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Biomedical subjects

O H Lowry

Publications and source records attributed to O H Lowry.

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

Heterogeneity in regard to enzymes and metabolites within individual muscle fibers.

The possibility of variability along individual rat and human muscle fibers was assessed for four enzymes and four substances responsive to stimulation. Heterogeneity was determined by the differences between ends of fiber segments several millimeters long and by fluctuations in successive samples along the entire length of such segments. Among the enzymes, a cytosolic enzyme, lactate dehydrogenase, varied the least: average coefficient of variation (CV) of 5%. This is only a little greater than the analytic error. On the other hand, the CV for a mitochondrial enzyme, fumarase, was 13%. A mixed cytosolic-mitochondrial enzyme, malate dehydrogenase, was intermediate (CV of 9%). The CV for glycogen phosphorylase, which is normally bound to glycogen particles, was 34% along one fiber segment. Among the nonenzyme components, average CVs in stimulated fibers were 34, 20, 7, and 7%, respectively, for glucose 6-phosphate, phosphocreatine, ATP, and malate. Major differences were not random but developed gradually over distances of 0.5-2 mm along the fibers, and in some cases significant correlations between enzyme and metabolite levels were demonstrable.

Adenosine Triphosphate↗
PubMed1984

Myoglobin levels in individual human skeletal muscle fibers of different types.

An attempt was made to determine the relationship of myoglobin content to specific fiber types in human muscle. Biopsies were obtained from biceps brachii, vastus lateralis, and gastrocnemius muscles of untrained subjects and from the vastus lateralis muscle of a highly trained athlete at peak training and at intervals of no training (detraining). Individual muscle fibers were assayed, by quantitative microanalytical methods, for myoglobin, lactate dehydrogenase, malate dehydrogenase, citrate synthase, beta-hydroxyacyl-coenzyme A dehydrogenase, and adenylokinase activities all on the same fiber. The enzyme levels were used to classify the fibers into type I or II. The results show that the content of myoglobin in human muscle does not differ greatly between fiber types in contrast to other species. The type II fibers contained, on the average, at least two-thirds as much myoglobin as type I fibers. The concentration of myoglobin did not change in either fiber type during detraining (84 days), despite marked changes in lactate dehydrogenase, adenylokinase and the three oxidative enzymes.

Adenylate Kinase↗
PubMed1984

Comparison of muscle fiber typing by quantitative enzyme assays and by myosin ATPase staining.

Fibers in cross sections of human and rat muscle were typed by using histochemical ATPase stains, and the results were compared with those of quantitative enzyme assays of fragments of the same fibers dissected from serial freeze-dried sections. Two enzymes previously used to assess the metabolic type were measured in each case: lactate dehydrogenase and either adenylokinase (human fibers) or malate dehydrogenase (rat fibers). With human fibers there was essentially complete agreement between ATPase staining and the metabolic enzyme assays in distinguishing types I and II fibers. The agreement was less consistent with regard to type IIA and IIB fibers. A number of ATPase type IIC fibers were identified in one human muscle, and were found to fall between ATPase types I and IIA on the basis of metabolic enzyme assay results. Rat-fiber ATPase types I, IIA, and IIB from the plantaris muscle were rather well segregated on a two-dimensional lactate dehydrogenase-malate dehydrogenase grid. In the rat soleus muscle, ATPase types I and IIA fibers were shifted to lower lactate dehydrogenase levels, with IIC fibers interposed between them.

3-Hydroxyacyl CoA Dehydrogenases↗
PubMed1984

The distribution of six enzymes of oxidative metabolism along the rat nephron.

