agents, and strongly inhibited by LaCl3 and phosphatidylglycerol. Nuclear porelamina complexes isolated from phosphorylated envelopes contained phosphoproteins of 7, 20, 51, 59, and 70 kDa. Incubation of pore-lamina complexes isolated from unlabeled envelopes with [gamma-32P]ATP resulted in 32P incorporation into the 20-, 51-, and 50-kDa proteins.

Membranes prepared from highly purified rat liver lysosomes contain endogenous protein-phosphorylation activities. The transfer of phosphate to membrane fractions from [gamma-32P]ATP was analyzed by gel electrophoresis under acidic denaturing conditions. Two phosphopeptides were detected, with molecular weights of 3,000 and 14,000. Phosphorylation of these proteins was unaffected by the addition of cAMP, cGMP, or the heat-stable inhibitor of cAMP-dependent protein kinase. No additional phosphorylation was observed when cAMP-dependent protein kinase was included in the reaction or when exogenous protein kinase substrates were added. The 14,000-dalton 32P-labeled product was formed rapidly in the presence of low concentrations (250 microM) of either Ca2+ or Mg2+. This product was labile under both acidic and alkaline conditions, suggesting that this protein contains an acyl phosphate, present presumably as a catalytic intermediate in a phosphotransferase reaction. The lower molecular weight species required a high concentration (5 mM) of Mg2+ for phosphorylation, and micromolar concentrations of Ca2+ stimulated the Mg2+-dependent activity. The addition of Ca2+ and calmodulin stimulated the phosphorylation reaction to a greater extent than with Ca2+ alone. This activity was strongly inhibited by 0.2 mM LaCl3 and to a lesser extent by 50 microM chlorpromazine or trifluoperazine. These results suggest that the 3000-dalton peptide may be phosphorylated by a Ca2+, calmodulin-dependent kinase associated with the lysosomal membrane.

The effect of insulin on 32Pi incorporation into phospholipids in various subcellular sites of isolated rat hepatocytes was investigated. After labeling the phospholipids of hepatocytes from rats previously starved for 24 h with 32Pi (10 mu Ci/10(6) cells) for 90 min, either saline or insulin (32 nM) was added. Following incubations of 1, 5, and 30 min, chilled cells were rapidly washed, homogenized in the presence of inhibitors of phospholipid degradation, and fractionated into the major subcellular organelles. Phospholipids were extracted from plasma membranes, microsomes, lysosomes, mitochondria, and nuclei with acidic chloroform:methanol. The aqueous deacylation products were separated by anion exchange high performance liquid chromatography, and the 32Pi incorporated into all the major diacylglycerophospholipids was determined. In parallel experiments, the specific radioactivity of 32Pi and [gamma-32P]ATP was determined. The results revealed that insulin had no effect on the turnover of the major phospholipids, including the polyphosphoinositides, of all subcellular compartments analyzed relative to the control. In addition, there were no significant differences in the amount and 32P labeling of cellular orthophosphate between saline- and insulin-treated cells. The specific radioactivity of [gamma-32P]ATP was increased by 20% after 30-min treatment with insulin, requiring appropriate correction of 32P-labeled phosphatidic acid, phosphatidylinositol 4-phosphate, and phosphatidylinositol 4,5-bisphosphate for estimation of mass changes at near steady-state labeling of cellular ATP.

The inhibitory effect of galactose on phagocyte function was investigated in normal and hypergalactosemic chicks by monitoring the in vitro killing of Escherichia coli by leukocytes and the in vivo clearance of colloidal 125I-labeled bovine serum albumin ([125I]BSA) from the circulation. Elevated levels of galactose (30 mM) significantly impaired the bactericidal activities of leukocytes from both control and hypergalactosemic chicks. However, the latter cells were more susceptible to the galactose-dependent inhibition. Leukocytes from hypergalactosemic chicks displayed near-normal bactericidal activity when assayed in vitro under simulated normal conditions in the absence of galactose. Mean corrected phagocytic indexes, obtained from data on the clearance of colloidal [125I]BSA, were calculated to be 0.358 and 0.299 for control and hypergalactosemic chicks, respectively. Moreover, increased concentrations of galactose significantly impaired the bactericidal activity of circulating leukocytes but did not significantly affect the phagocytic activity of the reticuoendothelial system.

