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R N Trelease

Publications and source records attributed to R N Trelease.

49 records · Page 3Linked to original sources
PubMed1979

Malate synthase activity in cotton and other ungerminated oilseeds: a survey.

Extracts from several species and varieties of ungerminated cotton seeds plus homogenates from 18 other oilseeds (representing 11 different families) were examined for malate synthase and isocitrate lyase activity. Malate synthase activities in the various cotton seeds ranged from 35 to 129% of the units per dry seed weight found in Deltapine 16 cotton. For other oilseeds, the range was from 0.3 to 58% of Deltapine 16 cotton. Castor bean (Ricinus communis L.) had the least activity per mg dry weight (12-fold lower than the next lowest species), while Pima cotton (Gossypium barbadense L.) had the highest level (8.53 units). On a per seed basis, these values were 15 and 747 nanomoles per minute.Malate synthase activity was measurable in all seed types examined, whereas isocitrate lyase could not be detected in any of the seeds. We suggest that synthesis of malate synthase during seed development is universal among oilseeds in the absence of glyoxylate-cycle-associated isocitrate lyase activity.

Journal Article↗
PubMed1978

Control of Enzyme Activities in Cotton Cotyledons during Maturation and Germination: II. Glyoxysomal Enzyme Development in Embryos.

The sequence of glyoxysomal enzyme development was investigated in cotyledons of cotton (Gossypium hirsutum L. cv. Deltapine 16) embryos from 16 to 70 days after anthesis (DAA). Catalase, malate dehydrogenase, and citrate condensing enzyme activities were barely detectable prior to 22 DAA, but showed dramatic increases from 22 to 50 DAA. Development of malate synthase activity, however, was delayed during this period, rising to peak activity from 45 to 50 DAA (just prior to desiccation) in the absence of any detectable isocitrate lyase activity. Substantial activities of all of these enzymes (except isocitrate lyase) persisted in the dry seeds. Isopycnic centrifugations on sucrose gradients demonstrated that the enzymes were compartmentalized within particles increasing in buoyant density with time of development (1.226 to 1.245 grams per cubic centimeter from 22 to 50 DAA). Of particular significance were the observations in 22-day embryos of smooth surfaced membrane dilations of rough endoplasmic reticulum having cytochemical catalase reactivity, and the demonstrations of catalase activities in microsomal fractions isolated throughout the 16- to 50-DAA period. Our data do not allow determination of the mechanism(s) for enzyme activation and/or addition to previously existing or newly formed microbodies, but do show that development and acquisition of enzyme activities within glyoxysomes occur sequentially and thus are not regulated in concert as previously thought.

Journal Article↗
PubMed1976

Subcellular localization of glyoxylate cycle enzymes in Ascaris suum larvae.

Evidence is presented on the particulate nature of glyoxylate cycle enzymes in metazoa with the use of 15-day old larvae of the nematode Ascaris suum. Homogenization procedures were developed to disrupt the resistant nematode cuticle. Malate synthase and isocitrate lyase, key enzymes of the glyoxylate cycle, consistently sedimented with mitochondrial enzymes in differential pellets while catalase, a major peroxisomal enzyme, was always soluble. Isopycnic sucrose gradient centrifugation of the differential pellet yielded two protein peaks: one at 1.18 g/cm3 (characteristic for mitochondria), and another at 1.23 g/cm3 (common for glyoxysomes and peroxisomes). Electron microscopy of these fractions revealed that the lighter peak consisted primarily of mitochondria, while the heavier band contained proteinaceous bodies termed "dense granules" morphologically resembling microbodies. SIgnificantly, both malate synthase and isocitrate lyase cosedimented with the mitochondrial marker enzymes in the lighter peak (1.18 g/cm3) and not with the dense granules. Further purification of mitochondria, accomplished by separating dense granules with a step gradient before isopycnic centrifugation, substantiated the evidence that microbodies (glyoxysomes) do not occur in these nematode larvae. Rough-surfaced membranes were alternatively considered as the subcellular site, but the evidence tends to favor localization of the glyoxylate bypass enzymes in the mitochondria.

Animals↗
PubMed1975

Cytochemical demonstration of malate synthase and glycolate oxidase in microbodies of cucumber cotyledons.

The cytochemical localizations of malate synthase (glyoxysomal marker) and glycolate oxidase (peroxisomal marker) have been examined in cotyledon segments and sucrose-gradient fractions from germinated cucumber (Cucumis sativus L.) seedlings. The seedlings were grown in the dark for 4 days, transferred to 4 hours of continuous light, then returned to the dark for 24 hours. Under these conditions, high specific activities for both glyoxysomal and peroxisomal enzymes are maintained in cotyledon homogenates and microbody-enriched fractions. Electron cytochemistry of the marker enzymes reveals that all or virtually all the microbodies observed in cotyledonary cells and sucrose-gradient fractions contain both enzymes. The staining in gradient fractions was determined from scoring a minimum of 600 photographed microbodies for each enzyme. After correcting for the number of particles stained for catalase reactivity (representing true microbodies), 94 and 97% of the microbodies were found stained for malate synthase and glycolate oxidase activity, respectively.The results from these studies provide pertinent information toward understanding the succession from glyoxysomal to peroxisomal metabolism in cotyledons of fatty seedlings. The coexistence of two separate microbody types functioning at different stages of development apparently is not the case. The localizations of both marker enzymes within one microbody type strongly suggest that the metabolic transition involves a change in enzyme complement within an ongoing population of microbodies.

