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H A Lardy

Publications and source records attributed to H A Lardy.

At least 109 records · Page 6Linked to original sources
PubMed1978

A vicinal dithiol containing an essential cysteine in phosphoenolpyruvate carboxykinase (guanosine triphosphate) from cytosol of rat liver.

The highly purified form of phosphoenolpyruvate carboxykinase (PEPCK) contained 13 thiols (all in the reduced state) per 72 000 daltons. Modification of the enzyme with equimolar 5,5'-dithiobis(2-nitrobenzoate) (Nbs2) caused rapid formation of a cystine disulfide bridge and an even more rapid loss of enzymatic activity. Formation of the cystine bridge proceeded about 25 times faster than formation of the analogous intramolecular disulfide of dithiothreitol induced by Nbs2. o-Iodosobenzoate, Cd2+, and the 2,3-dimercapto-1-propanol complex of arsenite were potent, time-dependent, irreversible inhibitors of PEPCK. The inactivation by arsenite-2,3-dimercapto-1-propanol and o-iodosobenzoate was first order with respect to both time and inhibitor concentration. The sum of these data indicates the existence in PEPCK of a critical cysteine that is in a vicinal dithiol grouping with a second cysteine. PEP protected against cystine bridge formation induced by equimolar Nbs2 but not against the extent of inactivation. In the presence of PEP, the modification by Nbs2 of one cysteine/mol of enzyme (k = 1.2 X 10(6) M-1 min-1 at pH 7.2) caused nearly complete inactivation. Replacing the bulky 5-thio-2-nitrobenzoate moiety with cyanide did not result in any reactivation. This critical, cyanylated cysteine was determined to be 44% of the distance from the amino terminus.

Animals↗
PubMed1978

Hyperglycaemic activity and metabolic effects of 3-aminopicolinic acid.

Administering 3-aminopicolinate to rats starved for 24h immediately initiated a progressive increase in blood glucose concentration. Hyperglycaemia was not the result of glycogenolysis, nor was it due to an inhibition of insulin release, since it caused marked hyperinsulinaemia. The rate of [6-(3)H]glucose disappearance from the blood of the intact rat was not altered by 3-aminopicolinate, indicating that it does not cause hyperglycaemia by inhibiting glucose utilization or by causing a redistribution of total body glucose. 3-Aminopicolinate increased the rate of fall in the specific radioactivity of blood [6-(3)H]-glucose, indicating dilution of the glucose pool by newly synthesized glucose. The rate of (14)C incorporation into blood glucose from [(14)C]alanine and [(14)C]lactate was increased 90 and 35% respectively, whereas that from [(14)C]glycerol and [(14)C]xylitol was either unaffected or slightly decreased by 3-aminopicolinate administration. Liver phosphoenolpyruvate of rats was increased to four to seven times the normal concentration 10min to 1h after injections of 50-300mg of 3-aminopicolinate/kg body wt. and the amounts of 2-phosphoglycerate and 3-phosphoglycerate were increased to three to four times normal. The high concentrations of liver phosphoenolpyruvate, 2-phosphoglycerate and 3-phosphoglycerate, as well as the enhancement of gluconeogenesis from lactate and alanine, but not from glycerol or xylitol, is compatible with an enhancement of gluconeogenesis at a step between pyruvate and the triose phosphates. After injections of 3-aminopicolinate, liver malate, citrate, aspartate, alanine, lactate and pyruvate were also increased, but to lesser extents than was phosphoenolpyruvate. The increases in some of these metabolites were approximated after an intravenous infusion of glucose, so their elevated concentration after 3-aminopicolinate administration could have been, in part, a consequence of the hyperglycaemia. The possibility is considered that 3-aminopicolinate stimulates gluconeogenesis in vivo by facilitating Fe(2+) activation of phosphoenolpyruvate carboxykinase as it does with the purified enzyme in vitro [MacDonald & Lardy (1978) J. Biol. Chem.253, 2300-2307]. In this effect 3-aminopicolinate may simulate the physiological role of the naturally occurring ferroactivator protein [Bentle & Lardy (1977) J. Biol. Chem.252, 1431-1440].

