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Biomedical subjects

H A Lardy

Publications and source records attributed to H A Lardy.

At least 73 records · Page 4Linked to original sources
PubMed1984

Mechanism of action of 2,5-anhydro-D-mannitol in hepatocytes. Effects of phosphorylated metabolites on enzymes of carbohydrate metabolism.

Isolated rat hepatocytes convert 2,5-anhydromannitol to 2,5-anhydromannitol-1-P and 2,5-anhydromannitol-1,6-P2. Cellular concentrations of the monophosphate and bisphosphate are proportional to the concentration of 2,5-anhydromannitol and are decreased by gluconeogenic substrates but not by glucose. Rat liver phosphofructokinase-1 phosphorylates 2,5-anhydromannitol-1-P; the rate is less than that for fructose-6-P but is stimulated by fructose-2,6-P2. At 1 mM fructose-6-P, bisphosphate compounds activate rat liver phosphofructokinase-1 in the following order of effectiveness: fructose-2,6-P2 much greater than 2,5-anhydromannitol-1,6-P2 greater than fructose-1,6-P2 greater than 2,5-anhydroglucitol-1,6-P2. High concentrations of fructose-1,6-P2 or 2,5-anhydromannitol-1,6-P2 inhibit phosphofructokinase-1. Rat liver fructose 1,6-bisphosphatase is inhibited competitively by 2,5-anhydromannitol-1,6-P2 and noncompetitively by 2,5-anhydroglucitol-1,6-P2. The AMP inhibition of fructose 1,6-bisphosphatase is potentiated by 2,5-anhydroglucitol-1,6-P2 but not by 2,5-anhydromannitol-1,6-P2. Rat liver pyruvate kinase is stimulated by micromolar concentrations of 2,5-anhydromannitol-1,6-P2; the maximal activation is the same as for fructose-1,6-P2. 2,5-Anhydroglucitol-1,6-P2 is a weak activator. 2,5-Anhydromannitol-1-P stimulates pyruvate kinase more effectively than fructose-1-P. Effects of glucagon on pyruvate kinase are not altered by prior treatment of hepatocytes with 2,5-anhydromannitol. Pyruvate kinase from glucagon-treated hepatocytes has the same activity as the control pyruvate kinase at saturating concentrations of 2,5-anhydromannitol-1,6-P2 but has a decreased affinity for 2,5-anhydromannitol-1,6-P2 and is not stimulated by 2,5-anhydromannitol-1-P. The inhibition of gluconeogenesis and enhancement of glycolysis from gluconeogenic precursors in hepatocytes treated with 2,5-anhydromannitol can be explained by an inhibition of fructose 1,6-bisphosphatase, an activation of pyruvate kinase, and an abolition of the influence of phosphorylation on pyruvate kinase.

Animals↗
PubMed1984

Regulation of calcium content in bovine spermatozoa.

Plasma membrane vesicles isolated from bovine epididymal and ejaculated spermatozoa have widely different capabilities for transporting Ca2+. Spermatozoa were ruptured by nitrogen cavitation, and the plasma membrane fraction was harvested after low speed and sucrose gradient centrifugation; purity was assessed by marker enzyme analyses, electron microscopy, and sedimentation properties. Plasma membrane vesicles isolated from epididymal sperm accumulate Ca2+ passively at a faster rate and to a greater extent than vesicles prepared from ejaculated sperm. Ca2+ transport across bovine sperm plasma membranes is an ATP-independent, Na+-dependent process that obligatorily exchanges intravesicular Na+ for external Ca2+. The rate of Na+/Ca2+ exchange is significantly lower in ejaculated sperm vesicles than in those of epididymal sperm. Bovine plasma membranes contain little or no Ca2+-dependent ATPase activity. It is suggested that, at the time of ejaculation, calcium flux into bovine sperm is prevented by the interaction of the plasma membrane with putative factors in seminal fluid that specifically interfere with Na+/Ca2+ exchange. We have isolated a protein from seminal plasma that prevents calcium accumulation by bovine epididymal sperm (Rufo, G. A., Jr., Singh, J. P., Babcock, D. F., and Lardy, H. A. (1982) J. Biol. Chem. 257, 4627-4632). A protein with properties resembling those of the seminal calcium transport inhibitor is found on the membrane vesicles from ejaculated sperm but not on membranes from epididymal sperm. We conclude that this protein binds strongly to the plasma membrane of bovine sperm and is responsible for preventing calcium uptake by ejaculated sperm.

