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J Giovanelli

Publications and source records attributed to J Giovanelli.

At least 37 records · Page 2Linked to original sources
PubMed1980

Lemna paucicostata Hegelm. 6746: DEVELOPMENT OF STANDARDIZED GROWTH CONDITIONS SUITABLE FOR BIOCHEMICAL EXPERIMENTATION.

Photoautotrophic and mixotrophic growth of Lemna paucicostata Hegelm. 6746 (formerly Lemna perpusilla Torr. 6746) was investigated to establish standardized conditions for biochemical studies. Optimal temperature for growth was 29 to 30 C. The medium used previously (Datko AH, Mudd SH, Giovanelli J 1977 J Biol Chem 252: 3436-3445) was modified by inclusion of NH(4)Cl, decreasing macronutrient and ethylenediamine tetraacetate concentration, increasing micronutrient concentration, and inclusion of bicarbonate (for photoautotrophic growth) or 2-(N-morpholino)ethanesulfonic acid (for mixotrophic growth) buffers. Varying the sulfate concentration between 14 and 1 millimolar had no effect on growth. For photoautotrophic growth in the new medium (medium 4), the effects of CO(2) concentration, light intensity, and pH were measured. Under the optimal conditions, a multiplication rate (MR) of 300 to 315, equivalent to a doubling time of 23 to 24 hours was obtained. Addition of glutamine or asparagine did not increase this MR. For mixotrophic growth in low light, the effects of sucrose concentration and pH were determined. Under optimal conditions, MR was 210. A concentration of sucrose less than maximal for growth was chosen for the medium for experiments which will include (14)C-labeling of intermediates. MR under these conditions was 184. Growth was equally good in medium 4 and in half-strength Hutner's medium when sulfate was high (0.4 to 1 millimolar), but better in medium 4 when sulfate was low (20 micromolar). Growth rates could be restored to normal in half-strength Hutner's with low sulfate by decreasing the molybdate concentration.By modifying medium 4 to contain very low amounts of sulfate, and by preconditioning medium and plants, it was shown that there was an increment in plant protein of approximately 2.5 micrograms per nanomole of added MgSO(4). Colonies undergoing sulfur limitation exhibited a slow growth rate and a high frond to colony ratio. Molybdate and selenate produced growth inhibition reversible by sulfate. Conditions were developed in which the plants could be maintained indefinitely in the presence of either molybdate or selenate in altered metabolic steady-states with lowered growth rates and protein per frond.

Journal Article↗
PubMed1980

Lemna paucicostata Hegelm. 6746: LIFE CYCLE AND CHARACTERIZATION OF THE COLONY TYPES IN A POPULATION.

The sequence of frond emergence and the intervals required for daughter colony separation have been determined for Lemna paucicostata Hegelm. 6746 growing under standardized conditions. After separation of a new mother colony, the first daughter colony is produced from the left meristematic pocket and separates after approximately 60 hours, the second daughter, produced from the right pocket, separates after a further 30 hours, and the third daughter, again from the left, after a further 40 hours. The pattern of alternating longer and shorter intervals for separation of daughters continues throughout the life of the mother colony.Protein contents of fronds and whole colonies were determined either by chemical methods or by labeling to isotopic equilibrium with (35)SO(4) (2-). The smallest fronds measured were 5 to 6% of the final area they would attain when fully expanded and contained 10% of the protein they would finally contain. Thus, most protein accumulation occurred during the phase of rapid frond expansion rather than in an earlier primordial stage. The specific rates of protein accumulation were 0.027 to 0.030 micrograms per microgram protein per hour during rapid frond expansion and relatively constant thereafter at 0.014 to 0.019 for whole colonies until at least four daughter colonies had separated.A substantial amount of (35)S is transferred directly from the mother frond to its attached daughter fronds. The results are consistent with the transfer of soluble compounds arising through turnover of protein in the mother frond.Tree diagrams and a mathematical treatment were utilized to relate the distribution of colony types and the over-all doubling time in a large population of Lemna colonies to the doubling times and modes of division of the individual colonies within that population. It was shown that a population with the growth properties described increases exponentially and that within a relatively few generations reaches an equilibrium population distribution which is independent of the initial proportions of the members of the population. The measured distribution of colony types producing odd-numbered as compared to those producing even-numbered daughters was the same as that predicted by the theoretical analyses. Once a mass culture of Lemna paucicostata has attained its equilibrium distribution of colony types, under our standard conditions the proportions of colonies in which the indicated daughter will be next to separate will be: 60%, first; 15%, second; 11%, third; 5%, fourth; 8% fifth or higher. Thus, about 91% of colonies will have produced no more than four daughter colonies. For at least this period the specific rate of protein accumulation is relatively constant and senescent changes in mother fronds are rare. In the equilibrium distribution, 39% of colonies will themselves be first daughters, 25% will be second daughters, 14% will be third daughters, 9% fourth, and 13% greater than fourth daughters. These combined results suggest that obtaining representative samples of colonies from such a population for biochemical studies should be relatively simple.