Animals↗
PubMed1978

Increased calcium-ion influx is a component of capacitation of spermatozoa.

Capacitation (modifications required for gamete fusion) is produced by incubating guinea-pig spermatozoa in vitro in a chemically defined medium. It is shown that during such incubation a net uptake of Ca2+ by the sperm occurs in two distinguishable phases. An initial loose association of Ca2+, possibly to surface sites, is unaffected by agents (Mg2+, inhibitors of mitochondiral function) that prevent or delay the exocytotic spermatozoal acrosome reaction. The time course of a secondary Ca2+ uptake parallels or slightly precedes the time course of the acrosome reaction. This parallelism is maintained during a variety of treatments that either expedite (local anaesthetics, ionophore A23187, Triton X-100) or delay (Mg2+, low external Ca2+) the acrosome reaction. We conclude that the secondary Ca2+ influx described herein apparently serves to link alterations of the spermatozoal membrane to subsequent contractile and secretory components of the capacitation sequence.

Acrosome↗
PubMed1978

Norepinephrine, vasopressin, glucagon, and A23187 induce efflux of calcium from an exchangeable pool in isolated rat hepatocytes.

Isolated rat hepatocytes do not actively accumulate Ca(2+) during prolonged incubation in vitro; however, these cells do exhibit a limited exchange of intracellular with extracellular Ca(2+). The exchangeable pool represents about 2 nmol of Ca(2+) per mg of protein. In medium containing either a low (20 muM) or high (1 mM) concentration of Ca(2+), the divalent cation ionophore, A23187 (at concentrations of 0.03-0.1 nmol/mg of protein), causes release of (45)Ca(2+) from this exchangeable pool but does not allow net influx of extracellular Ca(2+) detectable by the use of a Ca(2+)-sensitive electrode. Like A23187, the hormones norepinephrine, vasopressin, and glucagon (at concentrations that stimulate gluconeogenesis) each induces a similar net efflux of Ca(2+). Treatment with one hormone decreases the subsequent reponse to the others, whereas treatment with A23187 abolishes the hormonal effects upon both Ca(2+) release and gluconeogenesis. The action of norepinephrine, but not of glucagon, upon Ca(2+) efflux is prevented by the alpha-adrenergic antagonist, phenoxybenzamine. The action of norepinephrine is not prevented by the beta-adrenergic antagonist, propranolol. Together these results indicate that the release of Ca(2+) from a common pool of exchangeable Ca(2+) is important to the action of a variety of hormones on hepatocytes. This Ca(2+) pool in the isolated hepatocyte is characterized as being similar in size and having exchange kinetics that are comparable to those reported for the major intracellular pool of Ca(2+) in the intact liver. The possibility that this pool is intramitochondrial calcium is discussed.

Animals↗
PubMed1977

Pyruvate metabolism in bovine epididymal spermatozoa.

Treating bovine epididymal spermatozoa with rutamycin or rotenone inhibited both respiration and motility supported by endogenous substrates. When oxidative phosphorylation had been blocked with various inhibitors, pyruvate was metabolized to yield ATP and restored motility. Fructose, which is metabolized via glycolysis to yield ATP, was also able to resuscitate the cells. Other substrates tested (lactate, acetate, alpha-ketoglutarate, or glyoxylate) were unable to restore motility in rutamycin-treated cells. In the presence of pyruvate, the phosphorylation uncoupler, carbonylcyanide-p-trifluoromethyoxphenylhydrazone, reduced motility and ATP to common levels in untreated cells or cells treated with rutamycin or rotenone. Pyruvate is thus metabolized to produce ATP by a pathway independent of oxidative phosphorylation associated with the electron transport chain. 5-Methoxyindole-2-carboxylic acid, an inhibitor of lipoyldehydrogenase, prevented the increase of motility and ATP in rutamycin-treated cells, indicating that alpha-keto acid oxidation is involved in the production of ATP from pyruvate when rutamycin is present. With pyruvate present, bongkrekic acid, antimycin A, and anaerobiosis eliminated motility, reduced ATP to low levels, and also significantly reduced the rate of pyruvate metabolism. Acetate was produced from pyruvate only when cellular ATP concentrations were low. Decreases in free carnitine concentrations showed that pyruvate initially used was converted to acetylcarnitine. The results indicate that the intramitochondrial lactate dehydrogenase X, which is unique to spermatozoa, allows the NADH resulting from pyruvate oxidation to reduce other pyruvate molecules to lactate. Pyruvate thus competes with, and can substitute for, the NADH dehydrogenase of the electron transport chain. Pyruvate rapidly repletes the acetylcarnitine pool under a variety of conditions.