Animals↗
PubMed1984

The effect of nutritional state and hormones on the number of circulating red cells in Gallus domesticus.

When chickens are fasted, the liver becomes dark, whereas the spleen is bleached. Microscopic and spectrophotometric analysis indicated the colour resulted from red blood cells accumulating in enlarged sinusoids of the liver. In birds fasted for 48 hr the hematocrit increases 30%. Blood volume and red cell volume are not altered; the extra cells are not newly synthesised. The extra cells arise from the spleen, possibly under alpha-adrenergic control if given epinephrine by injection and under beta-adrenergic control during fasting as indicated by experiments with alpha and beta-receptor blocking agents.

Animal Nutritional Physiological Phenomena↗
PubMed1984

The influence of fasting on chicken liver metabolites, enzymes and mitochondrial respiration.

Chicken liver mitochondria were isolated in relatively pure form as indicated by electron microscopy and marker enzyme assay. The rate of respiration, respiratory control index and ADP/O ratios with several different substrates indicated that chicken liver mitochondria are more uncoupled than rat liver mitochondria. Chickens have ten-fold higher malate concentrations in liver than do rats, 2-oxoglutarate was also more abundant in chicken livers. Fasted birds had a five-fold increase in beta-hydroxybutyrate as compared with fed birds; whereas malate and lactate concentrations decreased. Fasted birds had increased levels of isocitrate dehydrogenase (NADP dependent) and lactate dehydrogenase in the cytosol, and increased malate dehydrogenase (NAD dependent), isocitrate dehydrogenase (NADP dependent) and malic enzyme activities in the mitochondria.

Animals↗
PubMed1983

Catecholamine stimulation of hepatic gluconeogenesis at the site between pyruvate and phosphoenolpyruvate.

Phosphoenolpyruvate carboxykinase has been implicated by Rognstad (Rognstad, R. (1979) J. Biol. Chem. 254, 1875-1878) as the rate-limiting step for gluconeogenesis from lactate on the basis of a linear Dixon plot (reciprocal rate of gluconeogenesis versus concentration of inhibitor, mercaptopicolinate). We have confirmed this result with isolated hepatocytes incubated in the absence, but not the presence, of bovine serum albumin. Nonlinear plots are likely the result of mercaptopicolinate binding to the albumin. Both norepinephrine and dibutyryl cyclic AMP decreased the slopes and intercepts of the Dixon plots, but a linear relationship was still obtained. When aminooxyacetate inhibited transaminase reactions sufficiently to depress gluconeogenesis, the resulting mercaptopicolinate inhibition plot was still linear in the presence or absence of norepinephrine. Thus, linearity in the Dixon plot does not assure that the enzyme at the site of inhibition is the rate-limiting step for a pathway. Flux through phosphoenolpyruvate carboxykinase does not appear to be hormonally regulated by changes in oxalacetate concentration since this compound was unchanged by norepinephrine or dibutyryl cyclic AMP. Ca2+ enhanced norepinephrine stimulation of gluconeogenesis from asparagine and glutamine and of ureogenesis from glutamine, indicating both mitochondrial and cytosolic sites of action for this hormone. The effects of catecholamines and cyclic AMP were most clearly distinguished by their influence on glutamate concentration when glutamine was the substrate. Dibutyryl cyclic AMP increased, but norepinephrine decreased glutamate. It is possible that decreased glutamate concentration is a reflection of a catecholamine-directed oxidation of mitochondrial NADPH.

