Biomedical subjects

H Beevers

Publications and source records attributed to H Beevers.

At least 73 records · Page 4Linked to original sources

PubMed1972

Intracellular distribution of enzymes of the cytidine diphosphate choline pathway in castor bean endosperm.

The occurrence and subcellular distribution of enzymes of the cytidine diphosphate choline pathway of lecithin synthesis have been examined. Choline kinase (EC 2.7.1.32) was completely soluble, while phosphorylcholine-cytidyl transferase (EC 2.7.7.15) and phosphorylcholine-glyceride transferase (EC 2.7.8.2) were associated with particulate fractions. Although components sedimenting at 10,000 to 100,000 x g contained both enzymes, phosphorylcholine-cytidyl transferase and particularly phosphorylcholine-glyceride transferase were present in the 10,000 x g pellet, which contained the major organelles, mitochondria, and glyoxysomes. When the crude homogenate was centrifuged on a sucrose density gradient, four major bands of particulate protein were recovered. A band at density 1.24 g/cm(3) contained the glyoxysomes and was devoid of phosphorylcholine-cytidyl transferase and phosphorylcholine-glyceride transferase activity. Enzyme activity was barely detectable in the mitochondria, at density 1.18 g/cm(2). Phosphorylcholine-glyceride transferase was found almost exclusively in a sharp band at density 1.12 g/cm(3), and phosphorylcholinecytidyl transferase was found in the uppermost band at density 1.08 g/cm(3). Thus, for the synthesis of lecithin in their membranes, the glyoxysomes and mitochondria depend on enzymes elsewhere in the cell; the final two steps in lecithin formation occur, apparently exclusively, in separate particulate cell components.

Carbon Isotopes↗

PubMed1972

Developmental studies on microbodies in wheat leaves : I. Conditions influencing enzyme development.

Catalase, glycolate oxidase, and hydroxypyruvate reductase, enzymes which are located in the microbodies of leaves, show different developmental patterns in the shoots of wheat seedlings. Catalase and hydroxypyruvate reductase are already present in the shoots of ungerminated seeds. Glycolate oxidase appears later. All three enzymes develop in the dark, but glycolate oxidase and hydroxypyruvate reductase have only low activities. On exposure of the seedlings to continuous white light (14.8 x 10(3) ergs cm(-2) sec(-1)), the activity of catalase is doubled, and glycolate oxidase and hydroxypyruvate reductase activities increase by 4- to 7-fold. Under a higher light intensity, the activities of all three enzymes are considerably further increased. The activities of other enzymes (cytochrome oxidase, fumarase, glucose-6-phosphate dehydrogenase) are unchanged or only slightly influenced by light. After transfer of etiolated seedlings to white light, the induced increase of total catalase activity shows a much longer lag-phase than that of glycolate oxidase and hydroxypyruvate reductase. It is concluded that the light-induced increases of the microbody enzymes are due to enzyme synthesis. The light effect on the microbody enzymes is independent of chlorophyll formation or the concomitant development of functional chloroplasts. Short repeated light exposures which do not lead to greening are very effective. High activities of glycolate oxidase and hydroxypyruvate reductase develop in the presence of 3-amino-1,2,4-triazole which blocks chloroplast development. The effect of light is not exerted through induced glycolate formation and appears instead to be photomorphogenetic in character.In senescing leaves excised from the plants decreases in activity of glycolate oxidase, and hydroxypyruvate reductase follow with some delay the decrease in chlorophyll content. The activity of catalase, however, is maintained at high levels, especially when the detached shoots are kept in light.

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PubMed1972

Developmental Studies on Microbodies in Wheat Leaves : II. Ontogeny of Particulate Enzyme Associations.

Crude particulate fractions from wheat leaves (Triticum vulgare L.) were separated on continuous sucrose density gradients, resulting in: broken chloroplasts, a mitochondrial fraction (indicated by cytochrome c oxidase), and microbodies. The visible band of the microbody fraction from adult leaves appears at a buoyant density of 1.25 grams per cm(3) and contains most of the activities of catalase, glycolate oxidase, and hydroxypyruvate reductase on the gradient. In the shoots of freshly soaked seeds, catalase is already highly particulate. During further development in light or in darkness, 40 to 60% of the total activities of catalase and glycolate oxidase and 25 to 40% of the total activity of hydroxypyruvate reductase are always found in the particulate fractions of the leaves. In young developmental stages, the peaks of the activity profiles of the microbody enzymes appear on sucrose gradients at relatively low densities, first between 1.17 to 1.20 grams per cm(3). During development in light, the buoyant density of the microbody fraction shifts to the final value of 1.25 grams per cm(3). However, even after 1 week of growth in the dark, the microbody fraction from etiolated leaves was observed at buoyant densitites 1.17 to 1.24 grams per cm(3) and did not appear as a defined visible band. A characteristic visible microbody band at a buoyant density 1.24 grams per cm(3) was found when the dark-grown seedlings received only three separate 5-minute exposures to white light. A similar peak was also obtained from light-grown leaves in which chloroplast development had been blocked by 3-amino-1,2,4-triazole.

