Pig Heart Muscle Research Articles

A sensitive method for the detection of aspartate: 2-oxoglutarate aminotransferase activity on polyacrylamide gels

A sensitive technique for the qualitative and semiquantitative determination of the activity of aspartate aminotransferase on polyacrylamide gels after electrophoresis is described. It relies on the ability of aspartate aminotransferase to produce SO 3 −− in the transamination between l-cysteine sulfinate and 2-oxoglutarate. The method is based on the reduction of nitroblue tetrazolium by SO 3 −− using phenazine methosulfate as a coupling agent. The method has been characterized using human and pig sera, crude homogenates and crystalline preparation from pig heart muscle, and bacterial crude extracts.

47] Short-chain and long-chain enoyl-CoA hydratases from pig heart muscle

47] Short-chain and long-chain enoyl-CoA hydratases from pig heart muscle

Relationship between the effect on calcium turnover and early cardiotoxicity of doxorubicin and 4'-epi-doxorubicin in guinea pig heart muscle.

Doxorubicin and 4'-epi-doxorubicin, two anthracycline derivatives with different cardiotoxic effects in experimental models, were found to decrease myocardial contractility in isolated guinea pig atria by significantly modifying calcium turnover. This effect seems to be mainly localized on the fast exchanging membrane-bound calcium, while these drugs do not significantly influence the intracellular stores of calcium. 4'-epi-doxorubicin, which induces a less negative inotropic effect than doxorubicin, produces a smaller inhibition of calcium turnover. This supports the hypothesis that the inhibition of calcium turnover and particularly of the fast exchanging calcium compartment is a general mechanism involved in the early anthracycline-induced cardiotoxicity.

Complete amino acid sequence of mitochondrial aspartate aminotransferase from pig heart muscle. Peptide ordering procedures and the complete sequence.

Complete amino acid sequence of mitochondrial aspartate aminotransferase from pig heart muscle. Peptide ordering procedures and the complete sequence.

Complete amino acid sequence of mitochondrial aspartate aminotransferase from pig heart muscle. Tryptic peptides.

The amino acid sequences of 39 tryptic peptides from carboxymethylated mitochondrial aspartate aminotransferase from pig heart muscle were analyzed. The peptides were purified by gel filtration, ion exchange column chromatography, paper chromatography, and high voltage paper electrophoresis, and their sequences were examined by manual Edman degradation, carboxypeptidase digestion, and fragmentation with thermolysin or chymotrypsin. These peptides accounted for 318 of the total 401 amino acid residues in the protein subunit.

Amino acid sequence of L-3-hydroxyacyl CoA dehydrogenase from pig heart muscle

Amino acid sequence of L-3-hydroxyacyl CoA dehydrogenase from pig heart muscle

Correlations between tertiary structure and energetics of coenzyme binding in pig heart muscle lactate dehydrogenase.

Fluorescence, equilibrium dialysis, and microcalorimetric measurements have been performed on complex formation between pig heart muscle lactate dehydrogenase (EC 1.1.1.27) and a series of systematically modified nicotinamide adenine dinucleotide analogues to provide quantitative data for a discussion on energy-structure-function correlations. As a result of these studies, one can draw the conclusion that estimates of the relative stability of enzyme-ligand complexes on the mere basis of structural information on the macromolecule and its complexes with the ligand are likely to neglect contributions to the energy and entropy parameters, which stem from such processes as changes in solvation and conformation of both the free ligand and the macromolecule in the reaction. Since the reaction parameters reflect the differences between these states, information on hydrogen bonding and hydrophobic interaction schemes of the liganded and unliganded macromolecule alone is principally insufficient.

Cardiotonic effect of phthalazinol (EG-626) in the isolated guinea pig by myocardium: mechanical and electrophysiological study.

EG-626, 7-ethoxycarbonyl-6, 8-dimethyl-4-hydroxymethyl-1 (2H)-phthalazinone, was found to be a considerably potent cardiotonic agent. It produced both the positive chronotropic and inotropic actions in the guinea pig heart muscle. Positive inotropic action of isoproterenol was potentiated by EG-626 at the concentration which did not produce a substantial positive inotropic action by itself. Cardiac action potential was not modified by EG-626 at concentrations sufficient to produce positive inotropic actions. EG-626 has a strong activity to produce slow responses in the depolarized myocardium, indicating that it can increase the density of the slow channels. The possibility was shown that the increase in the density of the slow channel may play an important role in the positive inotropic action of EG-626. The increase in the intracellular cyclic AMP due to the phosphodiesterase inhibition is tentatively most likely to be the cause of the EG-626 induced in the density of the slow channels.