Aminooxyacetic Acid↗
PubMed1983

The effect of substrate cycling on the ATP yield of sperm glycolysis.

The conversion of glucose to lactate via the Embden-Meyerhof pathway yields a maximum of 2 mol of ATP per mol of glucose degraded with lesser amounts of ATP obtained if metabolic intermediates are removed for biosynthesis or if substrate cycling occurs during carbohydrate degradation. Bull sperm are an ideal test system for a quantitative estimate of ATP yield because they contain enzymes necessary for potential substrate cycling and do not carry out significant biosynthesis. Experiments utilized specifically labeled glucose and fructose to test for substrate cycling between glucose in equilibrium glucose-6-PO4, fructose in equilibrium fructose-6-PO4, and fructose-6-PO4 in equilibrium bisphosphate. Flux through the pathway (low = less than or equal to 0.05; moderate = 0.1; high = greater than 0.5 mol of carbohydrate consumed per h/10(8) cells) was altered by changing incubation temperature and/or by adding metabolic effectors. The data (in situ rates of kinases and phosphatases and metabolite crossover plots) obtained under these conditions were used to establish if the generally accepted regulatory enzymes (hexokinase and phosphofructokinase) approach the overall flux through the glycolytic pathway, i.e. that of a classical kinetically limiting rate-determining step. In summary: (a) At low flux, the rates of phosphorylation greatly exceeded overall flux. This resulted in extensive substrate cycling at all points and a net ATP yield of less than or equal to 0 mol/mol of glucose initially phosphorylated. (b) At moderate flux, rate of hexokinase approached that of overall flux through the glycolytic pathway but "excessive" phosphofructokinase activity led to substrate cycling between fructose-6-PO4 and fructose 1,6-bisphosphate and resulted in a low net ATP yield (0-0.6 mol/mol of glucose). (c) At high flux, rates of phosphofructokinase and hexokinase approached that of the overall flux. Minimal substrate cycling occurred between fructose-6-PO4 and fructose 1,6-bisphosphate, and the net ATP yield approached 1.8 mol/mol of glucose. These experiments established that the theoretical stoichiometric ATP yield of the pathway was rarely achieved during these incubations because of extensive substrate cycling. Indeed, for cauda epididymal bull sperm isolated and incubated in vitro, the glycolytic pathway serves to generate lactate, an intermediate product that is further metabolized in the mitochondria to yield the ATP that is associated with degradation of exogenous carbohydrate.

Adenosine Triphosphate↗
PubMed1983

Regulation of gluconeogenesis by norepinephrine, vasopressin, and angiotensin II: a comparative study in the absence and presence of extracellular Ca2+1.

In hepatocytes isolated from fasted rats, vasopressin and angiotensin II stimulate the rate of gluconeogenesis from lactate or pyruvate in a Ca2+-dependent manner similar to that previously reported for norepinephrine. Actions of the peptide hormones on gluconeogenesis from glycerol or sorbitol, reduced substrates that require oxidation before they enter the gluconeogenic pathway at triosephosphate, also resemble those of norepinephrine. Stimulation of glucose production from these substrates is observed only in the presence of extracellular Ca2+. Actions of the peptide hormones on gluconeogenesis from dihydroxyacetone or fructose, the oxidized counterparts of glycerol and sorbitol, respectively, do not resemble those of norepinephrine. While norepinephrine enhances rates of glucose production from dihydroxyacetone or fructose in the absence of extracellular Ca2+, vasopressin and angiotensin II are ineffective either in the absence or presence of extracellular Ca2+. When the oxidation-reduction state in hepatocytes metabolizing dihydroxyacetone is altered by adding an equimolar concentration of ethanol (to provide cytosolic reducing equivalents), the results are similar to those obtained when cells are incubated with the reduced counterpart of dihydroxyacetone, glycerol, i.e., the peptide hormones cause an apparent increase in the rate of glucose production in a Ca2+-dependent manner. If, on the other hand, hepatocytes are incubated with glycerol or sorbitol and an equimolar concentration of pyruvate (to provide a cytosolic hydrogen acceptor), the peptide hormones, unlike norepinephrine, are ineffective in stimulating gluconeogenesis in the absence of extracellular Ca2+. These results indicate that whereas many of the actions of vasopressin and angiotensin II are similar to those of alpha 1-adrenergic agents, there are major differences in the manner in which the hormones act at various sites to regulate gluconeogenesis.