To determine whether the deleterious action of D-galactose upon phagocyte function could be related to inhibition of glucose uptake, the properties of glucose transport were investigated by following the incorporation of [G-3H]2-deoxyglucose into human and guinea pig polymorphonuclear leukocytes (PMN). Uptake of [G-3H]2-deoxyglucose by guinea pig PMN proceeded in vitro with a Km of 1.8 mM and Vmax of 0.67 nmol/min per 10(6) cells. This system was competitively inhibited by glucose and mannose but was not significantly affected by galactose, fructose, or 3-0-methylglucose. Maximal uptake of 2-deoxyglucose occurred at 41 degrees C, and phosphorylation was necessary for its intracellular concentration. Transport of 2-deoxyglucose, although not altered by uncouplers of oxidative phosphorylation, was sensitive to inhibitors of glycolysis. Preincubation of cells with 2 mM iodoacetate for 30 min significantly reduced the uptake of 2-deoxyglucose and the intracellular levels of adenosine-5'-triphosphate without decreasing cell viability. These results indicated that uptake of 2-deoxyglucose in guinea pig PMN occurred by facilitated diffusion with subsequent phosphorylation. Similar results were obtained with PMN isolated from human peripheral blood.

Microtubule protein prepared by cycles of assembly-disassembly contains a nucleoside diphosphate kinase (NDP kinase) activity. We have isolated the NDP kinase responsible for this activity from twice-polymerized bovine brain microtubule protein by a five-step chromatographic procedure. The molecular weight of this enzyme was 103,000 +/- 7,000 daltons as determined by sedimentation equilibrium experiments performed with a Beckman Airfuge. A doublet of subunit bands with molecular masses of about 18,000 daltons was detected by silver staining after gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis of this preparation. We conclude that the enzyme is a hexamer, although we cannot identify the mix of subunits. We were able to isolate only nanogram quantities of this enzyme, too little for extensive studies, so we isolated the enzyme directly from bovine brain without a preliminary microtubule protein isolation. The whole-brain NDP kinase was isolated by the same chromatographic steps as the enzyme from microtubule protein preparations. Both enzymes had a doublet of subunits at the same molecular weights and both were the same isozyme, chromatofocusing at a pH of 8.0. Both enzymes had similar kinetic properties and similar thermal inactivation profiles. These similar properties of the two enzymes suggest that they are identical. Both subunits of NDP kinase could be reversibly phosphorylated by ATP. Phosphorylation of the native enzyme created multiple, more acidic forms that retained activity. The isolation of this NDP kinase, which can copurify with microtubule protein through cycles of assembly-disassembly, will facilitate future studies on the role of this enzyme in the mechanism and regulation of microtubule assembly.

Polyclonal antibodies against pig liver thioltransferase were raised in a New Zealand rabbit. These antibodies completely neutralized the thioltransferase activity of the homogeneous enzyme and that in the crude cytosolic homogenate at an equivalent titer. The antibodies also cross-reacted equally with calf thymus glutaredoxin and calf liver thioltransferase, but not with Escherichia coli thioredoxin, suggesting that thioltransferase and glutaredoxin from the same species are identical. Immunoblotting analysis of the cytosolic proteins from 14 different pig tissues revealed that most pig tissues contain a 12-kDa protein which reacts with these antibodies. This protein is found in greater abundance in stomach, small intestine, liver, skeletal muscle, kidney, heart, lung, and cerebral cortex, whereas retina, cerebellum, spleen, pancreas, and thymus have low levels of the protein. No reactive protein was detected in the lens. The tissue distribution of the protein was also determined by assay of the enzyme activity and was generally in good agreement with that obtained from the immunoblotting survey. Pig liver thioltransferase was cleaved by trypsin, chymotrypsin, Staphylococcus aureus V8 protease, and cyanogen bromide. The selected peptides purified by reversed phase high performance liquid chromatography or ion exchange fast protein liquid chromatography were subjected to reaction with the polyclonal antibodies against pig liver thioltransferase. Four antigenically reactive fragments were detected by dot-blotting analysis. These peptides are located in the first 30-amino acid residues from the NH2 terminus and the sequence from amino acid residues 39-67, indicating that the active site of the enzyme, Cys22 and Cys25, is located on one of the antigenic determinant domains.

