Active enzyme sedimentation of pig heart fumarase.

Active band sedimentation studies of pig heart fumarase indicate that the enzyme is predominantly tetrameric at enzyme concentrations between 0.0125 and 0.25 mg/ml and at a fumarate concentration of 2.5 mM. At enzyme concentrations of 0.25--1.0 mg/ml and fumarate concentrations known to activate and inhibit the enzyme, the sedimentation band of fumarase becomes disperse and indicates the presence of polymers greater than tetramers.

Malate dehydrogenase. Kinetic studies of substrate activation of supernatant enzyme by L-malate.

At pH 8.0 in 0.05 M Tris-acetate buffer at 25 degrees C, homogeneous supernatant malate dehydrogenase exhibits substrate activation by L-malate. The turnover number, Michaelis constant for L-malate, and Michaelis constant for NAD are: 0.46 X 10(4) min(-1), 0.036 mM, and 0.14 mM, respectively, for nonactivated enzyme and 1.1 X 10(4) min(-1), 0.2mM, and 0.047 mM for the same series of constants in activated enzyme. Nonactivating behavior is observed at concentrations between 0.02 and 0.15 mM L-malate and activating behavior is observed between 0.15 and 0.5 mM L-malate. L-Malate activation is compared with similar activation of mitochondrial malate dehydrogenase. While it is not possible to exclude unequivocally all mechanisms, the data seem to be consistent with the occurrence of a fundamentally ordered bi bi mechanism, possibly involving activation through the allosteric binding of L-malate. It is concluded that the data are consistent with a form of the "reciprocating compulsory order mechanism" in which nonactivated enzyme reflects catalysis by one subunit and activated catalysis expresses the coordinated activity of two subunits. The allosteric interaction and the "reciprocating mechanism/ are not mutually exclusive.

The effect of dietary fat or cholesterol and cholic acid on the rate of synthesis of rat liver glucose-6-P dehydrogenase.

The effect of dietary fat or cholesterol and cholic acid on the rate of synthesis of rat liver glucose-6-P dehydrogenase was measured by isolating hepatocytes from the livers of rats fed diets containing various amounts and types of lipid. Hepatocyte proteins were labeled with [3H]leucine and glucose-6-P dehydrogenase was immunoprecipitated with a specific antiserum. The immunoprecipitates were electrophoresed on sodium dodecyl sulfate-polyacrylamide gels and the radioactivity in the glucose-6-P dehydrogenase subunit was used to calculate the relative rate of synthesis of the enzyme. It is concluded that the activity of glucose-6-P dehydrogenase is primarily regulated by alterations in the rate at which the enzyme is synthesized. Dietary fat may decrease the rate of degradation of glucose-6-P dehydrogenase.

Malate dehydrogenase, circular dichroism difference spectra of porcine heart mitochondrial and supernatant enzymes, binary enzyme-coenzyme, and ternary enzyme-coenzyme-substrate analog complexes.

Circular dichroism spectra and circular dichroism difference spectra, generated when porcine heart mitochondrial and supernatant malate dehydrogenase bind coenzymes or when enzyme dihydroincotinamide nucleotide binary complexes bind substrate analogs, are presented. No significant changes are observed in protein chromophores in the 200- to 240-nm spectral range indicating that there is apparently little or no perturbation of the alpha helix or peptide backbone when binary or ternary complexes are formed. Quite different spectral perturbances occur in the two enzymes with reduced coenzyme binding as well as with substrate-analog binding by enzyme-reduced coenzyme binding. Comparison of spectral perturbations in both enzymes with oxidized or reduced coenzyme binding suggests that the dihydronicotinamide moiety of the coenzyme interacts with or perturbs indirectly the environment of aromatic amino acid residues. Reduced coenzyme binding apparently perturbs tyrosine residues in both mitochondrial malate dehydrogenase and lactic dehydrogenase. Reduced coenzyme binding perturbs tyrosine and tryptophan residues in supernatant malate dehydrogenase. The number of reduced coenzyme binding sites was determined to be two per 70,000 daltons in the mitochondrial enzyme, and the reduced coenzyme dissociation constants, determined through the change in ellipticity at 260 nm, with dihydronicotinamide adenine dinucleotide binding, were found to be good agreement with published values (Holbrook, J. J., and Wolfe, R. G. (1972) Biochemistry 11, 2499-2502) obtained through fluorescence-binding studies and indicate no apparent extra coenzyme binding sites. When D-malate forms a ternary complex with malate dehydrogenase-reduced coenzyme complexes, perturbation of both adenine and dihydronicotinamide chromophores is evident. L-Malate binding, however, apparently produces only a perturbation of the adenine chromophore in such complexes. Since the coenzyme has been found to bind in an open conformation on the surface of the enzyme and the substrate analogs bind at or very near the dihydronicotinamide moiety binding site, protein conformational changes are implicated during ternary complex formation with D-malate which can effect the adenine chromophore at some distance from the substrate binding site.

Categorization Methodology: an Approach to the Collection and Analysis of Certain Classes of Qualitative Information.

