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N E Tolbert

Publications and source records attributed to N E Tolbert.

At least 109 records · Page 6Linked to original sources
PubMed1973

Activation of polyphenol oxidase of chloroplasts.

Polyphenol oxidase of leaves is located mainly in chloroplasts isolated by differential or sucrose density gradient centrifugation. This activity is part of the lamellar structure that is not lost on repeated washing of the plastids. The oxidase activity was stable during prolonged storage of the particles at 4 C or -18 C. The Km (dihydroxyphenylalanine) for spinach leaf polyphenol oxidase was 7 mm by a spectrophotometric assay and 2 mm by the manometric assay. Polyphenol oxidase activity in the leaf peroxisomal fraction, after isopycnic centrifugation on a linear sucrose gradient, did not coincide with the peroxisomal enzymes but was attributed to proplastids at nearly the same specific density.Plants were grouped by the latency properties for polyphenol oxidase in their isolated chloroplasts. In a group including spinach, Swiss chard, and beet leaves the plastids immediately after preparation from fresh leaves required a small amount of light for maximal rates of oxidation of dihydroxyphenylalanine. Polyphenol oxidase activity in the dark or light increased many fold during aging of these chloroplasts for 1 to 5 days. Soluble polyphenol oxidase of the cytoplasm was not so stimulated. Chloroplasts prepared from stored leaves were also much more active than from fresh leaves. Maximum rates of dihydroxyphenylalanine oxidation were 2 to 6 mmoles x mg(-1) chlorophyll x hr(-1). Equal stimulation of latent polyphenol oxidase in fresh or aged chloroplasts in this group was obtained by either light, an aged trypsin digest, 3-(4-chlorophenyl)-1, 1-dimethylurea, or antimycin A. A variety of other treatments did not activate or had little effect on the oxidase, including various peptides, salts, detergents, and other proteolytic enzymes.Activation of latent polyphenol oxidase in spinach chloroplasts by trypsin amounted to as much as 30-fold. The trypsin activation occurred even after the trypsin had been treated with 10% trichloroacetic acid, 1.0 n HCl or boiled for 30 minutes. No single peptide from the digested trypsin was found to be the sole activating factor. About 0.25 mug of trypsin activated 50% the polyphenol oxidase activity in a standard chloroplast assay containing 2.1 mug of chlorophyll. Treatment of spinach chloroplasts with tris buffer or ethylenediamine tetraacetate extracted the ATPase activity, but the polyphenol oxidase activity remained with the broken plastids. However these treatments increased the latent polyphenol oxidase activity 50- to 100-fold.Chloroplasts from a second group of plants, including alfalfa, wheat, oats, peas, and sugarcane leaves, oxidized dihydroxyphenylalanine at a rate of 11 to 120 mumoles x mg(-1) chlorophyll x hr(-1). Polyphenol oxidase in these chloroplasts required a low intensity of red light for activity. Fifty or 75% activation of the oxidase in wheat chloroplasts required 4 to 6 foot candles of light and more light was required for alfalfa chloroplasts. Blue or far red light were ineffective. Trypsin was inhibitory. Upon aging chloroplasts from wheat leaves, but not alfalfa or peas, for 5 to 7 days at 4 C the total polyphenol oxidase activity did not increase, but the activation characteristics changed to those of chloroplasts from the spinach group. Chloroplasts from a third group of plants, including bean, tomato, and corn leaves, slowly oxidized dihydroxyphenylalanine in the dark and exhibited no latency.

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PubMed1973

The occurrence of glycolate dehydrogenase and glycolate oxidase in green plants: an evolutionary survey.

Homogenates of various lower land plants, aquatic angiosperms, and green algae were assayed for glycolate oxidase, a peroxisomal enzyme present in green leaves of higher plants, and for glycolate dehydrogenase, a functionally analogous enzyme characteristic of certain green algae. Green tissues of all lower land plants examined (including mosses, liverworts, ferns, and fern allies), as well as three freshwater aquatic angiosperms, contained an enzyme resembling glycolate oxidase, in that it oxidized l- but not d-lactate in addition to glycolate, and was insensitive to 2 mm cyanide. Many of the green algae (including Chlorella vulgaris, previously claimed to have glycolate oxidase) contained an enzyme resembling glycolate dehydrogenase, in that it oxidized d- but not l-lactate, and was inhibited by 2 mm cyanide. Other green algae had activity characteristic of glycolate oxidase and, accordingly, showed a substantial glycolate-dependent O(2) uptake. It is pointed out that this distribution pattern of glycolate oxidase and glycolate dehydrogenase among the green plants may have phylogenetic significance.Activities of catalase, a marker enzyme for peroxisomes, were also determined and were generally lower in the algae than in the land plants or aquatic angiosperms. Among the algae, however, there were no consistent correlations between levels of catalase and the type of enzyme which oxidized glycolate.

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PubMed1972

Isolation of Plastids from Sunflower Cotyledons during Germination.

Plastids from cotyledons of sunflower (Helianthus annus L.) seedlings, germinated in the dark or in the light, were isolated by isopycnic sucrose density gradient centrifugation. At all stages of development the whole plastids contained triose phosphate isomerase, NADPH-glyoxylate reductase, and l-dihydroxyphenylalanine oxidase, which were used as marker enzymes. At the beginning of germination the isopycnic density of whole plastids (proplastids) was about 1.22 g cm(-3). During development of proplastids into etioplasts in the dark, their isopycnic density increased to 1.26 g cm(-3). During exposure of germinating seedlings to white light for 2 days, the isopycnic density of whole plastids decreased from 1.26 to 1.22 g cm(-3). These changes in isopycnic density of plastids on sucrose density gradients are consistent with changes in the plastid ultrastructure caused by the protein-rich prolamellar body or by the lipid-rich thylakoids. Broken plastids (thylakoids), determined by the main peak of chlorophyll, increased in isopycnic density from less than 1.14 to about 1.17 g cm(-3) during illumination. During germination no major changes occurred in the isopycnic density of mitochondria. Microbodies had an isopycnic density of 1.24 g cm(-3) in very early stages of germination, and their density increased to 1.265 g cm(-3), when glyoxysomal enzymes reached maximum development.