The intracellular site of vasopressin-induced phosphoinositide breakdown in rat hepatocytes was investigated. After 45 s of vasopressin treatment of hepatocytes prelabeled with 32Pi, the levels of 32P-labeled phosphatidylinositol 4-phosphate (PI-P) and phosphatidylinositol 4,5-bisphosphate (PI-P2) in the plasma membrane decreased by approximately 40%, then gradually returned to near control levels after 10 min of treatment. Only small changes in the levels of [32P] PI-P and [32P]PI-P2 were observed in the other subcellular fractions, and were attributed to contamination of these fractions by plasma membranes. The level of 32P-labeled phosphatidylinositol in the plasma membrane decreased by 15% after 45 s of vasopressin treatment and then increased above control levels at later times while 32P-labeled phosphatidic acid levels in the plasma membrane gradually increased to 2-fold greater than control after 5 min of treatment. Using 32P-labeled plasma membranes obtained from prelabeled hepatocytes, it was found that PI-P and PI-P2 were rapidly degraded by a calcium-dependent polyphosphoinositide-specific phosphodiesterase. The enzyme was activated by physiological concentrations (200 nM) of free calcium when assayed at low ionic strength, but the calcium requirement shifted to micromolar concentrations under isosmotic, intracellular-like, ionic conditions. Addition of vasopressin (200 nM) to the 32P-labeled plasma membranes stimulated the breakdown of 20% of the [32P]PI-P2 present in the plasma membranes in 1 min when assayed under isosmotic conditions in the presence of 2 nM MgCl2 and approximately 200 nM free calcium. This suggests that the phosphoinositide-specific phosphodiesterase is not active under normal cellular conditions, but is activated upon the addition of vasopressin to the intact cell.

To determine the relative contributions of glucose, insulin, dexamethasone, and triiodothyronine to the induction of hepatic glucose-6-phosphate dehydrogenase, hepatocytes isolated from normal or adrenalectomized rats, either fasted or fed, were examined in culture. Addition of insulin (42 milliunits/ml, 0.9 microM) and dexamethasone (1 microM) to hepatocytes obtained from 3-day-fasted rats and cultured for 48 h in serum-free Dulbecco's medium resulted in a 7- to 11-fold increase in Glc-6-P dehydrogenase specific activity compared with a 2- to 3-fold increase in activity in control cultures incubated without added hormones. The effects of insulin and dexamethasone were independent of DNA synthesis, dose-dependent, and additive; each contributing about one-half of the total response. Medium glucose was neither sufficient nor necessary for the insulin- or dexamethasone-stimulated increase in Glc-6-P dehydrogenase specific activity. Addition of triiodothyronine (10 microM) preferentially blocked the dexamethasone-stimulated increase in Glc-6-P dehydrogenase specific activity. Insulin failed to stimulate the induction of Glc-6-P dehydrogenase in hepatocytes obtained from normal fed rats or from fasted and fed adrenalectomized rats. However, insulin caused a significant increase in the Glc-6-P dehydrogenase specific activity of these cells when dexamethasone was concurrently added to the culture medium.

Percutaneous liver biopsies and blood samples were obtained from 80 multiparous dairy cows in nine Michigan herds. Biopsies and samples were obtained serially over the peripartum period. Thirty-nine cows received 17 g of supplemental myoinositol in the diet to test its use as a possible lipotropic substance and 41 received a placebo. Liver biopsies were assayed for triglyceride (TG) and total myoinositol content. Serum was assayed for dextran precipitable cholesterol and non-esterified fatty acids (NEFA). Inositol supplementation had no effect on any of the lipid variables. There was a significant herd effect on liver inositol, serum dextran precipitable cholesterol and NEFA concentrations. Serum NEFA and liver TG concentrations increased in the immediate postpartum period, while dextran precipitable cholesterol decreased. A significant herd X period interaction existed for liver TG and serum dextran precipitable cholesterol concentrations. Liver TG and serum NEFA concentrations were positively correlated. Excessive infiltration of bovine liver with lipid at calving appears to be an exaggerated manifestation of normal metabolic changes.