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PubMed1972

The problem of reduced nicotinamide adenine dinucleotide oxidation in glyoxysomes.

NADH is generated in glyoxysomes both in the glyoxylate cycle and in beta-oxidation. No system has yet been described which would oxidize NADH in these organelles. A series of oxidants which might function by coupling NADH oxidation to O(2) through endogenous carriers in the glyoxysomes was examined. Oxidation was brought about by ferricyanide or dichlorophenol-indophenol, but it was shown that this "diaphorase" activity is probably a contaminant. Hydroxypyruvate reductase (NAD-linked) is present in the glyoxysomes, and at very high substrate concentrations (>10 mm) this enzyme can also transfer electrons from NADH to glyoxylate. However, it is most unlikely that this concentration of glyoxylate is ever approached in glyoxysomes, where the malate synthetase would compete on much superior terms. The maximum rates of NADH oxidation observed in the presence of ferricyanide or glyoxylate are only a fraction of those required to reoxidize NADH at the rate occurring in vivo.

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PubMed1972

Microbody enzymes and carboxylases in sequential extracts from c(4) and c(3) leaves.

A seven-step sequential grinding procedure was applied to leaves of Atriplex rosea, Sorghum sudanense, and Spinacia oleracea to study the distribution of carboxylases and microbody enzymes. In the extracts from C(4) species there were 7- to 10-fold reciprocal changes in specific activities of ribulose-1, 5-diphosphate carboxylase and phosphoenolpyruvate carboxylase. No such changes occurred in sequential extracts from spinach. No inhibitors of ribulose-1, 5-diphosphate carboxylase were detected when the mesophyll extracts of Sorghum were assayed together with spinach extracts. These results reaffirm the conclusion of others that phosphoenolpyruvate carboxylase is largely confined to the mesophyll in these species and ribulose-1, 5-diphosphate carboxylase to the bundle sheath. The specific activities of glycolate oxidase and hydroxypyruvate reductase in bundle sheath extracts were two to three times those in mesophyll fractions. Catalase behaved similarly in Atriplex rosea but in Sorghum the specific activity was virtually the same in all fractions. From the relative amounts of these enzymes present, and comparison with the data obtained from spinach, it is concluded that typical leaf peroxisomes are present in the bundle sheaths of both C(4) species and in the mesophyll of Atriplex rosea. The relative enzyme activities in the mesophyll of Sorghum suggest that the microbodies there are of the non-specialized type found in many nongreen tissues. The activities of the microbody enzymes in the bundle sheath of Sorghum seem quite inadequate to support photorespiration.

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PubMed1971

Gluconeogenesis in the castor bean endosperm: I. Changes in glycolytic intermediates.

The control points of the Embden-Meyerhof-Parnas pathway in germinating castor bean (Ricinus communis) endosperms are sought in two ways: (a) by measuring the amounts of various glycolytic intermediates at intervals during the germination; (b) by determining the crossover points appearing during anoxia.A significant departure from thermodynamic equilibrium between substrates and products is found at the level of fructose 1,6-diphosphatase and phosphofructokinase. A definite shift of this ratio is observed at the onset of active gluconeogenesis. The concentrations of phosphoenolpyruvate and 3-phosphoglyceric acid increase at the same time. Another departure from the expected equilibrium is also observed at the level of the pyruvate kinase.The imposition of anoxia on 5-day-old endosperms reveals two crossover points, at the level of the same enzymes. It is therefore concluded that they regulate the glycolytic flow.The maximal glycolytic flow, however, is only 1/10 of the gluconeogenic one. To account for this high gluconeogenic efficiency, it is postulated that gluconeogenesis and glycolysis occur in separate intracellular regions. The consistent departure from equilibrium between adenylates observed during the early stages of anoxia supports the concepts that the pools of glycolytic and gluconeogenic intermediates are indeed compartmented and that the two processes are independently regulated.

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PubMed1971

Uricase and allantoinase in glyoxysomes.

In fat-degrading tissues of seedlings of seven different plant species examined, uricase activity (urate:O(2) oxidoreductase, EC 1.7.33) was associated with particulate fractions. After equilibrium density centrifugation on sucrose density gradients the enzyme activity was recovered in the glyoxysomal band (density: 1.25 grams per cubic centimeter). Allantoinase is also present in glyoxysomes but, equally, in the proplastid region (density: 1.22 grams per cubic centimeter). Xanthine oxidase, xanthine dehydrogenase, allantoicase, and urease were not detected in glyoxysomes from castor bean endosperm. Uricase in these particles shows its maximal activity at pH 8.9. The apparent K(m) is 7.4 mum. Urate concentrations greater than 120 mum as well as certain other purine compounds inhibit the enzyme. Cyanide at a concentration of 10 mum is a potent inhibitor. 2,6-Dichlorophenolindophenol did not substitute for oxygen as electron acceptor.

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PubMed1971

Influence of cycloheximide on the synthesis and utilization of amino acids in suspension cultures.