Cysteine sulfinate transamination activity of aspartate aminotransferases

Aspartate aminotransferases from pig heart cytosol and mitochondria, Escherichia coli B and Pseudomonas striata accepted L-cysteine sulfinate as a good substrate. The mitochondrial isoenzyme and the Escherichia enzyme showed higher activity toward L-cysteine sulfinate than toward the natural substrates, L-glutamate and L-aspartate. The cytosolic isoenzyme catalyzed the L-cysteine sulfinate transamination at 50% the rate of L-glutamate transamination. The Pseudomonas enzyme had the same reactivity toward the three substrates. Antisera against the two isoenzymes and the Escherichia enzyme inactivated almost completely cysteine sulfinate transamination activity in the crude extracts of pig heart muscle and Escherichia coli B, respectively. These results indicate that cysteine sulfinate transamination is catalyzed by aspartate aminotransferase in these cells.

Rapid purification of pig heart fumarase by general ligand chromatography

A rapid method for the purification of fumarase from pig heart muscle has been developed using general ligand chromatography with adenosine triphosphate as ligand. Fumarase exhibited distinctive elution patterns from several types of nucleotide-agarose matrices, which may prove of value in distinguishing putative isozymic forms. Fumarase purified from both soluble and particulate fractions of cardiac tissue appeared to be identical in terms of subunit molecular weight, electrophoretic mobility on cellulose acetate, substrate kinetics, and inactivation by several inhibitors. When fresh cardiac tissue was suspended in sucrose medium, both fumarase and citrate synthase were released from the mitochondria to about the same extent (10%). However, fumarase release was increased approximately three-fold, while the release of titrate synthase increased only slightly, when tissue which had been frozen and thawed was suspended in sucrose medium.

Tris buffer: A new method for studying pulsus alternans in isolated heart muscle

A regular alternation of strong and weak heart beats, i.e. pulsus alternans, could be induced in isolated heart muscle of guinea pigs or cats when the commonly used buffering agent Tris (hydroxymethyl) aminomethane was used instead of bicarbonate. Alternans developed spontaneously in Tris-buffered atria of guinea pigs when the Ca 2+ concentration of the bathing medium was decreased from 2.5 to 1.0 m m, or when the Mn 2+ concentration was increased from 0 to 0.5 m m; alternans persisted in Tris-bathed atria when stimulation frequency was changed from 1.0 Hz to 0.4, 0.8 and 1.6 Hz. Alternation of contractions similarly occurred in Tris-buffered ventricular papillary muscles of cats, especially when these preparations were challenged with even a temporary (1 to 2 min) increase in stimulation frequency. Alternans was not produced in bicarbonate-buffered heart muscle with the inotropic interventions studied. Present findings identify a previously unrecognized aspect of the cardiac effects of substituting Tris buffer for bicarbonate, and provide a model for studying the important cardiac phenomenon of pulsus alternans in heart muscle isolated from in vivo influences.

The nature of the substrate inhibition in lactate dehydrogenases as studied by a spin-labeled derivative of NAD +

The formation of the ternary complex of lactate dehydrogenase ( l-lactae:NAD + oxidoreductase, EC 1.1.1.27) from pig heart and skeletal muscle with the adduct of pyruvate to NAD +, spin-labeled at N 6 was studied by ultraviolet spectroscopy and ESR techniques. According to ultraviolet measurements we found identical binding characteristics for the natural coenzyme and its spin-labeled analog. The rate by which the ESR signal of free spin-labeled NAD + decreased upon addition of pyruvate to the binary complexes was substantially different in the two isozymes. With the heart type an initial drop followed by a further linear decrease, zero order in the enzyme and coenzyme concentration was observed. In case of the skeletal muscle isozyme no immediate reaction and a first order process occurred. The initial reaction can be attributed to a non-covalent enzyme/spin-labeled NAD +/pyruvate complex with a dissociation constant for pyruvate of 11 ± 1 mM , thus explaining the well-known substrate inhibition in the heart isozyme above 2 mM pyruvate. The further reaction is then determined by the buffer dependent enolization of pyruvate. In the muscle isozyme formation of the covalent adduct is not assisted by prior binding of pyruvate in a non-covalent ternary complex and therefore the rate depends on the binary complex concentration.