Using quantitative methods, citrate synthase (CS), fumarase, beta-hydroxyacyl-coenzyme A (CoA) dehydrogenase (beta OAC), 3-keto-acid CoA transferase (KCT), malic dehydrogenase (MDH), and malic enzyme were measured in seven defined parts of the nephron and in thin limb and papilla areas dissected from freeze-dried microtome sections of rat kidney. The results not only show a wide range of activity along the nephron for each of the enzymes, but that the proportions between the enzymes vary markedly among the different parts of the nephron. This suggests the existence of major regional differences in the capacity to oxidize specific metabolites. The ratio between two citrate cycle enzymes, fumarase and CS, was 4- or 5-fold higher in proximal segments than in the glomerulus or thin limb areas. The ratio between beta OAC (an enzyme of fatty acid oxidation) and CS was 3- to 5-fold higher in the middle proximal segments than in glomeruli or thin limb and papilla areas. The key enzyme for ketone body metabolism, KCT, was essentially confined to the thick tubule segments. Malic enzyme, in contrast to the other five enzymes, was highest in the proximal straight segments. New methods, sufficiently sensitive for this histochemical study, are described for malic enzyme and 3-keto-acid CoA transferase.

3-Hydroxyacyl CoA Dehydrogenases↗
PubMed1983

An enzymatic method for inosine 5'-monophosphate in the femtomole range.

A method for measuring inosine 5'-monophosphate (IMP) by enzymatic generation of NADPH is described. Procedures are given for direct fluorometric assay in the nanomole range and indirect measurement with amplification by enzymatic cycling in the pico- and femtomole ranges. The most sensitive procedure represents a nearly 50,000-fold increase in sensitivity over enzymatic methods now available. Specificity of the assay was greatly enhanced by the use of the antibiotic coformycin, a potent inhibitor of adenosine deaminase (EC 3.5.4.4). This enzyme was found to be a major contaminant of one of the necessary enzymes, phosphoglucomutase (EC 2.7.5.1). The use of the method is illustrated by measurements of IMP in single stimulated and control rat muscle fibers.

Adenosine Deaminase Inhibitors↗
PubMed1983

Role of nicotinamide adenine dinucleotide in ethanol-induced depressions in testicular steroidogenesis.

It is rapidly becoming accepted, without direct evidence, that a change in the NAD+/NADH ratio in the testes produced by the metabolism of ethanol is the principal mechanism involved in its now well-established effects on testicular steroidogenesis. The purposes of the present studies were 2-fold: (1) to examine whether, in fact, in vivo or in vitro ethanol exposure alters the NAD+/NADH ratio in the testes; and (2) to examine the validity of previous reports in which it was found that NAD+ prevented the effects of ethanol on testicular steroidogenesis under in vitro conditions. With regard to the first objective, we found that a large dose of ethanol (2.5 g/kg) markedly reduced gonadotropin-stimulated testicular steroidogenesis in vivo in the male rat, but it did not alter the NAD+ and NADH concentrations in the testes. Similarly, extremely high ethanol concentrations (200 mM) substantially suppressed hMG-stimulated testosterone biosynthesis in in vitro Leydig cell preparations but no change in NAD+ concentration occurred; NADH levels were very low in the Leydig cell preparations (less than 2% of NAD+ levels), but did not appear to change as a function of ethanol exposure. Finally, in contrast to previously published results, we found that NAD+ (1 mM) did not prevent the in vitro effects of ethanol on cAMP-stimulated testicular steroidogenesis. Consequently, our results fail to support the hypothesis that acute in vivo or in vitro ethanol administration inhibits the biosynthesis of testosterone by altering the NAD+/NADH ratio in the testes.

Acetaldehyde↗
PubMed1983

Correcting a potential defect in an enzymatic cycle for NADP.

An enzymatic cycle for NADP which uses as one of its enzymes glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides has occasionally caused trouble due to failure to completely heat-kill this enzyme before the indicator step. It was found that a very small increase in pH was the cause of this. It was also found that the other two proteins present in the reagent greatly increase heat inactivation of the enzyme. The inactivation problem is completely overcome by keeping the pH below 7.2.

Fluorometry↗
PubMed1983

Diversity of metabolic patterns in human brain tumors: enzymes of energy metabolism and related metabolites and cofactors.