Cells from 4-days old suspension cultures of Paul's Scarlet rose were incubated with acetate-U-(14)C for 10 minutes. After washing, cells were incubated for 2 hours in growth medium in the presence and absence of cycloheximide. The (14)C content of individual amino acids in the soluble form and in protein were determined at the end of the 10 minute pulse and at intervals thereafter in control cells and those treated with cycloheximide. During the period following the pulse there was a 3-fold increase in the (14)C content of protein in control cells; no such increase occurred in the presence of cycloheximide.In the control cells there was a net increase in the (14)C content of eight individual amino acids during this period. For six of these, the corresponding increases in the presence of cycloheximide were curtailed by the following amounts: arginine 98%, lysine 94%, isoleucine 93%, threonine 82%, valine 49%, and proline 36%. This is interpreted to mean that the synthesis of each of these amino acids was slowed down when their incorporation into protein was prevented.

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PubMed1971

Isolation of microbodies from plant tissues.

Specialized microbodies have previously been isolated and characterized from fatty seedling tissues (glyoxysomes) and leaves (leaf peroxisomes). We have now examined 11 other plant tissues, including tubers, fruits, roots, shoots, and petals, and find that all contain particulate catalase, a distinctive common enzyme component of microbodies. On linear sucrose gradients the catalase activity peaks sharply at a higher equilibrium density (1.20 to 1.25 gram per cm(3) in the various tissues) than the mitochondria (1.17 to 1.20). Only small amounts of protein are recovered in the fractions containing catalase, although a definite band is visible in preparations from some tissues, e.g., potato. As in the preparations from castor bean endosperm and spinach leaves for which comparable data are provided, the distribution of glycolate oxidase and uricase follows closely that of catalase on the gradients. The preparations from potato lack glyoxylate reductase and the transaminases, typical enzymes of leaf peroxisomes, and the distinctive enzymes of glyoxysomes are missing. Nonspecialized microbodies with limited enzyme composition can thus be isolated from a variety of plant tissues.

Journal Article↗

PubMed1970

Developmental studies on glyoxysomes in Ricinus endosperm.

The development of glyoxysomes and their associated enzymes, isocitrate lyase and malate synthetase, was studied in the endosperm of castor bean seeds during germination and early growth in darkness. The protein content of the glyoxysome fraction, separated by sucrose density centrifugation, increased linearly from day 2 to day 4 and declined subsequently, while maximum enzyme activities were reached at day 5. The specific activities of the enzymes in the glyoxysomes increased until day 5 and remained constant thereafter. At all stages of germination the only organelle with isocitrate lyase activity was the glyoxysome, but at the earlier stages a greater portion of the total activity was recovered in the soluble form. Malate synthetase was found primarily in the glyoxysomes after day 4, but at earlier stages part of the activity appeared at regions of lower density on the sucrose gradient. It was shown that this particulate malate synthetase activity was due to glyoxysomes broken during preparation, and that, as a result of this breakage, isocitrate lyase was solubilized. We conclude that both enzymes are housed in the glyoxysome in vivo throughout the germination period, and that the rise and fall in enzyme activities in phase with fat breakdown correspond to the net production and destruction of this organelle.

Carbon Isotopes↗

PubMed1970

Acetate metabolism in cell suspension cultures.

Cell suspension cultures of Paul's Scarlet rose were grown over a 14-day period, during which a 50-fold increase in fresh weight occurred. Three phases could be recognized from weight, DNA determinations, and microscopic examination. From days 0 to 7, cell division was accompanied by cell expansion; from days 7 to 10, only cell expansion occurred; and from days 10 to 14, there was no further growth.When acetate-1-(14)C was supplied continuously to 4-day and 12-day cells, (14)C was readily incorporated into lipid, CO(2), organic acids, amino acids, and protein. In the older cells, relatively greater amounts of (14)C were recovered in organic and amino acids, and accumulation of (14)C in these components continued after a steady rate of incorporation of (14)C into protein and (14)CO(2) had been established.Glutamate-(14)C was the most heavily labeled compound recovered after a pulse of acetate-1-(14)C in both cell types and was depleted when acetate-1-(14)C was removed, particularly rapidly in 4-day cells. (14)C was rapidly lost from the malate-(14)C labeled during the pulse of acetate-1-(14)C in 4-day cells, whereas malate-(14)C continued to increase in 12-day cells after the pulse. Glutamate-(14)C was shown to be the source of the (14)C accumulating in malate in 12-day cells.Several other soluble amino acids were labeled during a pulse of acetate-(14)C in both cell types. After the pulse most of the (14)C lost from these was recovered in the corresponding amino acid in protein, showing that only protein precursor pools had been labeled. The behavior of asparagine was exceptional since its (14)C content increased after the pulse and no turnover was apparent. The kinetics of labeling of aspartate, malate, and CO(2) showed that oxaloacetate generated in the tricarboxylic acid cycle following the pulse was not in equilibrium with aspartate.

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PubMed1969