A new methodology has been developed for the study of certain classes of qualitative information. The methodology is composed of two major techniques: the F-sort task for data collection and latent partition analysis for data summarization. In this paper, similarities and differences of the methodology in relation to existing techniques are discussed. This includes a review of the historical antecedents and development of the methodology as well as a listing of recent applications of the methodology, which have been in several fields of psychology and education. Following that review, a detailed presentation is given of how the methodology was applied to studying teachers' views of facilitating student learning in the classroom. In the data analysis, some previously unpublished enhancements of latent partition analysis are introduced -- for basic categorization statistics, recovery of subgroup information, and linkage to multidimensional scaling. This complete and substantively intriguing example is intended to illustrate the power and utility of the methodology in exploring important research topics. Then, examinations are made of the main components of the F-sort technique and of latent partition analysis. The intent is to lay out detailed frameworks for designing and interpreting research that applies the categorization methodology. The methodology has not previously been given an integrated description. Finally, discussion concerns the uses of the methodology and the importance of standardized application of the procedures.

Effect of age and dietary fat level on fatty acid oxidation in the neonatal pig.

A total of 35 pigs were obtained by cesarean section, placed in individual sterile isolators, and randomly allotted to treatment groups. Thirty pigs received purified, isoenergetic liquid diets containing 2 or 32% butterfat (dry matter basis) and were killed at 1, 7, or 21 days of age. Five pigs were killed at 2 hours post delivery and received no diet. Twenty-one-day old pigs showed a tendency for higher weight gain and feed consumption when consuming the 32% fat diet although the differences were not significant. The rate of oxidation of [U-14C]palmitate to CO2 and acid soluble products was measured in homogenates of liver, kidney, heart, and leg muscle (biceps femoris) from pigs 0, 1, 7, and 21 days of age. The relative rates of oxidation of [U-14C]myristate, [U-14C]palmitate, and [U-14C]stearate were measured in homogenates of liver from 7-day old pigs. Palmitate oxidation was stimulated by carnitine in all four tissues and the rate of carnitine-stimulated palmitate oxidation to acid soluble products in heart and to CO2 in liver was higher in tissues from pigs consuming the 32% fat diet. The rate of palmitate oxidation increased with age in liver, kidney and leg muscle tissues and was maximum at 21 days in kidney and leg muscle and at 7 days in liver. The rate of palmitate oxidation in heart tended to decrease with animal age. In homogenates of liver from 7-day old pigs, palmitate was oxidized at a faster rate than stearate or myristate. The activities of carnitine palmitoyltransferase (CPT) (EC 2.3.1a) and succinate dehydrogenase (EC 1.3.99.1) in mitochondria isolated from liver, kidney, heart, and leg muscle did not vary considerably with age although CPT activity tended to be higher in those tissues from pigs consuming the high fat diet. Changes in the rate of palmitate oxidation with age tended to parallel changes in the level of mitochondrial protein per g of wet tissue and suggested an increased ability to utilize fat as an energy substrate during early development of the neonatal pig.

Malate dehydrogenase, inhibition of pig heart supernatant enzyme by iodoacetamide.

Pig heart supernatant malate dehydrogenase is alkylated by 250 mM iodoacetamide at 37 degrees C in pH 7.5 Tris/acetate buffer which is 0.05 M with respect to acetate to form enzyme with 1,3-dicarboxamidomethyl histidine, 3-carboxamidomethyl histidine, 1-carboxamidomethyl histidine, carboxamidomethyl cysteine, and carboxamidomethyl methionine. 1,3-Dicarboxamidomethyl histidine forms with a stoichiometry of 1/enzyme subunit at a rate equaling the rate (approximately 1.24 +/- 0.09 X 10(-2) min-1) of enzyme inactivation by iodoacetamide. All other amino acid derivatives form at a slower rate initially or continuously. The enzyme is protected against inactivation by iodoacetamide through NADH binding, suggesting that there is an "essential" histidine residue at or near the active site.

Malate dehydrogenase, anticooperative NADH, and L-malate binding in ternary complexes with Supernatant pig heart enzyme.

Supernatant malate dehydrogenase from pig heart, a dimeric protein containing two very similar or identical subunits, shows negatively cooperative (anticooperative) interactions between NADH binding sites in the presence, but not in the absence, of 0.1 M L-malate. This behavior is observed consitently whether the technique used employs protein fluorescence quenching, NADH fluorescence enhancement, or ultrafiltration dialysis. Fluorescence titration shows that L-malate is also anticooperatively bound in the presence of saturating concentrations of NADH. The data are consistent with an "induced asymmetry" model in which conformational change accompanies the formation of the ternary complex. Two of the three chromatographically resolvable forms of the enzyme have been tested and found to have anticooperative behavior.

Effect of dietary fat level on growth and lipogenesis in the colostrum deprived neonatal pig.

Thirty male neonatal pigs were obtained by cesarean section and randomly allotted from littermate groups to three diets containing 2%, 17%, and 32% fat on a dry matter basis. Butterfat was used to replace glucose in the isoenergetic liquid, semipurified diets. Each pig was placed in a sterile isolator and fed ad libitum five times daily. After 14 days, pigs were weighed, killed, and samples of liver and backfat were obtained. The carcass of each pig was ground and samples obtained for determination of total body fat and protein. An increase in the level of dietary fat resulted in a significant increase in 14 day weight gain and a tendency for improved feed efficiency. These results demonstrate not only that the neonatal pig can utilize semipurified liquid diets high in butterfat content, but also that energy from butterfat appears to be used as efficiently as energy from glucose from growth purposes. Increasing dietary fat level decreased the activity of fatty acid synthetase and citrate cleavage enzyme in adipose tissue and liver, and decreased the activity of malic enzyme in adipose tissue. The specific activities of these three enzymes were higher in adipose tissue than in liver.