Angiotensin II↗
PubMed1983

Regulation of carbohydrate metabolism by 2,5-anhydro-D-mannitol.

In hepatocytes isolated from fasted rats, 2,5-anhydromannitol inhibits gluconeogenesis from lactate plus pyruvate and from substrates that enter the gluconeogenic pathway as triose phosphate. This fructose analog has no effect, however, on gluconeogenesis from xylitol, a substrate that enters the pathway primarily as fructose 6-phosphate. The sensitivity of gluconeogenesis to 2,5-anhydromannitol depends on the substrate metabolized; concentrations of 2,5-anhydromannitol required for 50% inhibition increase in the order lactate plus pyruvate less than dihydroxyacetone less than glycerol less than sorbitol less than fructose. The inhibition by 2,5-anhydromannitol of gluconeogenesis from dihydroxyacetone is accompanied by an increase in lactate formation and by two distinct crossovers in gluconeogenic-glycolytic metabolite patterns-i.e., increases in pyruvate concentrations with decreases in phosphoenolpyruvate and increases in fructose-1,6-bisphosphate concentrations with little change in fructose 6-phosphate. In addition, 2,5-anhydromannitol blocks the ability of glucagon to stimulate gluconeogenesis and inhibit lactate production from dihydroxyacetone. 2,5-Anhydromannitol decreases cellular fructose 2,6-bisphosphate content in hepatocytes; therefore the effects of the fructose analog are not mediated by fructose 2,6-bisphosphate, a naturally occurring allosteric regulator. 2,5-Anhydromannitol also inhibits gluconeogenesis in hepatocytes isolated from fasted diabetic rats, but higher concentrations of the analog are required.

Animals↗
PubMed1983

Potassium-dependent increases in cytosolic pH stimulate metabolism and motility of mammalian sperm.

Sperm cytosolic pH, determined by the spectral properties of intracellular carboxyfluorescein, is decreased rapidly by the diffusion and subsequent dissociation of the uncharged weak acids pyruvic, lactic, or hydroxybutyric and is increased by diffusion and subsequent intracellular protonation of the weak base NH3. Metabolic and kinetic activity increases dramatically when intracellular pH is elevated above 6.8-6.9 by addition of 50 mM NH4Cl to sperm suspended in a 120 mM NaCl medium. Respiratory stimulation is not observed upon comparable additions of 50 mM Li+ or K+ or when the pH of the medium is increased from 6.5 to 8.2. However, increases of the external pH to 7.8-8.2 in medium employing 120 mM KCl result in increased metabolic and kinetic activity, comparable to the maximal stimulation induced by the phosphodiesterase inhibitor caffeine. An increase in cytosolic pH from 6.3-6.6 to 6.8 occurs concomitant with the respiratory stimulation induced by KCl in alkaline media. No change in cytosolic pH follows addition of caffeine. Cyclic AMP-dependent protein kinase activity ratios, determined in cellular extracts, are increased by caffeine treatment but are not elevated by 120 mM KCl, by alkaline pH, or by their combination. These observations indicate that cytosolic pH plays a role in the regulation of motility and metabolism of mammalian sperm that is not mediated by cyclic AMP but that may be under control of a plasma membrane voltage-dependent proton channel. However, H+ fluxes across vesicles prepared from sperm membranes are unaffected by variation in the magnitude of the transvesicular K+ concentration gradient.