Fatty liver syndrome or hepatic lipidosis (HL) is a condition thought to contribute to an increased incidence of peripartum disease, reduced response to therapy and decreased fertility in dairy cows. This syndrome is characterized by excess triglyceride (TG) accumulation in the liver and apparent decreased hepatic lipoprotein output. In lactating rats, a similar condition results from feeding an inositol-deficient diet. It is also characterized by excess hepatic TG accumulation and decreased hepatic lipoprotein output. Myo-inositol is a necessary component of the phospholipid phosphatidyl-inositol, which is an important membrane constituent. Myo-inositol occurs in feed mainly as the inositol hexaphosphate phytic acid. Phytic acid is undigestible by the monogastric but rumen phytases are assumed to adequately hydrolyze it. In early lactation dairy cows, lipid mobilization is intense, and the myo-inositol requirement may exceed the dietary supply or availability. Myo-inositol is being tested in a field trial as a potential lipotropic agent for dairy cows. Preliminary results suggest no lipotropic benefit from added myo-inositol.

A protein, previously designated thioltransferase (Askelof, P., Axelsson, K., Eriksson, S., and Mannervik, B. (1974) FEBS Lett. 38, 263-267) was purified to homogeneity as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and flatbed gel isoelectric focusing. The preparative procedure, a modification of that of Axelsson et al. (Axelsson, K., Eriksson, S., and Mannervik, B. (1978) Biochemistry 17, 2978-2984) and Hatakeyama et al. (Hatakeyama, M., Tanimoto, Y., and Mizoguchi, T. (1984) J. Biochem. (Tokyo) 95, 1811-1818) was faster and higher-yielding than the previous procedures. The purified enzyme has a molecular weight of 11,700 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and a pI of 8.8. The amino acid composition of thioltransferase is reported, and it closely resembles that of calf thymus glutaredoxin. The optimal pH for this enzyme was 8.5 when S-sulfocysteine was used as a substrate. The plots of the activity of thioltransferase as a function of S-sulfocysteine and 2-hydroxyethyl disulfide concentrations showed sigmoidal relationships. The K0.5 for S-sulfocysteine was 0.6 mM. The enzyme was very sensitive to sulfhydryl alkylating reagents. Preincubation of the enzyme with disulfide compounds prevented the enzyme from inactivation by iodoacetamide but inhibited the thioltransferase activity in the absence of iodoacetamide. The results suggest that the active center of thioltransferase is cysteine dependent and that substrates may form mixed disulfides with the enzyme. Based on the iodoacetamide inactivation and disulfide protection of thioltransferase activity, a model for the catalytic mechanism of the thiol-disulfide oxidoreduction is proposed.

SummaryMyotis lucifugus, simultaneously exhibits enlarged accessory sex glands and atrophied testis during hibernation. Evidence is presented for presence of an unidentified androgenic substance in the interscapular brown fat obtained from the bat during the hibernation season. The active substance isolated from the nonsaponifiable matter of one gram of brown fat elicits a growth response (rat seminal vesicle assay) equivalent to that produced by 676 μg of testosterone. The presence of an androgenic factor in brown fat suggests a unique role for this tissue in the sexual cycle of the bat.We wish to acknowledge the valuable assistance of Messrs. Charles R. Cook and Robert Kolek.

Summary1. Δ7-Cholestenol or its acetate injected into the blood stream of the rat disappeared rapidly, less than half remaining in blood after 2 hours. At this time 34% of the injected Δ7-cholestenol was found in the lungs, spleen, and liver; 26% remained in these organs after 24 hours. 2. A substantial portion of the Δ7-cholestenol remaining was in the ester form. Injected Δ7-cholestenol acetate disappeared without any corresponding accumulation of free sterol. 3. Administration of free Δ7-sterol resulted in transient increases in the cholesterol content of blood, liver, lungs, and spleen. No comparable increases followed the injection of Δ7-cholestenol acetate.