Adenosine Diphosphate↗
PubMed1977

Mitochondrial metabolism of pyruvate in bovine spermatozoa.

Treatment with the polyene antibiotic, filipin, renders the spermatozoan cell membrane permeable to small molecules, but not to the intracellular enzymes aldolase and lactate dehydrogenase. Pyruvate (10 mM) as the sole substrate was metabolized very slowly. L-Carnitine increased pyruvate metabolism 3- to 4-fold and allowed limited rates of oxidative phosphorylation. When spermatozoa treated with filipin were supplemented with malate, there was a rapid, almost linear rate of pyruvate metabolism which was slightly increased by L-carnitine. In the absence of malate, 20 to 30% of the pyruvate used was reduced to lactate; this increased to 57% in the presence of malate. Without malate, about 90% of the pyruvate metabolized was converted to lactate and acetate or L-acetylcarnitine. Rutamycin or rotenone increased both the rate of pyruvate use and the delta lactate/deltapyruvate ratio. Under all treatments, L-carnitine consistently reduced the percentage of pyruvate converted to lactate by about 10%; part of the pyruvate was preferentially shunted into L-acetylcarnitine rather than lactate. The mitochondrial inhibitors, rotenone or rutamycin, did not change the amount of pyruvate that was converted to metabolites other than lactate, or L-acetylcarnitine, or both. Pyruvate-supported State 3 respiration was linear only if L-carnitine, or malate, or both, were added to the incubation medium. Added malate was necessary to produce a rapid State 3 respiratory rate and was also required for significant respiratory activity in the presence of rotenone or rutamycin. From cells metabolizing [2-14C]pyruvate (1.4 mM), 14C-labeled acid-extractable metabolites were separated by ion exchange column chromatography. All of the [2-14C]pyruvate (+/-5%) used was recovered in 14C-labeled metabolites and 14CO2. In the presence of malate, citrate accumulation was significant, and was always large in comparison to flux through the citric acid cycle. Glutamate, beta-hydroxybutyrate, acetoacetate, fumarate, aspartate, and alpha-ketoglutarate did not accumulate in significant amounts. Some 14C-labeled succinate was produced but only in the presence of malate. Alkaline hydrolysis of a fraction containing carnitine esters yielded acetate and a compound tentatively identified as beta-hydroxybutyrate or lactate. As in intact cells, intramitochondrial lactate dehydrogenase competes successfully with the electron transport system for the NADH generated by pyruvate metabolism. The role of lactate and L-carnitine, and conclusions suggested by the accumulation of certain metabolites are discussed in relation to control of citric acid cycle activity.

Acetates↗
PubMed1977

P-enolpyruvate carboxykinase ferroactivator. Purification and some properties.