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PubMed1978

Homocysteine biosynthesis in green plants. Physiological importance of the transsulfuration pathway in Chlorella sorokiniana growing under steady state conditions with limiting sulfate.

The physiological roles of the transsulfuration and direct sulfhydration pathways in Chlorella sorokiniana growing under steady state photoautotrophic conditions with limiting sulfate were studied by following the patterns of assimilation of 35SO4(2-) into sulfur amino acids. The labeling patterns expected of each pathway were defined by means of models based on the rates of net synthesis of the terminal pools of GSH, protein cysteine, and protein methionine. The labeling patterns observed are entirely consistent with the transsulfuration pathway and inconsistent with the direct sulfhydration pathway. By analysis of the amounts of radioactivity present in key intermediates at labeling times as short as 1 s, it was demonstrated that direct sulfhydration makes no detectable contribution to homocysteine biosynthesis, and if operative contributes no more than approximately 3% of the total homocysteine biosynthesized. From the combined determinations of the initial rates of labeling and net rates of synthesis of the various sulfur amino acids, a tentative working model is presented that summarizes our best current estimates of the major fluxes of sulfur in the experimental system. The labeling data further showed that soluble cysteine consists of at least two pools. One pool, termed "rapidly turning over" cysteine comprises less than 1% of the total soluble cysteine, and is the precursor of GSH, protein cysteine, and, almost certainly, cystathionine. The other pool, "slowly turning over" cysteine, appears to be in equilibrium with "rapidly turning over" cysteine, but not to be further metabolized.

Chlorella↗
PubMed1978

Phytostat for the growth of lemna in semicontinuous culture with low sulfate.

An apparatus is described by means of which Lemna perpusilla 6746 was grown photoautotrophically at a series of constant concentrations of inorganic sulfate, as low as 0.26 mum. Theoretical considerations relevant to this system are discussed and examples of the operation of the apparatus are presented. The data obtained were used to calculate sulfate uptake by the plant colonies as a function of sulfate concentration. The apparatus should be useful for the production of relatively large quantities of Lemna, grown under highly uniform conditions, with or without limitation of one or more nutrient(s). Other small vegetatively reproducing aquatic plants could be similarly studied. Uptake studies could be carried out on a variety of plant materials.

Journal Article↗
PubMed1978

Sulfur-containing Compounds in Lemna perpusilla 6746 Grown at a Range of Sulfate Concentrations.