A protein, previously designated thioltransferase (Askelof, P., Axelsson, K., Eriksson, S., and Mannervik, B. (1974) FEBS Lett. 38, 263-267) was purified to homogeneity as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and flatbed gel isoelectric focusing. The preparative procedure, a modification of that of Axelsson et al. (Axelsson, K., Eriksson, S., and Mannervik, B. (1978) Biochemistry 17, 2978-2984) and Hatakeyama et al. (Hatakeyama, M., Tanimoto, Y., and Mizoguchi, T. (1984) J. Biochem. (Tokyo) 95, 1811-1818) was faster and higher-yielding than the previous procedures. The purified enzyme has a molecular weight of 11,700 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and a pI of 8.8. The amino acid composition of thioltransferase is reported, and it closely resembles that of calf thymus glutaredoxin. The optimal pH for this enzyme was 8.5 when S-sulfocysteine was used as a substrate. The plots of the activity of thioltransferase as a function of S-sulfocysteine and 2-hydroxyethyl disulfide concentrations showed sigmoidal relationships. The K0.5 for S-sulfocysteine was 0.6 mM. The enzyme was very sensitive to sulfhydryl alkylating reagents. Preincubation of the enzyme with disulfide compounds prevented the enzyme from inactivation by iodoacetamide but inhibited the thioltransferase activity in the absence of iodoacetamide. The results suggest that the active center of thioltransferase is cysteine dependent and that substrates may form mixed disulfides with the enzyme. Based on the iodoacetamide inactivation and disulfide protection of thioltransferase activity, a model for the catalytic mechanism of the thiol-disulfide oxidoreduction is proposed.

The active site cysteine of pig liver thioltransferase was identified as Cys22. The kinetics of the reaction between Cys22 of the reduced enzyme and iodoacetic acid as a function of pH revealed that the active site sulfhydryl group had a pKa of 2.5. Incubation of reduced enzyme with [1-14C]cysteine prevented the inactivation of the enzyme by iodoacetic acid at pH 6.5, and no stable protein-cysteine disulfide was found when the enzyme was separated from excess [1-14C]cysteine, suggesting an intramolecular disulfide formation. The results suggested a reaction mechanism for thioltransferase. The thiolated Cys22 first initiates a nucleophilic attack on a disulfide substrate, resulting in the formation of an unstable mixed disulfide between Cys22 and the substrate. Subsequently, the sulfhydryl group at Cys25 is deprotonated as a result of micro-environmental changes within the active site domain, releasing the mixed disulfide and forming an intramolecular disulfide bond. Reduced glutathione, the second substrate, reduces the intramolecular disulfide forming a transient mixed disulfide which is then further reduced by glutathione to regenerate the reduced enzyme and form oxidized glutathione. The rate-limiting step for a typical reaction between a disulfide and reduced glutathione is proposed to be the reduction of the intramolecular disulfide form of the enzyme by reduced glutathione.

Isolated rat hepatocytes were incubated with 32Pi for various times and then fractionated into plasma membranes, mitochondria, nuclei, lysosomes, and microsomes by differential centrifugation and Percoll density gradient centrifugation. The phospholipids were isolated and deacylated by mild alkaline treatment. The glycerophosphate esters were separated by anion exchange high pressure liquid chromatography and assayed for radioactivity. It was found that plasma membranes, mitochondria, nuclei, lysosomes, and microsomes displayed similar rates of 32P incorporation into the major phospholipids, phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylglycerol, and phosphatidic acid. This suggests that the phospholipids of these organelles are undergoing rapid turnover and replacement with newly synthesized phospholipids from the endoplasmic reticulum. However, the plasma membrane fraction incorporated 32P into phosphatidylinositol 4-phosphate (DPI) and phosphatidylinositol 4,5-bisphosphate (TPI) at rates 5-10 and 25-50 times, respectively, faster than any of the other subcellular fractions. Although the plasma membrane is the primary site of 32P incorporation into DPI and TPI, this study also demonstrates that significant incorporation of 32P into DPI occurs in other subcellular sites, especially lysosomes.

To determine the relative contributions of glucose, insulin, dexamethasone, and triiodothyronine to the induction of hepatic glucose-6-phosphate dehydrogenase, hepatocytes isolated from normal or adrenalectomized rats, either fasted or fed, were examined in culture. Addition of insulin (42 milliunits/ml, 0.9 microM) and dexamethasone (1 microM) to hepatocytes obtained from 3-day-fasted rats and cultured for 48 h in serum-free Dulbecco's medium resulted in a 7- to 11-fold increase in Glc-6-P dehydrogenase specific activity compared with a 2- to 3-fold increase in activity in control cultures incubated without added hormones. The effects of insulin and dexamethasone were independent of DNA synthesis, dose-dependent, and additive; each contributing about one-half of the total response. Medium glucose was neither sufficient nor necessary for the insulin- or dexamethasone-stimulated increase in Glc-6-P dehydrogenase specific activity. Addition of triiodothyronine (10 microM) preferentially blocked the dexamethasone-stimulated increase in Glc-6-P dehydrogenase specific activity. Insulin failed to stimulate the induction of Glc-6-P dehydrogenase in hepatocytes obtained from normal fed rats or from fasted and fed adrenalectomized rats. However, insulin caused a significant increase in the Glc-6-P dehydrogenase specific activity of these cells when dexamethasone was concurrently added to the culture medium.