P-enolpyruvate carboxykianse ferroactivator was purified from rat liver cytosol to apparent homogeneity. This protein has an S20 of 4.69. High speed sedimentation equilibrium indicated a molecular weight of 82,000; that obtained by gel filtration was 126,000. this discrepancy in molecular weight by the two methods may indicate that this protein has a high axial ratio. A subunit molecular weight of 23,600 was obtained by sodium dodecyl sulfate electrophoresis. Synthesis of P-enolpyruvate is stimulated 3-fold when P-enolpyruvate carboxykinase is incubated in the presence of ferroactivator plus Fe2+; activity with Fe3+, Mn2+, Co2+, Cd2+, Mg2+, or Ca2+ was not affected by the ferroactivator. P-enolpyruvate synthesis by carboxykinase was activated by Mn2+ in the absence of ferroactivator. Quinolinic acid strongly inhibits carboxykinase activated by ferroactivator and Fe2+, but does not inhibit the enzyme activated by Mn2+. In the reverse reaction, ferroactivator and Fe2+ stimulated oxalacetate synthesis only briefly and carboxykinase activity rapidly returned to control rates. Increased oxalacetate synthesis by carboxykinase that had been incubated with ferroactivator and Co2+ was sustained. Stimulation of P-enolpyruvate synthesis by enzyme incubated in the presence of sulfate and Fe2+ rapidly diminished as a function of incubation time. Under similar conditions, P-enolpyruvate carboxykinase ferroactivator maintained the enzyme in the Fe2+-activated state for at least 4 h. The amounts of purified ferroactivator required to stimulate P-enolpyruvate carboxykinase maximally and the amount present in liver cytosol are sufficient to activate the enzyme maximally in vivo if adequate Fe2+ were present. It is possible that the rate of P-enolpyruvate synthesis in gluconeogenic tissues may be regulated by the availability of intracellular Fe2+ to the ferroactivator and P-enolpyruvate carboxykinase.

Amino Acids↗
PubMed1977

Studies on the kinetic mechanism of oxidative phosphorylation.

The kinetics of the synthesis of ATP from ADP and Pi by beef heart submitochondrial particles were examined. When Pi was the variable substrate positive cooperativity was observed, whereas if ADP was varied, linear double reciprocal plots were obtained. The analog of Pi, thiophosphate, was a noncompetitive inhibitor of ATP synthesis with respect to ADP, while the analog of ADP, AMP (CH2)P, was an uncompetitive Pi leads to ATP exchange inhibitor. The kinetics of the initial velocity isotopic exchanges of oxidative phosphorylation were also examined. When the Pi leads to ATP exchange was examined, it was found that if ADP concentration was held constant while ATP and Pi concentrations were varied at a constant ratio, linear double reciprocal plots were obtained. However, if Pi concentration was held constant and ADP and ATP concentrations were varied at constant ratio, apparent substrate inhibition was observed. The 2, 4-dinitrophenol-sensitive ADP leads to ATP exchange showed linear double reciprocal plots regardless of which components were varied. These results are interpreted to indicate that in the direction of ATP synthesis, the reaction is ordered, with Pi adding to the enzyme before ADP addition.

Adenosine Diphosphate↗
PubMed1976

Effect of inosine 5' -(beta, gamma-imido) triphosphate and other nucleotides on beef heart mitochondrial ATPase.

The effects of various substrates and alternative substrates on the hydrolytic activity of beef heart mitochondrial ATPase was examined. It was found that ATP or ADP, ITP hydrolysis showed positive cooperativity. IDP inhibited ITP hydrolysis and caused positive cooperativity. When ITP was present during an ATP hydrolysis assay, the rate of ATP hydrolysis was stimulated. IDP had no effect on ATP hydrolysis rates. A nonhydrolyzable ITP analog, inosine 5'-(beta, gamma-imido)triphosphate (IMP-P(NH)P), was synthesized and purified. It was found to be a potent competitive inhibitor of ITP and GTP hydrolytic activity. However, this beta-gamma-imido-bridged ITP analog was found to change the ITP and GTP hydrolysis kinetics from linear to positively cooperative. This compound inhibited ATP hydrolysis at substrate concentrations of 100 muM and lower, and stimulated ATP hydrolysis at substrate concentrations between 100 muM and 2 mM. IMP-P(NH)P had no effect on ATP hydrolysis when the substrate concentration was above 2 mM. In the presence of the activating anion, bicarbonate, IMP-P(NH)P inhibited ATP hydrolysis competitively, and induced positive cooperativity. IMP-P(NH)P had no effect on the ATP equilibrium Pi exchange, the ITP equilibrium Pi exchange, or ATP synthesis catalyzed by beef heart submitochondrial particles.

Adenine Nucleotides↗
PubMed1976

Effect of cations and anions on the steady state kinetics of energy-dependent Ca2+ transport in rat liver mitochondria.