Ammonium Chloride↗
PubMed1982

The development and application of a radioimmunoassay for rat phosphoenolpyruvate carboxykinase.

We employed a newly developed radioimmunoassay to quantitate P-enolpyruvate carboxykinase protein directly in liver and kidney of intact rats. The radioimmunoassay was dependent upon the presence of sodium dodecyl sulfate in the competitive binding reaction mixture. In conjunction with assayable activity measurements, the radioimmunoassay results also made it possible to assess the average specific activity of the enzyme. In the fed state, liver P-enolpyruvate carboxykinase was 0.89 microM (micromoles/kg of tissue) and the kidney enzyme was 1.90 microM. Following a 48-h fast, the enzyme in liver increased to 2.83 microM and that in kidney to 6.83 microM. Although liver enzyme concentration increased 3-fold, total liver enzymic activity was increased only about 30%. The discrepancy was due to the decrease in liver weight which occurred during fasting. The kidneys, which do not lose weight during fasting, had a 65% elevation of total organ activity. Total organ enzyme activity was predominantly dependent on changes in enzyme mass. In the fed state, the kidney/liver enzyme mass ratio was 0.44, in the fasted state it was 0.68. Variation in enzyme concentration, enzyme half-life, and response to tryptophan and chronic triamcinolone administration all gave evidence for organ-specific regulation.

Animals↗
PubMed1982

Purification and properties of Oxidized and reduced forms of Hepatic fructose 1,6-bisphosphatase.

D-Fructose 1,6-bisphosphate 1-phosphohydrolase (EC 3.1.3.11) was isolated from rat liver in two forms: "A," isolated in the presence, and "B," isolated in the absence of dithiothreitol. Both forms had an apparently identical molecular weight of approximately 37,000/subunit and the same Km for fructose 1,6-bisphosphate of 2 microM. However, the Ki of the AMP inhibition of form A was 140 microM and of form B, 370 microM. With form B the same inhibition as with form A was reached by incubating the enzyme with dithiothreitol. The two forms of the enzyme differed in their total, as well as in their number of fast reacting thiol groups. Form A was the more reduced form, exhibiting 22.4 thiol groups/molecule, 2.5 of them fast reacting with 5,5'-dithiobis-(2-nitrobenzoic acid). Only 0.5 fast reacting groups and a total of 19.2 were found with form B. The fast reacting thiol groups disappeared when assayed in the presence of AMP. It is suggested that a redox reaction alters a site that influences the inhibitory action of AMP, so as to regulate the activity of fructose 1,6-bisphosphatase.

Adenosine Monophosphate↗
PubMed1982

Purification and characterization of a rat liver ferroactivator with catalase activity.

A rat liver protein with both phosphoenolpyruvate carboxykinase ferroactivator activity and catalase activity has been purified to near-homogeneity. The protein has a native molecular weight of 240,000 and is composed of four identical subunits containing ferriprotoporphyrin IX prosthetic groups. The visible spectrum has absorbance maxima at 403, 500, 530, and 620 nm; it is not reduced by dithionite. The spectrum, physical properties, and specific activity are almost identical with those of catalases from other sources, and the protein has been tentatively identified as rat liver catalase. The protein exhibited partial reactivity in double immunodiffusion plates to antiserum prepared against rat liver ferroactivator isolated by a previous method (Bentle, L. A., and Lardy, H. A. (1977) J. Biol. Chem. 252, 1431-1440) raising the possibility that the original ferroactivator and rat liver catalase are structurally related. Inactivation of catalase by 3-amino-1,2,4-triazole was accompanied by loss of ferroactivator activity as well. The apparent specific activity of ferroactivator, as well. The apparent specific activity of ferroactivator, whether heme-containing or not, can be increased between 2- and 100-fold by the inclusion of bovine serum albumin, HCO3-, or a combination of the two in the incubation.