Percutaneous liver biopsies and blood samples were obtained from 80 dairy cows in nine Michigan herds over the peripartum period. Thirty-nine cows were fed 17 g of supplemental inositol and 41 were fed a placebo. Liver biopsies were assayed for total myoinositol and triglyceride (TG) concentrations. Blood samples were assayed for serum dextran precipitable cholesterol, nonesterified fatty acids (NEFA), insulin, thyroxine (T4), free (FT4), triiodothyronine (T3) and free T3 (FT3) concentrations. Serum concentrations of insulin and the thyroid hormones decreased near parturition, with lowest concentrations occurring in the immediate postpartum period. Concentrations of T3 correlated well with T4, and the concentrations of free thyroid hormones reflected concentrations of total thyroid hormones. The percentage of hormone in the free fraction remained constant over time. Serum insulin, T3 and T4 were negatively correlated with serum NEFA and liver TG concentrations. Thyroid hormone concentrations were positively correlated with serum dextran precipitable cholesterol concentrations. Inositol supplementation was associated with reduced circulating T3 and FT3 concentrations, but not T4 and FT4 concentrations. Changes in hormone concentrations at parturition and their relationship to liver TG and serum NEFA concentrations were consistent with a metabolic adaptation by the dairy cow to the negative energy balance of early lactation.

SummarySubcutaneous injections of L-phenylalanine and DL-p-chlorophenylalanine in developing rats resulted in reduced body weight, brain weight, and concentration of 2′,3′-cyclic nucleotide 3′-phosphohydrolase—a myelin-enriched protein. These reductions were less marked when injections were reduced by half; when pups were allowed to recover after age 13 days, no differences were observed at 40 days of age. Brain hexokinase and cytochrome c oxidase development was not affected by the injection treatments.

Membranes prepared from highly purified rat liver lysosomes contain endogenous protein-phosphorylation activities. The transfer of phosphate to membrane fractions from [gamma-32P]ATP was analyzed by gel electrophoresis under acidic denaturing conditions. Two phosphopeptides were detected, with molecular weights of 3,000 and 14,000. Phosphorylation of these proteins was unaffected by the addition of cAMP, cGMP, or the heat-stable inhibitor of cAMP-dependent protein kinase. No additional phosphorylation was observed when cAMP-dependent protein kinase was included in the reaction or when exogenous protein kinase substrates were added. The 14,000-dalton 32P-labeled product was formed rapidly in the presence of low concentrations (250 microM) of either Ca2+ or Mg2+. This product was labile under both acidic and alkaline conditions, suggesting that this protein contains an acyl phosphate, present presumably as a catalytic intermediate in a phosphotransferase reaction. The lower molecular weight species required a high concentration (5 mM) of Mg2+ for phosphorylation, and micromolar concentrations of Ca2+ stimulated the Mg2+-dependent activity. The addition of Ca2+ and calmodulin stimulated the phosphorylation reaction to a greater extent than with Ca2+ alone. This activity was strongly inhibited by 0.2 mM LaCl3 and to a lesser extent by 50 microM chlorpromazine or trifluoperazine. These results suggest that the 3000-dalton peptide may be phosphorylated by a Ca2+, calmodulin-dependent kinase associated with the lysosomal membrane.

D-myo-Inositol 1,4,5-trisphosphate has been previously demonstrated to act as a second messenger for the hormonal mobilization of intracellular calcium in rat liver. In this study, the breakdown of D-myo-inositol 1,4,5-trisphosphate by a phosphatase activity was characterized. Using partially purified subcellular fractions, it was found that D-myo-inositol 1,4,5-trisphosphate phosphatase (I-P3ase) specific activity was highest in the plasma membrane fraction, while D-myo-inositol 1,4-bisphosphate phosphatase specific activity was highest in the cytosolic and microsomal fractions. The plasma membrane I-P3ase was Mg2+-dependent with optimal activity observed at 0.5-1.5 mM free Mg2+. The enzyme had a neutral pH optimum, suggesting that it was neither an acid nor alkaline phosphatase. Neither LiCl nor NaF inhibited the I-P3ase activity. However, both L-cysteine and dithiothreitol stimulated the activity 2-fold. Spermine (2.0 mM) inhibited the I-P3ase activity by 50%, while putrescine and spermidine had little or no effect.