Biopsies from 15 human gliomas, five meningiomas, four Schwannomas, one medulloblastoma, and four normal brain areas were analyzed for 12 enzymes of energy metabolism and 12 related metabolites and cofactors. Samples, 0.01-0.25 microgram dry weight, were dissected from freeze-dried microtome sections to permit all the assays on a given specimen to be made, as far as possible, on nonnecrotic pure tumor tissue from the same region. Great diversity was found with regard to both enzyme activities and metabolite levels among individual tumors, but the following generalities can be made. Activities of hexokinase, phosphorylase, phosphofructokinase, glycerophosphate dehydrogenase, citrate synthase, and malate dehydrogenase levels were usually lower than in brain; glycogen synthase and glucose-6-phosphate dehydrogenase were usually higher; and the averages for pyruvate kinase, lactate dehydrogenase, 6-phosphogluconate dehydrogenase, and beta-hydroxyacyl coenzyme A dehydrogenase were not greatly different from brain. Levels of eight of the 12 enzymes were distinctly lower among the Schwannomas than in the other two groups. Average levels of glucose-6-phosphate, lactate, pyruvate, and uridine diphosphoglucose were more than twice those of brain; 6-phosphogluconate and citrate were about 70% higher than in brain; glucose, glycogen, glycerol-1-phosphate, and malate averages ranged from 104% to 127% of brain; and fructose-1,6-bisphosphate and glucose-1,6-bisphosphate levels were on the average 50% and 70% those of brain, respectively.

Adolescent↗
PubMed1983

Effects of detraining on enzymes of energy metabolism in individual human muscle fibers.

Muscle biopsies were obtained from three cyclists and four runners at the end of 10-24 mo of intensive training and after intervals of detraining up to 12 wk. Control samples came from four untrained persons and four former athletes. Macro mixed fiber samples were assayed for lactate dehydrogenase, adenylate kinase, glycogen phosphorylase, citrate synthase, malate dehydrogenase, beta-hydroxyacyl-CoA dehydrogenase, succinate dehydrogenase, beta-hydroxybutyrate dehydrogenase, creatine kinase, hexokinase, 1-phosphofructokinase, fructosebisphosphatase, protein, and total creatine. In the case of three trained persons and two controls, the first six of the enzymes were also measured in individual fibers. Before detraining, enzymes of oxidative metabolism were substantially higher than in controls, and differences in levels between type I and type II fibers were smaller. During detraining, oxidative enzymes were decreased in both fiber types but the type II fibers did not fall to control levels even after 12 wk. Phosphorylase increased with detraining in both fiber types. The same is true for lactate dehydrogenase and adenylate kinase, except in the case of the type I fibers of one individual. Among the other six enzymes (measured in mixed fiber samples), only hexokinase was consistently affected (decreased) by detraining.

Adenylate Kinase↗
PubMed1983

Quantitative histochemistry of canine cardiac Purkinje fibers.

Single canine Purkinje cells were isolated by microdissection and analyzed quantitatively for six enzymes of energy metabolism. Subendocardial Purkinje cells were clearly distinguishable morphologically and biochemically from adjacent myocardium and had enzyme levels comparable with extramural Purkinje cells. Oxidative enzymes, citrate synthase, malate dehydrogenase, and beta-hydroxyacyl CoA dehydrogenase were 40-60% lower in Purkinje cells than in myocardium. Lactate dehydrogenase was also 40% lower, but the other glycolytic enzymes, hexokinase and phosphofructokinase, were similar in level in myocardium and Purkinje cells. The results of this study show that it is completely practicable to apply quantitative histochemical analysis to biochemical study of the cardiac conducting system.

3-Hydroxyacyl CoA Dehydrogenases↗
PubMed1982

Relationships in different parts of the nephron between enzymes of glycerol metabolism and the metabolite changes which result from large glycerol loads.

