The substrate specificity, kinetics, and mechanism of glycerate-3-kinase from spinach leaves

Glycerate-3-kinase (EC 2.7.1.31) from spinach leaves shows absolute specificity for D-glycerate as phosphate acceptor, yielding 3-phosphoglycerate as a product. ATP complexed with either Mg2+ or Mn2+ is the preferred phosphate donor. The enzyme has Km (D-glycerate) = 0.25 mM, Km (Mg-ATP) = 0.21 mM, Vmax = 300 mumol min-1 mg protein-1, and a turnover number = 12,000 X min-1. The equilibrium constant for the reaction is approximately 300 at pH 7.8. Pyrophosphate, 3-phosphoglycerate and ribulose 1,5-bisphosphate are the strongest inhibitors among the phosphorylated and nonphosphorylated metabolites tested; however, their regulatory role in vivo is questioned. Substrate kinetics, as well as product and analog inhibition data, are consistent with a sequential random mechanism. The distinct characteristic of the glycerate kinase-catalyzed reaction is the formation of a dead-end complex between the enzyme, D-glycerate, and 3-phosphoglycerate.     

Translocation of photoassimilate from leaves of two popyploid genotypes of tall fescue differing in photosynthetic rates

The photosynthetic rate of a decaploid genotype (1-16-2) of tall fescue ( Festuca arundinacea Schreb.) is about twice that of a common hexaploid genotype (V6-802) (Plant Physiol. 72: 16–21, 1983). Translocation of photosynthate out of the leaves is a possible means of regulating carbon assimilation. To evaluate this possibility, we have examined a) translocation velocity, b) time course of translocation from leaves, c) photoassimilate partitioning pattern into whole plants in pulse and chase experiments, and d) interveinal distances between two ploidy genotypes. Most of the ¹⁴C accumulated in sucrose, and the labelled carbon moved down the leaf blades at similar velocities (6 to 10 cm h⁻¹) in both genotypes. Recent ¹⁴C assimilate was rapidly translocated from the fed area of the leaf blade. For example, the decaploid and the common hexaploid had translocated 40 and 26% of the ¹⁴C, respectively, at 6 h, and 79 and 49% of the ¹⁴C, respectively, at 24 h. Partitioning of ¹⁴C among plant organs was considerably different between the genotypes after a 24 h chase. For example, out of the total ¹⁴C recovered from the whole plant, the decaploid had retained 40% in the labelled leaf with 10, 33 and 29% in other leaves, stem bases and roots, respectively; whereas the hexaploid had retained 91% in the labelled leaf with 4, 3 and 2% in other leaves, stem bases and roots, respectively. However, the higher rate of translocation was correlated with greater interveinal distances in the decaploid genotype. These results suggested that the higher translocation percentage in the decaploid than the hexaploid genotype was due to greater sink activity.     

Purification and Characterization of the Pea Chloroplast Pyruvate Dehydrogenase Complex : A Source of Acetyl-CoA and NADH for Fatty Acid Biosynthesis

The pyruvate dehydrogenase complex has been purified 76-fold, to a specific activity of 0.6 μmoles per minute per milligram protein, beginning with isolated pea (Pisum sativum L. var Little Marvel) chloroplasts. Purification was accomplished by rate zonal sedimentation, polyethyleneglycol precipitation, and ethyl-agarose affinity chromatography. Characterization of the substrates as pyruvate, NAD⁺, and coenzyme-A and the products as NADH, CO₂, and acetyl-CoA, in a 1:1:1 stoichiometry unequivocally established that activity was the result of the pyruvate dehydrogenase complex. Immunochemical analysis demonstrated significant differences in structure and organization between the chloroplast pyruvate dehydrogenase complex and the more thoroughly characterized mitochondrial complex. Chloroplast complex has a higher magnesium requirement and a more alkaline pH optimum than mitochondrial complex, and these properties are consistent with light-mediated regulation in vivo. The chloroplast pyruvate dehydrogenase complex is not, however, regulated by ATP-dependent inactivation. The properties and subcellular localization of the chloroplast pyruvate dehydrogenase complex are consistent with its role of providing acetyl-CoA and NADH for fatty acid synthesis.     