The intracellular site of vasopressin-induced phosphoinositide breakdown in rat hepatocytes was investigated. After 45 s of vasopressin treatment of hepatocytes prelabeled with 32Pi, the levels of 32P-labeled phosphatidylinositol 4-phosphate (PI-P) and phosphatidylinositol 4,5-bisphosphate (PI-P2) in the plasma membrane decreased by approximately 40%, then gradually returned to near control levels after 10 min of treatment. Only small changes in the levels of [32P] PI-P and [32P]PI-P2 were observed in the other subcellular fractions, and were attributed to contamination of these fractions by plasma membranes. The level of 32P-labeled phosphatidylinositol in the plasma membrane decreased by 15% after 45 s of vasopressin treatment and then increased above control levels at later times while 32P-labeled phosphatidic acid levels in the plasma membrane gradually increased to 2-fold greater than control after 5 min of treatment. Using 32P-labeled plasma membranes obtained from prelabeled hepatocytes, it was found that PI-P and PI-P2 were rapidly degraded by a calcium-dependent polyphosphoinositide-specific phosphodiesterase. The enzyme was activated by physiological concentrations (200 nM) of free calcium when assayed at low ionic strength, but the calcium requirement shifted to micromolar concentrations under isosmotic, intracellular-like, ionic conditions. Addition of vasopressin (200 nM) to the 32P-labeled plasma membranes stimulated the breakdown of 20% of the [32P]PI-P2 present in the plasma membranes in 1 min when assayed under isosmotic conditions in the presence of 2 nM MgCl2 and approximately 200 nM free calcium. This suggests that the phosphoinositide-specific phosphodiesterase is not active under normal cellular conditions, but is activated upon the addition of vasopressin to the intact cell.

Treatment of isolated rat liver nuclear envelopes with 1% Triton X-100 solubilized 20-30% of the nuclear envelope protein and 85% of the ATPase activity. Chromatography on DEAE-Sepharose in 1% Triton X-100 at pH 7.5 removed all of the chemically measurable phospholipid from the bound ATPase activity; however, endogenously synthesized phosphatidylinositol [4-32P]phosphate (PIP) co-chromatographed with the ATPase activity on this column. Further purification by chromatography on heparin-agarose removed all of the [32P]PIP and RNA from the ATPase; however, the recovery of ATPase activity from this column was very low. RNA, polyadenylic acid, and polyguanylic acid stimulated the delipidated ATPase activity 4-6-fold. This activity was not further stimulated by the addition of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, or phosphatidic acid, but was stimulated 2-fold by the addition of phosphatidylinositol. Addition of 40 microM PIP and 50 micrograms of RNA/ml resulted in a 25-fold stimulation of basal ATPase activity. Phosphatidylinositol 4,5-bisphosphate, in the presence of RNA, was also able to stimulate ATPase activity but to a lower extent than that of PIP. Therefore, the nuclear envelope-associated ATPase appears to require interaction with a polynucleotide and PIP to express full activity. PIP, which is actively metabolized in the nuclear envelope, may therefore be involved in the regulation of the activity of this enzyme.

To account for enhanced susceptibility to infection among galactosemics, the acute effects of D-galactose on metabolic and functional activities of phagocytic cells in vitro were investigated. Human and guinea pig polymorphonuclear leukocytes (PMN) when incubated in medium containing 30 mM galactose displayed substantially less killing of Escherichia coli than when incubated in medium with 5 mM glucose. Impaired bactericidal activity was dependent upon galactose concentration but could be partially averted by supplementing the galactose-containing medium with 15 mM glucose. Phagocytic activities of guinea pig PMN and peritoneal macrophages were assayed by following ingestion of 32P-labeled E. coli and were also depressed by elevated galactose. Galactose was readily epimerized to glucose by resting PMN, and this conversion was stimulated by phagocytosis. Incubation of macrophages in the presence of galactose resulted in depletion of intracellular levels of adenosine 5' -triphosphate as well as other metabolities.