Coronary vasospastic action of thromboxane A 2 in isolated, working guinea pig hearts

The effects of thromboxane A 2 (TXA 2) on isolated, working guinea pig heart and left ventricular papillary muscle preparations were investigated. TXA 2 was generated by the enzymatic conversion of prostaglandin H 2 (PGH 2) with indomethacin-treated horse platelet microsomes (IPM). TXA 2 caused dose-dependently a considerable decrease in the coronary flow, left ventricular systolic pressure and left ventricular dp/dt in the perfused heart, while the similar effects of PGH 2 were transient and weaker than those of TXA 2. These effects of TXA 2 disappeared after the TXA 2-generating system was left standing at 37°C for 3 min. When IPM in the TXA 2-generating system was pretreated with a TXA 2 synthetase inhibitor, L-8027, the enhanced cardiac responses induced by the mixture of IPM and PGH 2 disappeared. TXA 2 had no direct effect on the contractile force of the papillary muscle. These results suggest that TXA 2 has a specific coronary vasospastic action without a direct inotropic effect.

Buoyant densities of dissolved and crystalline lactate dehydrogenase from pig heart and pig muscle

Buoyant densities of the H4 and M4 isoenzymes of lactate dehydrogenase from pig, in both dissolved and crystalline cross-linked states, have been determined at neutral pH in concentrated CsCl solutions by equilibrium centrifugations in an ultracentrifuge. In both states, buoyant densities of the M4-LDH were lower than those of the H4-LDH, presumably due to the binding of counterions and hydration water by ten additional charged amino acid residues present on the M4-LDH. The cross-linked crystals had lower buoyant densities than enzymes in dissolved state consistent with the lower density of the cross-linking reagent. The variations in buoyant densities observed in CsCl solutions are in agreement with a previously described model for the water-and ion-binding to proteins which assigns almost all of the bound water to the charged amino acid side chain residues. In Cs2SO4 solutions the buoyant densities of the H4-LDH in both physical states were less than in CsCl, probably because strongly hydrated sulphate ions bind to the charged amino acid residues.

A crystallographic investigation of citrate synthase from pig and chicken heart muscle

Citrate synthase (EC 4. 1. 3. 7.) from pig heart and chicken heart muscle can be crystallized from 10 mM phosphate buffer (pH 7. 0–7. 5) and 10–15% PEG4000 solution. The space group is P4 12 12 with one subunit/asymmetric unit for the pig heart enzyme. The chicken heart citrate synthase purified from Blue-dextran as well as ATP-Sepharose affinity chromatography both crystallize in space group P2 12 12 with one molecule/asymmetric unit, but they have different unit cell dimensions. The a-axis differs by about 17 Å between these two crystal forms, while b- and c-axis dimensions are virtually identical.

Purification and properties of a pig heart thiolase with broad chain length specificity and comparison of thiolases from pig heart and Escherichia coli.

A thiolase (acetyl CoA acyltransferase, EC 2.3-1.16) which acts on substrates of various chain lengths (thiolase I) has been purified from pig heart muscle 366-fold to near homogeneity as judged by gel electrophoresis. Its molecular weight was estimated to be 200,000 in the absence and 46,000 in the presence of sodium dodecyl sulfate. Kinetic measurements with acetoacetyl coenzyme A, 3-ketohexanoyl-CoA, 3-ketooctanoyl-CoA, and 3-ketodecanoyl-CoA yielded apparent Km values of 16, 8.3, 2.4, and 1.8 micron, respectively, whereas apparent Vmax values of 65 to 69 mumol/min/mg were obtained with all substrates except for acetoacetyl-CoA, with which a value of 26.5 mumol/min/mg was observed. Antibodies prepared against this thiolase were used to demonstrate that thiolase I and acetoacetyl-CoA thilase (thiolase II) from pig heart mitochondria are immunologically unrelated. The antibodies cross-reacted, however, with thiolase I from beef heart. Kinetic constants (Km, Vmax) were also determined for thiolases I and II from Escherichia coli, as were the native and subunit molecular weights of E. coli thiolase II. Although the E. coli thiolases were found to be immunologically distinct from the pig heart enzymes, their physical and kinetic properties are strikingly similar to those of the heart thiolases. In view of this finding and in view of the known physiological functions of the E. coli thiolases, it is proposed that thiolase I from pig heart is only involved in fatty acid metabolism, whereas thiolase II functions solely in ketone body degradation.