Journal Article↗
PubMed1975

Ultrastructure of developing Ascaris larvae undergoing lipid to carbohydrate interconversion.

Triglyceride utilization is correlated with glycogen resynthesis in developing Ascaris suum larvae. Glycogen content, determined by amyloglucosidase hydrolysis at 2-day intervals, decreases sharply during embryonation from 15% (per mg dry weight) at day 4 to 2% at day 12. Thereafter glycogen increases 3-fold through day 20, marking the resynthesis period. Triglyceride lipid droplets are confirmed primarily to the posterior half of the larvae and mark the site of the interconversion. Although they serve as precursors to glycogen, only a small diminution was observed ultrastructurally. Glycogen accumulation, on the other hand, correlates well with increases determined biochemically. alpha-glycogen builds up among lipid droplets, while beta-glycogen concentrates in the cytoplasm of somatic muscle cells paralleling myofibril development. Dense granules, restricted to the lipid body region, are considered as the possible subcellular site for the enzymatic conversion.

Ascaris↗
PubMed1974

Cytochemical localization of malate synthase in glyoxysomes.

Cytochemical staining techniques for microbodies (peroxisomes) are limited at present to the enzymes catalase and alpha-hydroxy acid oxidase, and neither technique can distinguish glyoxysomes from other microbodies. Described here is a procedure using ferricyanide for the cytochemical demonstration by light and electron microscopy of malate synthase activity in glyoxysomes of cotyledons from fat-storing cucumber and sunflower seedlings. Malate synthase, a key enzyme of the glyoxylate cycle, catalyzes the condensation of acetyl CoA with glyoxylate to form malate and release free coenzyme A. Localization of the enzyme activity is based on the reduction by free CoA of ferricyanide to ferrocyanide, and the visualization of the latter as an insoluble, electron-opaque deposit of copper ferrocyanide (Hatchett's brown). The conditions and optimal concentrations for the cytochemical reaction mixture were determined in preliminary studies using a colorimetric assay developed to measure disappearance of ferricyanide at 420 nm. Ultrastructural observation of treated tissue reveals electron-opaque material deposited uniformly throughout the matrix portion of the glyoxysomes, with little background deposition elsewhere in the cell. The reaction product is easily visualized in plastic sections by phase microscopy without poststaining. Although the method has been applied thus far only to cotyledons of fat-storing seedlings, it is anticipated that the technique will be useful in localizing and studying glyoxylate cycle activity in a variety of tissues from both plants and animals.

Copper↗
PubMed1971

Microbodies (Glyoxysomes and Peroxisomes) in Cucumber Cotyledons: Correlative Biochemical and Ultrastructural Study in Light- and Dark-grown Seedlings.

The changes in activities of glyoxysomal and peroxisomal enzymes have been correlated with the fine structure of microbodies in cotyledons of the cucumber (Cucumis sativus L.) during the transition from fat degradation to photosynthesis in light-grown plants, and in plants grown in the dark and then exposed to light. During early periods of development in the light (days 2 through 4), the microbodies (glyoxysomes) are interspersed among lipid bodies and contain relatively high activities of glyoxylate cycle enzymes involved in lipid degradation. Thereafter, these activities decrease rapidly as the cotyledons expand and become photosynthetic, and the activity of glycolate oxidase rises to a peak (day 7); concomitantly the microbodies (peroxisomes) become preferentially associated with chloroplasts.In seedlings grown in the dark for 10 days, the reserve lipid and the glyoxylate cycle enzyme activities persist for a longer time than in the light; correlated with this, there is a continued association of the microbodies with the lipid bodies. When these dark-grown seedlings are then exposed to 51 hours of the light-dark cycle, peroxisomal marker enzymes increase rapidly in activity, and the microbodies become appressed to chloroplasts. We conclude that the characteristic association observed between glyoxysomes and lipid bodies reflects their mutual involvement in net gluconeogenesis through the conversion of fatty acids to carbohydrate, while the close spatial relationship observed between peroxisomes and chloroplasts at later stages of development reflects their mutual involvement in glycolate metabolism.Although glyoxysomal enzyme activities are dropping rapidly while peroxisomal enzyme activities are increasing rapidly during the transition period in the light, the electron microscopic evidence does not indicate that glyoxysomes are being degraded or peroxisomes are being formed. Since in the dark-grown seedlings the activities of peroxisomal enzymes remain low and do not increase as they do in the light, an opportunity is afforded to compare quantitatively any changes in numbers of microbodies per cell with the changes in activities of glyoxysomal enzymes. It is found that the magnitude of the decrease in numbers of microbodies is considerably less than that of the decrease in glyoxysomal enzyme activities between days 4 and 10. When the cotyledons are exposed to light, peroxisomal enzyme activities increase greatly, but again there is no ultrastructural evidence for the synthesis of a new population of microbodies to accommodate this increase. These results allow us to conclude that the developmental transition from glyoxysomal to peroxisomal function almost certainly does not involve the actual replacement of one population of microbodies by another. Rather, the transition probably occurs within existing particles, either by a sequential functioning of two different kinds of microbodies or by a change in enzyme complement within a single population. Our findings with both light- and dark-grown cotyledons favor the latter possibility. The cytoplasmic invaginations into microbodies seen during greening of both light-grown cotyledons and etiolated cotyledons exposed to light may be morphological manifestations of the mechanism by which the microbodies lose or gain enzymes.

Journal Article↗