Nuclear envelopes were prepared from purified rat liver nuclei by lysis with heparin, digestion with deoxyribonuclease I (DNase I), or sonication. The envelopes were fractionated by centrifugation on sucrose density gradients and analyzed for protein kinase activity using endogenous and exogenous protein substrates and [gamma-32 P]ATP. The protein kinase activity toward endogenous proteins was markedly affected by the method used to isolate the envelopes, with sonication producing a preparation with very low activity. At least 12 phosphoproteins in nuclear envelopes isolated by the heparin or DNase I method were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. A 32P-labeled material migrating with an apparent Mr = 3000 was extracted with chloroform:methanol:HCl and was identified as a mixture of phospholipids. Total 32P incorporation into nuclear envelopes peaked at 5 min of incubation, followed by a decrease in labeled products. This decrease was due to both phosphoprotein phosphatase activity and degradation of the lipid products. The highest protein kinase activity toward endogenous proteins was expressed with [gamma-32P]ATP in the presence of MgCl2; however, some phosphorylation also occurred with MnCl2, CoCl2, NiCl2, and [gamma-32P]GTP in the presence of MgCl2. Nuclear envelope protein phosphorylation was unaffected by cyclic nucleotides and calmodulin, slightly inhibited by CaCl2, MnCl2, CoCl2, disulfides, and sulfhydryl alkylating agents, and strongly inhibited by LaCl3 and phosphatidylglycerol. Nuclear porelamina complexes isolated from phosphorylated envelopes contained phosphoproteins of 7, 20, 51, 59, and 70 kDa. Incubation of pore-lamina complexes isolated from unlabeled envelopes with [gamma-32P]ATP resulted in 32P incorporation into the 20-, 51-, and 50-kDa proteins.

A disaccharide was isolated from rat mammary tissue and determined to be 6-O-beta-galactopyranosyl myo-inositol (6-beta-galactinol) on the basis of combined gas-liquid chromatography-mass spectrometry of the permethylated and peracetylated derivatives as well as previously reported chemical and enzymatic evidence. 6-beta-Galactinol, also found in rat milk, increased during lactation, and on the 18th day represented approximately 17% of the total non-lipid neutral myo-inositol. The sugar was absent in all other rat tissues examined, suggesting a unique association with the process of lactation. This novel galactoside of one human subject on the basis of paper and gas-liquid chromatography.

Liver lysosomes from Triton-injected or normal rats were found to rapidly incorporate 32P from [gamma-32P]ATP into a lipid component of the membrane, in vitro. The lipid was identified as phosphatidylinositol 4-phosphate based on its chromatographic behavior on Silica Gel H thin layer plates as compared with standard phosphoinositides. The deacylation product, glyceryl-phosphorylinositol phosphate, was compared with standards in chromatographic and electrophoretic systems to further substantiate the identification of the radioactive material. A trace of phosphatidylinositol 4,5-bisphosphate was also found. The properties of the lysosomal membrane phosphatidylinositol kinase were examined using both endogenous lipid and exogenous phosphatidylinositol as substrate. The enzyme was active at neutral pH in the presence of 20 mM MgCl2. The addition of 0.4% Triton X-100 stimulated the enzyme activity toward endogenous substrate, and the highest activity was observed in the presence of detergent and 1 mM phosphatidylinositol. Degradation of the product was seen only in the presence of Triton X-100. The specific activity of the lysosomal phosphatidylinositol kinase is comparable to the detergent-stimulated activity of liver microsomes and plasma membrane, the previously recognized sources of this enzyme in the liver cell.

The complete amino acid sequence of pig liver thioltransferase has been determined. The homogeneous protein was cleaved by trypsin, chymotrypsin, Staphylococcus aureus V8 protease, and cyanogen bromide. The resulting peptides were purified by reversed-phase high performance liquid chromatography and ion-exchange fast protein liquid chromatography. Sequencing of the fragments was achieved with either automated Edman degradation or fast atom bombardment-mass spectrometry. Pig liver thioltransferase is a single polypeptide with 105 amino acid residues and an acetylated glutamine N terminus. The protein has 2 cysteine pairs with sequences of -Cys-Pro-Phe-Cys- and -Cys-Ile-Gly-Gly-Cys-, the first pair of which (Cys22 and Cys25) is located at the potential active site of the enzyme. The sequence of pig liver thioltransferase displays close homology (82%) with calf thymus glutaredoxin, suggesting that they belong to the same evolutionary family.