Lemna perpusilla 6746, grown photoautotrophically at a series of sulfate concentrations ranging from 0.32 to 1,000 mum, was labeled to radioisotopic equilibrium with (35)SO(4) (2-). Sulfur-containing compounds were isolated and purified from the colonies. Radioactivity in each compound was a measure of the amount of that compound present in the tissue. The following compounds were identified and quantitated: inorganic sulfate, glutathione, homocyst(e)ine, cyst(e)ine, methionine, S-methylmethionine sulfonium, S-adenosylmethionine, S-adenosylhomocysteine, cystathionine, chloroformsoluble (presumed to be sulfolipid), protein cyst(e)ine, and protein methionine. gamma-Glutamylcyst(e)ine, erythro- and threo-thiothreonine, and S-methylcysteine were not detected. No volatile (35)S compounds were formed during plant growth at 1,000 mum sulfate, nor were significant amounts of (35)S compounds excreted into the medium.The amount of each component present in colonies grown over the 3,000-fold range of medium sulfate was relatively constant except for inorganic sulfate. This increased about 30-fold from the lowest to the highest medium sulfate concentration. The total soluble sulfur amino acids increased about 1.5- to 2-fold, due primarily to an increased amount of glutathione. Protein cyst(e)ine and protein methionine were the major organic sulfur compounds in Lemna, and the amounts of these compounds remained virtually constant despite the variation in external sulfate concentration.Procedures for the analysis of S-adenosylmethionine, S-methylmethionine sulfonium, and S-adenosylhomocysteine are presented.

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PubMed1974

Homoserine esterification in green plants.

EXTRACTS OF PHYLOGENETICALLY DIVERSE PLANS WERE SURVEYED FOR THEIR ABILITY TO SYNTHESIZE THE FOLLOWING HOMOSERINE ESTERS WHICH ARE POTENTIAL PRECURSORS FOR METHIONINE AND THREONINE SYNTHESIS IN GREEN PLANTS: O-acetyl-, O-oxalyl-, O-succinyl-, O-malonyl-, and O-phosphohomoserine. Synthesis of O-acylhomoserine esters was detected only in Pisum sativum L. and Lathyrus sativus L. Extracts of P. sativum, a plant known to accumulate O-acetylhomoserine, catalyzed the specific synthesis of this ester from homoserine and acetyl-CoA. Extracts of L. sativus, a plant known to accumulate O-oxalylhomoserine, catalyzed the specific synthesis of this ester from homoserine and oxalyl-CoA. None of the other plants surveyed, including representatives of the green algae, horsetails, gymnosperms, and angiosperms, catalyzed the synthesis of any of the O-acylhomoserine esters studied. In contrast, synthesis of O-phosphohomoserine by the reaction catalyzed by homoserine kinase was demonstrated in extracts of all plants examined, including the two exceptional legumes.These results suggest that, among the five homoserine esters studied, O-phosphohomoserine is the major activated homoserine derivative in plants. Direct confirmation of the dominant physiological role of O-phosphohomoserine in the synthesis of cystathionine in the transsulfuration pathway of methionine biosynthesis in plants has recently been provided (Datko, A. H., Giovanelli, J., and Mudd, S. H. 1974. J. Biol. Chem. 249: 1139-1155).

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PubMed1973

beta-Cystathionase In Vivo Inactivation by Rhizobitoxine and Role of the Enzyme in Methionine Biosynthesis in Corn Seedlings.

Rhizobitoxine has previously been shown to inactivate irreversibly beta-cystathionase isolated from spinach. In the present studies, rhizobitoxine was shown to inhibit partially beta-cystathionase of spinach and corn seedlings in vivo. An activity of 30 to 40% of normal remained in toxin-treated seedlings of both spinach and corn. Possible reasons for the partial inhibition are discussed.Rhizobitoxine-treated and control corn seedlings were allowed to assimilate (35)SO(4) (2-) for 3 or 6 hours, and the radioactivity incorporated into sulfur amino acids at these times was studied. The most striking effect of rhizobitoxine was an increase (up to 22-fold) in radioactive cystathionine. Accumulation of radioactivity in methionine was only slightly inhibited by rhizobitoxine treatment. The results strongly suggest that the transsulfuration pathway contributes to methionine biosynthesis, and that metabolism via this pathway is impaired, but not entirely eliminated, by rhizobitoxine treatment.The present data do not permit decisions about the relative contributions of the transsulfuration and the direct sulfhydration pathways to methionine biosynthesis, or whether the pathological effects of rhizobitoxine are due chiefly to inhibition of beta-cystathionase.

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