α-Mannosidase-1 mutants of Dictyostelium dkoideum: Early aggregation-essential genes regulate enzyme precursor synthesis, modification, and processing

The lysosomal enzyme alpha-mannosidase-1 is one of the earliest developmentally controlled gene products in Dictyostelium discoideum. Although this enzyme is synthesized throughout the first 20 h of development, it is not required for complete morphogenesis, since structural gene (manA) mutants lacking activity develop normally. We isolated six strains deficient in alpha-mannosidase-1 activity which, unlike structural gene mutants, fail to aggregate. Fruiting revertants of these strains accumulate wild-type levels of alpha-mannosidase-1 activity, suggesting that both the enzymatic and morphological defects are caused by single mutations in nonstructural genes essential for early development. Direct genetic evidence for mutations outside of the structural gene was obtained by complementation analysis. We used alpha-mannosidase-1-specific monoclonal antibodies to analyze the biochemical defects in these mad (alpha-mannosidase-1-deficient) mutants. All mad mutants show a significantly reduced relative rate of enzyme precursor biosynthesis. The mad-404 mutation results in a complete lack of precursor biosynthesis, as well as a lack of functional alpha-mannosidase-1 mRNA. In some cases, however, the enzymatic defect results from improper post-translational modification which affects precursor processing. We conclude that a small number of aggregation-essential genes are involved in regulating the synthesis, modification, and processing of alpha-mannosidase-1 during development.     

Cellular location of heat-labile enterotoxin in Escherichia coli.

We demonstrated that both the A and B subunits of heat-labile enterotoxin from Escherichia coli are located in the periplasm. The toxin was shown to form aggregates in Tris-EDTA buffers which are routinely used for isolating membranes. The aggregates pellet upon centrifugation, and this may explain why several previous investigators have concluded that enterotoxin is associated with membranes.     

Assay of glutamine phosphoribosylpyrophosphate amidotransferase using [1-C]phosphoribosylpyrophosphate

Glutamine phosphoribosylpyrophosphate amidotransferase (EC 2.4.2.14) catalyzes the transfer of the amide group of glutamine to 5-phospho-α- -ribose-1-pyrophosphate. It is the first enzyme committed to the synthesis of purines by the de novo pathway. Previous assays of enzyme activity have either measured the phosphoribosylpyrophosphate-dependent disappearance of radioactive glutamine or have linked this reaction to subsequent steps in the purine pathway. A new assay for activity of the enzyme by directly measuring the synthesis of the product of the reaction, 5-β-phosphoribosyl-1-amine, using [1-¹⁴C]phosphoribosylpyrophosphate as substrate is described. Substrate and product are separated by thin-layer chromatography and identified by autoradiography. Glutamine or ammonia may be used as substrates; the apparent K_m values of the human lymphoblast enzyme are 0.46 m for glutamine and 0.71 m for ammonia. GMP is a considerably more potent inhibitor of the human lymphoblast enzyme than is AMP; 6-diazo-5-oxo- -norleucine inhibits only glutamine-dependent activity and has no effect on ammonia-dependent activity.     

Photosynthesis in Tall Fescue : IV. Carbon Assimilation Pattern in two Genotypes of Tall Fescue Differing in Net Photosynthesis Rates