The divalent cation ionophore A23187 has been used to investigate the kinetics of energy-dependent Ca2+ uptake by rat liver mitochondria under steady state conditions. During A23187-induced cyclic Ca2+ flux, the free Ca2+ concentration is adjusted using [ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA) buffers. The rate of Ca2+ transport, which is inferred from the rate of succinate oxidation, is a function of the free Ca2+ concentration in the medium. The kinetics are sigmoidal with the free Ca2+ concentration at half-maximal respiratory stimulation (K0.5) equal to 3.1 +/- 0.4 muM at 25 degrees. The maximal Ca2+-stimulated respiratory rate (Vmax) is a function of the ionic composition of the medium. Magnesium and Mg2+ plus phosphate produced a parallel stimulation of the maximal respiration rate whether activated by Ca2+ uptake or by the uncoupler carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP). In the absence of A23187, Ca:O rations of 4.0 were obtained under most experimental conditions. Magnesium is a potent competitive-like inhibitor, increasing the K0.5 for Ca2+ to 30.0 muM at 2.0 mM MgCl2. Magnesium dramatically decreases the apparent affinity for Ca2+ but does not appear to alter the kinetic mechanism. In contrast, the alkali metal cations are weak inhibitors, at most doubling the K0.5 for Ca2+; however, they antagonized Mg2+ inhibition with an order of effectiveness Li+ greater than or equal to Na+ greater than K+ greater than Rb+ =Cs+. Phosphate and acetate increased the Vmax slightly without altering the K0.5 for Ca2+. Phosphate did not influence the inhibitory effects of Mg2+ or Mg2+ plus K+. This study suggests that during steady state conditions, the maximal rate of Ca2+ accumulation is primarily electron transport-limited. The results are also discussed in terms of a possible physiological role for Mg2+ and K+ in the intracellular regulation of energy-dependent mitochondrial Ca2+ transport in liver.

Animals↗
PubMed1976

Action of ionophore A23187 at the cellular level. Separation of effects at the plasma and mitochondrial membranes.

Bovine epididymal spermatozoa incubated aerobically in vitro in the presence of 0.1 to 0.2 mM CaCl2 accumulate 25 to 50 nmol of calcium/10(8) cells. The addition of low concentrations of the ionophore A23187 (0.01 to 0.5 nmol/mg of sperm protein) induces efflux of this accumulated calcium. At high ionophore concentrations (0.5 to 5.0 nmol/mg of sperm protein), calcium release is followed by an influx of up to 25 nmol of calcium/10(8) cells that is not dependent on mitochondrial energization. A selective increase in the permeability of the sperm plasma membrane produced by treatment with the polyene antibiotic, filipin, results in the release of that calcium which is accumulated in the presence of high concentrations of A23187. Sperm first treated with filipin possess the ability to accumulate and retain calcium (in the presence of an oxidizable substrate) but release Ca2+ without subsequent reaccumulation after the addition of 3 nmol of A23187/mg of protein. These observations are explained by the existence of competing calcium pumps operating within the mitochondrial and plasma membranes of the spermatozoan. Treatment with high concentrations of A23187 allows calcium influx into a non-mitochondrial compartment of the sperm cell as a consequence of the equilibration of this cation across both mitochondrial and plasma membranes. The amount of calcium uptake and its sensitivity to filipin indicate that calcium binding to soluble, intracellular components is also involved. The ability of low concentrations of A23187 to induce calcium efflux is explained as a result of the continued operation of the plasma membrane pump coincident with ionophore-induced decay of the concentration gradient across the mitochondrial membrane. This hypothetical action of low levels of the ionophore on the mitochondria is supported by the observation of net movements of calcium with filipin-treated cells and the respiratory responses and movements of phosphate and membrane-associated calcium with intact sperm. It is suggested that the basis of this apparent selectivity of ionophore action lies in the relative activities and kinetic properties of the competing calcium pumps in the plasma and mitochondrial membranes of these cells. Ionophore-induced influx of calcium into the extramitochondrial space results in a stimulation of respiration and kinetic activity of the sperm. This activation of motility is observed also with cells made entirely dependent upon glycolysis (by treatment with respiratory inhibitors) and suggests a direct involvement of calcium in the regulation of flagellar function.

Animals↗