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PubMed1971

3-Phosphoglycerate Phosphatase in Plants: II. Distribution, Physiological Considerations, and Comparison with P-Glycolate Phosphatase.

3-Phosphoglycerate phosphatase and phosphoglycolate phosphatase were found in leaves of all 52 plants examined. Activities of both phosphatases varied widely between 1 to 20 micromoles per minute per milligram chlorophyll. Plants were grouped into two categories based upon the relative ratio of activity of 3-phosphoglycerate phosphatase to phosphoglycolate phosphatase. This ratio varied between 2:1 to 4:1 in the C(4)-plants except corn leaves which had a low level of 3-phosphoglycerate phosphatase. This ratio was reversed and varied between 1:2 to 1:6 in all C(3)-plants except one bean variety which had large amounts of both phosphatases. By differential grinding procedures for C(4) plants a major part of the 3-phosphoglycerate phosphatase was found in the mesophyll cells and P-glycolate phosphatase in the bundle sheath cells. Phosphoglycolate phosphatase, but not 3-phosphoglycerate phosphatase, was located in chloroplasts of C(3)- and C(4)- plants. Formation of 3-phosphoglycerate phosphatase increased 4- to 12-fold during greening of etiolated sugarcane leaves. This cytosol phosphatase displayed a diurnal variation in sugarcane leaves by increasing 50% during late daylight hours and early evening. It is proposed that the soluble form of 3-phosphoglycerate phosphatase is necessary for carbon transport between the bundle sheath and mesophyll cells during photosynthesis by C(4)-plants. In C(3)- and C(4)-plants this phosphatase initiates the conversion of 3-phosphoglycerate to serine which is an alternate metabolic pathway to glycolate metabolism and photorespiration.

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PubMed1971

3-Phosphoglycerate Phosphatase in Plants: III. Activity Associated with Starch Particles.

A particulate form of 3-phosphoglycerate phosphatase represents about 20% of this activity in spinach (Spinacia oleracea var. Longstanding Bloomsdale) leaves. By differential and isopycnic sucrose density gradient centrifugation, all the particulate activity was found in starch grains that pelleted through 2.5 m sucrose. This particulate phosphatase was extremely stable, had a pH optimum of 5.8, and an apparent Michaelis constant (3-phosphoglycerate) of 9 x 10(-4)m. No cation requirement for activity could be demonstrated, and the enzyme was inhibited by 0.5 mm Zn(2+) or Cu(2+). The enzyme was most active in catalyzing the hydrolysis of 3-phospho-d-glycerate, but it was not substrate specific. The phosphatase from the starch grains could not be removed by washing, dialysis, homogenization, or treatment with a French pressure cell, but it was solubilized by prolonged sonication or by addition of 0.25 m MgCl(2), when the particles were suspended in 0.8 m sucrose. The solubilized enzyme was partially purified. The properties of the enzyme on the particles or after solubilization were similar to those previously described for the cytosol form of this phosphatase. It is conjectured that the phosphatase of the starch grain regulates glucan synthesis by controlling a 3-phosphoglycerate pool which is an effector for ADP-glucose pyrophosphorylase.

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PubMed1971

Development of Microbodies in Sunflower Cotyledons and Castor Bean Endosperm during Germination.

In cotyledons of sunflower seedlings glyoxysomal and peroxisomal enzymes exhibit different rates of development during germination. The total activity of isocitrate lyase, a glyoxysomal marker enzyme, rapidly increased during the first 3 days, and then decreased 89% by day 9. Exposure to light accelerated this decrease only slightly. The specific activity of glyoxysomal enzymes (malate synthetase, isocitrate lyase, citrate synthetase, and aconitase) in the microbody fraction from sucrose density gradients increased between days 2 and 4 about 2- to 3-fold, and thereafter it remained about constant in light or darkness.Total activity of the peroxisomal enzymes increased slowly in the dark during the first 4 days of germination and thereafter remained at a constant level of activity in the dark or increased 2-fold in 24 hours of light. The specific activties of glycolate oxidase, hydroxypyruvate reductase, and serine-glyoxylate aminotransferase in the isolated microbody fraction increased about 10-fold between days 2 and 4 in the dark and then remained constant or increased again 10-fold after an additional 48 hours in the light.The total activity of the common microbody marker, catalase, developed similarly to isocitrate lyase, but decreased only 72% by day 9. The specific activities of enzymes (catalase, malate dehydrogenase, and aspartate aminotransferase) common to both microbody systems were 10- to 1000-fold greater than those of other enzymes. It is proposed that malate and aspartate may be involved in hydrogen transport between microbodies and other cellular sites.Glutamate-glyoxylate aminotransferase was very active in microbodies from castor bean endosperm and sunflower cotyledons. The specific activity of this aminotransferase developed similarly to glyoxysomal enzymes in the dark but further increased in the light, as did peroxisomal enzymes.The microbody fraction of castor bean endosperm germinated in the dark for 5 days contained both glyoxysomal and peroxisomal enzymes of similar specific activity.Adjacent to the microbody fraction on sucrose gradients from sunflower cotyledons were etioplasts at slightly lower densities and protein bodies at similar and higher densities. Their presence in the microbody fractions resulted in artificially low specific activities.

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