Thermodynamic Studies of Binary and Ternary Complexes of Pig‐Skeletal‐Muscle Lactate Dehydrogenase

The thermodynamics of formation of the various intermediary complexes in the reaction catalyzed by pig skeletal muscle lactate dehydrogenase (EC 1.1.1.27) has been investigated in 0.2 M potassium phosphate buffer, pH 7, over the temperature range 10–36°C. As with the analogous experiments on pig heart muscle lactate dehydrogenase [Schmid, F., Hinz, H.‐J. & Jaenicke, R. (1976) Biochemistry, 15, 3052–3059] the catalytic process was approximated by employing the substrate‐analogous inhibitors oxamate and oxalate instead of pyruvate and lactate, respectively, in the two reactions forming the ternary complexes lactate dehydrogenase · NADH · oxamate and lactate dehydrogenase · NAD · oxalate. The standard Gibbs free energy for association of oxamate with the binary lactate dehydrogenase · NADH complex has been derived from fluorescence titrations, while the corresponding value for the binding of oxalate to the lactate dehydrogenase · NAD complex resulted from ultraviolet difference spectroscopy studies. All reaction enthalpies stem from direct calorimetric measurements. The prominent feature of the calorimetric studies is the pronounced variation with temperature of binding enthalpies, showing a negative temperature coefficient of −1901 ± 46 (− 7954 ± 193) cal (J) · (mol of enzyme)−1· K−1 for the association of oxamate and the lactate dehydrogenase · NADH complex and of −1484 ± 80 (6209 ± 335) cal (J) · (mol of enzyme)−1· K−1 for the reaction between lactate dehydrogenase · NAD and oxalate. These negative Δcp values involved in the binding reactions are suggestive of structural alterations of the enzyme induced by ligand binding, which consist of a limited immobilization of the protein with respect to vibrational modes of freedom and/or partial reversal of hydrophobic hydration. These effects are somewhat larger for the pig skeletal muscle enzyme than for the pig heart muscle enzyme.

Purification and Properties of Pig Heart Muscle Pyruvate Dehydrogenase

Purification and Properties of Pig Heart Muscle Pyruvate Dehydrogenase

Inhibition by lanthanum of the Na ++K + activated, ouabain-sensitive adenosinetriphosphatase enzyme

The effect of La 3+ on the activity of a Na + + K +—activated, ouabain-sensitive ATPase enzyme isolated as a microsomal fraction from guinea pig heart muscle has been investigated. Ten to 100 μ mLa 3+ has a dose-dependent, uncompetitive inhibitory effect, approximately 100 fold greater than that caused by equimolar concentrations of Ca 2+. The activity of the ouabain-insensitive ATPase enzyme is not affected. The same concentrations of La 3+ failed to alter the 3+H-ouabain binding capacity of the microsomal fraction. These results are interpreted to mean that La 3+ and ouabain interact with different receptor sites and that La 3+ has other actions apart from its well recognized Ca 2+—antagonist activity.

Thermodynamic studies of binary and ternary complexes of pig heart lactate dehydrogenase

The thermodynamics of the reaction catalyzed by pig heart muscle lactate dehydrogenase (LDH; EC1.1.1,27) have been studied in 0,2 M potassium phosphate buffer, pH 7, over the temperature range of 10 to 35 degrees C by using oxamate and oxalate to simulate the corresponding reactions of the substrates pyruvate and lactate, respectively. The various complexes formed are characterized by Gibbs free energies, enthalpies, and entropies. The Gibbs free energies were determined by equilibrium dialysis investigations, fluorescence titrations, and ultraviolet difference spectroscopy, while the reaction enthalpies stem from direct calorimetric measurements, Formulas are given for both the temperature dependence of the equilibrium constants and the variation with temperature of the enthalpies involved in the four reactions between LDH and NADH or NAD, LDH-NADH and oxamate, and LDH-NAD and oxalate. All reactions show a marked negative temperature coefficient, deltacp, of the binding enthalpies indicating partial refolding to be associated with binary and ternary complex formation. This interpretation appears very probable in view of recent x-ray crystallographic studies on lactate dehydrogenase from dogfish, which demonstrate a volume decrease to occur on binding of oxamate to the LDH-NADH complex. The validity of the thermodynamic parameters, as derived with substrate analogues, for the actual catalytic reaction, gains strong support from the agreement between the sum of the heats involved in the four intermediary reactions reported in this study and direct determinations of the overall enthalpy associated with the catalytic process published in the literature.