Amino Acids↗
PubMed1982

Purification and characterization of a calcium transport inhibitor protein from bovine seminal plasma.

Previous work (Babcock, D. F., Singh, J. P., and Lardy, H. A. (1979) Dev. Biol. 69, 85-93) has shown that bovine seminal fluid contains a component(s) which is capable of preventing or delaying accumulation of extracellular calcium by ejaculated sperm. We report here the component is proteinaceous in nature and has been purified to apparent homogeneity. Conventional purification techniques were employed including ammonium sulfate fractionation, ion-exchange and gel permeation chromatography. The inhibitor protein is a single peptide of Mr = 9,600-10,500 as determined by gel permeation chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Isoelectric focusing in thin layer agarose gels indicates the protein has a pI = 8.3 which is consistent with a determined amino acid composition rich in basic residues. The protein shows no affinity for periodic acid-Schiff reagent and is therefore assumed to contain no carbohydrate. This protein can be distinguished from other previously characterized seminal plasma proteins and it is assumed that this protein is responsible for delaying uptake of calcium into ejaculated sperm by altering a component(s) of the sperm plasma membrane which serves to actively transport calcium into these cells.

Amino Acids↗
PubMed1982

Ca2+-mediated activation of phosphoenolpyruvate carboxykinase occurs via release of Fe2+ from rat liver mitochondria.

The addition of calcium chloride to rat liver homogenates resulted in activation of phosphoenolpyruvate carboxykinase by as much as 50%. The enhanced activity was inhibited by quinolinic acid; it was not additive with activation by FeCl2, and stimulation was prevented by 1,10-phenanthroline. Activation by calcium was lost when the particulate fractions of liver were removed, but an activating system could be reconstituted with isolated mitochondria, purified P-enolpyruvate carboxykinase, and purified ferroactivator. Iron-loaded mitochondria were more responsive to calcium than controls. A release of Fe2+ from washed mitochondria could be detected spectrophotometrically when 25-75 nmol of Ca/mg of protein were added to the mitochondrial suspension. If Ca2+ was buffered with ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, the threshold of Ca2+ necessary for release of Fe2+ was approximately 10(-7) M, with peak response between 5 X 10(-7) and 10(-6) M. Total Fe2+ detected was normally 20-30 pmol of Fe2+/mg of protein. The synthetic activator of P-enolpyruvate carboxykinase, 3-aminopicolinic acid, as well as other picolinic acid derivatives, is capable of withdrawing Fe2+ associated with the mitochondrial fraction; after incubation with mitochondria, 3-aminopicolinate will activate phosphoenolpyruvate carboxykinase in the absence of exogenous metal.

Animals↗
PubMed1982

Inhibition of phosphoenolpyruvate carboxykinase by streptonigrin.

Streptonigrin, an antibiotic with antineoplastic activity, inhibited rat liver phosphoenolpyruvate carboxykinase with an I50 of 0.3 microM when excess FeCl2 was present. No inhibition occurred in the absence of added metal ion. Inhibition was partial and noncompetitive versus ITP and oxalacetic acid. The enzyme was more susceptible to inhibition by streptonigrin in the absence of substrates. Fe2+ supported inhibition by streptonigrin to a greater extent than did Fe3+, while Mn2+ activated the enzyme in the presence of streptonigrin. For maximum inhibition, at least a 3-fold molar excess of iron over streptonigrin was required. The methyl ester of streptonigrin was also an inhibitor (I50 = 4 microM) while the fragment containing the C and D rings was not, indicating that inhibition did not depend solely on the presence of the picolinic acid moiety. When oxalacetate synthesis was measured, streptonigrin plus iron had no more effect on enzymatic activity than iron alone, and Mn2+ was capable of stimulating the streptonigrin-Fe2+ inhibited enzyme.

Animals↗