Glycerol metabolism in 9 defined parts of rat nephron was studied by measurement of glycerol kinase and observation of effects of large glycerol loads on related metabolites and on ATP and total adenylate. Glycerol kinase with glycerol as substrate was highest in the proximal convoluted tubule, slightly lower in straight portions, 20 to 25 times lower in distal segments, and almost indetectable elsewhere. With dihydroxyacetone as substrate, enzyme distribution differed little except for 30% lower activity in proximal convoluted segments. Methods of measurement with 10-40 ng of freeze-dried tissue are described. Glycerol loads produced large accumulations of glycerol 3-phosphate in proximal segments and in distal convoluted tubules. In the control nephron, fructose bisphosphate plus triose phosphates was 1 times higher in distal straight and convoluted segments than in proximal segments. Increases with glycerol loads were limited to proximal straight tubules (2-fold) and the thin limb area (60%). Glucose-6-P increased 3-fold in the late portion of the proximal straight tubule but not elsewhere. ATP and total adenylate were markedly depleted no phosphorylated intermediates accumulated. Levels of Pi were decreased in whole cortex and medullar following glycerol loads.

Adenine Nucleotides↗
PubMed1982

The distribution of glutamate, GABA and aspartate in the nucleus tractus solitarius of the cat.

This report describes the distribution of glutamate, GABA and aspartate in the nucleus tractus solitarius (NTS), area postrema, dorsal vagal nucleus and hypoglossal nucleus. Glutamate levels were particularly high in the dorsal, medial and commissural nuclei of the NTS. Section of the IXth and Xth cranial nerves caused a bilateral decrease in glutamate (ipsilateral side = 48%; contralateral side = 36%) and ipsilateral decrease in GABA (27%) in the dorsal and medial NTS. The ipsilateral dorsal vagal nucleus showed a 48% decrease in glutamate and a 30% decrease in GABA after axotomy.

Animals↗
PubMed1982

Metabolite changes in individual rat muscle fibers during stimulation.

Rat plantaris and soleus muscles were stimulated intensely in vivo for 1 and 15 min, freeze-clamped, and freeze-dried, and individual fibers were dissected free. Fibers, assigned to four groups on the basis of lactate dehydrogenase and malate dehydrogenase, were each separately analyzed for ATP, P-creatine, glycogen, glucose, glucose-6-phosphate (glucose-6-P), lactate, citrate, and malate. Some fibers were also analyzed for fructose 1,6-phosphate, total adenylate and total creatine. Although each group as a whole showed significant and often large differences in control composition and response to stimulation, individual fibers varied enough to create an almost continuous spectrum of metabolite levels from one extreme to the other. The data suggest that the slowest twitch fibers were the most active in the control state. Stimulation for 1 min caused a small increase in ATP in all groups with a large decrease in P-creatine in "fast white" fibers and a modest decrease in the rest. After 15-min stimulation, fast white fibers had lost 60% of initial ATP and 97% of initial P-creatine, whereas in other fiber types these compounds underwent little further change. Metabolite changes with stimulation were also greatest in fast white fibers. Glucose-6-P rose 15-fold in 1 min, then fell to below control by 15 min when glycogen had been exhausted; lactate rose two to six times more than in other types. Glucose rose in all groups to levels at 15 min, compatible with equilibrium with blood plasma.

Adenosine Triphosphate↗
PubMed1982

Distribution of enzymes of adenylate and guanylate nucleotide metabolism in rat nephron.

In a previous study of discrete segments of rat nephron, we reported the levels of high-energy adenylate and guanylate phosphates to be highest in the distal straight and convoluted tubules. Those findings stimulated the study of the distribution of seven enzymes involved in the following metabolic pathways of these nucleotides [Formula: see text]. The patterns of distribution of enzymes in each pathway differed greatly. The phosphodiesterases, 1 and 2, were high in glomeruli and distal tubular segments and low in proximal segments. Adenylate kinase, 3, in contrast, was high in glomeruli, proximal segments, thick ascending limb of Henle, and distal convoluted tubules. Guanylate kinase levels, 4, however, were similar in all segments. The pattern of nucleosidediphosphate kinase, 5, was high in proximal convoluted, thick ascending limb, and distal convoluted tubules. The pattern of the degradative enzyme, 5'-nucleotidase, 6, whose levels were highest in proximal segments, was opposite from that of AMP deaminase, 7, highest in the distal nephrons. These dissimilar patterns underscore the extent of nephron heterogeneity.

3',5'-Cyclic-AMP Phosphodiesterases↗