We previously reported that the net photosynthetic rate of a decaploid genotype (I-16-2) of tall fescue (Festuca arundinacea Schreb.) was 32 to 41 versus 22 milligrams CO₂ per square decimeter per hour in a hexaploid genotype (V6-802) (Randall, Nelson, Asay Plant Physiol 59: 38-41). The high rate was later correlated with increases in total ribulose 1,5-bisphosphate carboxylase protein (17%) and activity (27%) (Joseph, Randall, Nelson Plant Physiol 68: 894-898). This report characterizes photosynthesis with respect to light saturation and early products of photosynthesis in an attempt to identify regulatory metabolic site(s) in these two genotypes. Analysis of the early products of photosynthesis indicated that both genotypes fixed CO₂ via the Calvin-Benson cycle with phosphoglyceric acid as the initial primary product. Both genotypes had similar ¹⁴C-labeled intermediates. Sucrose was the primary sink of ¹⁴CO₂ assimilation. After 10 min of ¹⁴CO₂ assimilation with attached leaves, sucrose accounted for 89% (decaploid) and 81% (hexaploid) of the total ¹⁴C incorporated. In 10 min, this amounted to 1.3 (decaploid) and 0.8 (hexaploid) μmol [¹⁴C]sucrose formed g fresh weight⁻¹ and reflected the observed differences in photosynthetic rates. There was limited labeling of starch (1%) and fructan (1%). Results of total nonstructural carbohydrates and P_i analysis also demonstrated sucrose was the predominant carbohydrate in fescue leaves. Quantitative differences in sucrose and P_i between the two genotypes may reflect changes in partitioning and this possibility is discussed.     

Soybean leaf urease: Comparison with seed urease

Soybeans, Glycine max (L.) Merr., from ureides for transport of nitrogen from the root nodule to the shoot. The most direct routes for ureide utilization include the degradation of ureide-derived urea to NH₃ and CO₂. Ureolytic activity was found in leaf disks of soybean and exhbited optimal activity at pH 7 in the presence of a high concentration of urea (250 m M ). In vitro studies showed neither urea amidolyase nor urea dehydrogenase activity in soybean leaves and the ureolytic activity was characterized as urease. Several biochemical properties of soybean leaf urease were determined and compared to seed urease properties. Soybean leaf urease differed from that of seed in five ways: pH optima (5.25 and 8.75), apparent K_m (0.8 m M ), no inhibition by hydroxyurea, faster electrophoretic mobility and no cross-reactivity with soybean seed urease antibodies. The data suggest that urease is the primary urea metabolizing enzyme present in soybean leaves. The properties of soybean leaf urease support the conclusion that a unique isozyme of urease is present in leaf tissue.     

Regulation of Legumin Levels in Developing Pea Seeds under Conditions of Sulfur Deficiency: Rates of Legumin Synthesis and Levels of Legumin mRNA

It was shown previously that when peas (Pisum sativum L.) are grown with suboptimal sulfur supply the level of legumin (the more S-rich of the two major seed storage proteins) in the mature seed is selectively reduced (Randall, Thomson, Schroeder, 1979 Aust J Plant Physiol 6: 11-24). This paper reports a study of the cellular mechanisms involved in regulating legumin synthesis under these conditions. Pulse and pulse-chase labeling experiments were carried out with excised, immature cotyledons from normal and S-deficient plants. Legumin was isolated from cotyledon extracts by immunochromatography, and the proportion of legumin synthesis relative to total protein synthesis was determined. Results showed that reduced legumin accumulation could largely be accounted for by a greatly reduced level of legumin synthesis (80-88% reduction) rather than by a major increase in legumin breakdown.

Legumin mRNA levels were assayed by two methods. In vitro translation of polysomal RNA from cotyledons of normal and S-deficient plants indicated a reduction of 60 to 70% in synthesis of legumin-related products by preparations from S-deficient plants. A legumin cDNA clone was constructed, characterized, and used to measure the levels of legumin mRNA in polysomal and total RNA preparations from developing cotyledons. Legumin mRNA levels were reduced by 90% in preparations from S-deficient plants.

When restored to an adequate S supply, S-deficient plants (or pods taken from such plants) recovered normal levels of legumin synthesis (in vivo and in vitro) and of legumin mRNA. These results indicate that reduced legumin accumulation under conditions of S deficiency is primarily a consequence of reduced levels of legumin mRNA.