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UDP-glucose pyrophosphorylase (EC 2.7.7.9) has been highly purified from the plant fraction of soybean (Glycine max L. Merr. cv Williams) nodules. The purified enzyme gave a single polypeptide band following sodium docecyl sulphate polyacryla-mide gel electrophoresis, but was resolved into three bands of activity in non-denaturing gels. The enzyme appeared to be a monomer of molecular weight between 30 and 40 kDa. UDP-glucose pyrophosphorylase had optimum activity at pH 8.5 and displayed typical hyperbolic kinetics. The enzyme had a requirement for divalent metal ions, and was highly specific for the substrates pyrophosphate and UDP-glucose in the pyrophosphorolysis direction, and glucose-1-phosphate and UTP in the direction of UDP-glucose synthesis. The Km values were 0.19 mM and 0.07 mM for pyrophosphate and UDP-glucose, respectively, and 0.23 mM and 0.11 mM for glucose-1-phosphate and UTP. The maximum velocity in the pyrophosphorolysis direction was almost double that for the reverse reaction. UDP-glucose pyrophosphorylase did not appear to be subject to a high degree of fine control, and activity in vivo may be regulated mainly by the availability of the substrates.

Proteome analyses were carried out on commercial wheat germ of mature grain from the biscuit-making wheat cultivar, Rosella. Wheat germ protein extracts were fractionated by two-dimensional gel electrophoresis across two different immobilised pH gradients: pH 4.0-7.0 and 6.0-9.0. A total of 612 individual protein spots were excised from the gels and characterised by peptide mass fingerprinting. From these analyses, 347 individual proteins were identified from protein sequence database interrogation, and 301 different types of protein were catalogued according to protein function. The remaining 265 protein spots gave poor or no matches to proteins in the databases and were not identified in this study. Six different classes of enzymes were identified in the germ, many of them having roles in the mobilisation of energy reserves for germination. Abundantly expressed enzyme classes include the oxidoreductases, transferases and hydrolases. A comparison was also made between the major protein classes expressed in the germ and protein classes expressed in the endosperm from previous proteomic work. This study contributes significantly to our knowledge of protein expression and heterogeneity in the germ of wheat grain and forms the basis for future studies in regard to the characterisation of proteins during the initial stages of germination.

Analysis of the content and distribution of organic acids in chickpea plants (Cicer arietinum L.) showed that malonate was the most abundant acid in roots and nodules, whereas malate was the main acid in leaves and stems. The highest concentration of malonate in roots was in the apices. Malonate metabolism did not appear to be directly related to abiotic stress. We suggest that malonate has a role as a defensive chemical in roots and nodules of chickpeas.

To investigate why Rhizobium sp. (Cicer) strain CC 1192 cells accumulate poly-R-3-hydroxybutyrate in the free-living state but not as bacteroids in nodules on chickpea (Cicer arietinum L.) plants, we have examined the kinetic properties of acetyl coenzyme A (acetyl-CoA) acetyltransferase (also known as acetoacetyl-CoA thiolase and 3-ketothiolase [EC 2.3.1.9]) from both types of cells. The enzyme had a native molecular mass of 180 (plusmn) 4 kDa, and the subunit molecular mass was 44 (plusmn) 1 kDa. The seven amino acids from the N terminus were Lys-Ala-Ser-Ile-Val-Ile-Ala. Thiolysis and condensation activity of the enzyme from free-living CC 1192 cells were optimal at pHs 7.8 and 8.1, respectively. The relationship between substrate concentrations and initial velocity for the thiolysis reaction were hyperbolic and gave K(infm) values for acetoacetyl-CoA and CoA of 42 and 56 (mu)M, respectively. The maximum velocity in the condensation direction was approximately 10% of that of the thiolysis reaction. With highly purified preparations of the enzyme, a value of approximately 1 mM was determined for the apparent K(infm) for acetyl-CoA. However, with partially purified enzyme preparations or when N-ethylmaleimide was included in reaction mixtures the apparent K(infm) for acetyl-CoA was close to 0.3 mM. In the condensation direction, CoA was a potent linear competitive inhibitor with an inhibition constant of 11 (mu)M. The much higher affinity of the enzyme for the product CoA than the substrate acetyl-CoA could have significance in view of metabolic differences between bacteroid and free-living cells of CC 1192. We propose that in free-living CC 1192 cells, the acetyl-CoA/CoA ratio reaches a value that allows condensation activity of acetyl-CoA acetyltransferase, but that in CC 1192 bacteroids, the ratio is poised so that the formation of acetoacetyl-CoA is not favored.

The enzymatic capacity for metabolism of poly-(beta)-hydroxybutyrate (PHB) has been examined in nitrogen-fixing symbioses of soybean (Glycine max L.) plants, which may accumulate substantial amounts of PHB, and chickpea (Cicer arietinum L.) plants, which contain little or no PHB. In the free-living state, both Bradyrhizobium japonicum CB 1809 and Rhizobium sp. (Cicer) CC 1192, which form nodules on soybean and chickpea plants, respectively, produced substantial amounts of PHB. To obtain information on why chickpea bacteroids do not accumulate PHB, the specific activities of enzymes of PHB metabolism (3-ketothiolase, acetoacetyl-coenzyme A reductase, PHB depolymerase, and 3-hydroxybutyrate dehydrogenase), the tricarboxylic acid cycle (malate dehydrogenase, citrate synthase, and isocitrate dehydrogenase), and related reactions (malic enzyme, pyruvate dehydrogenase, and glutamate:2-oxoglutarate transaminase) were compared in extracts from chickpea and soybean bacteroids and the respective free-living bacteria. Significant differences were noted between soybean and chickpea bacteroids and between the bacteroid and free-living forms of Rhizobium sp. (Cicer) CC 1192, with respect to the capacity for some of these reactions. It is suggested that a greater potential for oxidizing malate to oxaloacetate in chickpea bacteroids may be a factor that favors the utilization of acetyl-coenzyme A in the tricarboxylic acid cycle over PHB synthesis.

The kinetic mechanism of NAD-dependent glyceraldehyde-3-phosphate dehydrogenase [D-glyceraldehyde-3-phosphate:NAD+ oxidoreductase (phosphorylating), EC 1.2.1.12] from the host cytosolic fraction of soybean (Glycine max L. Merr. cv. Williams) root nodules has been investigated by steady-state initial velocity studies and inhibition studies with products and product analogs. The results were consistent with the enzyme having a bi-uni-uni-uni ping-pong mechanism in which NAD+ and phosphate interacted sequentially with the enzyme, followed in turn by the release of 1,3-bisphosphoglycerate, the addition of D-glyceraldehyde-3-phosphate, and the release of NADH. At pH 7.2, NAD+ bound to the enzyme in a rapid-equilibrium fashion, whereas at pH 8.8 there was rapid-equilibrium addition of both NAD+ and phosphate to the enzyme. NADH was a strong competitive inhibitor with respect to NAD+ and phosphate. The enzyme was very labile, especially above pH 8, but the rate of loss of activity was considerably reduced in the presence of NAD+ and phosphate. The possible role of these properties in the regulation of NAD-dependent glyceraldehyde-3-phosphate dehydrogenase in the host cytosol of soybean nodules is discussed.

Eight surface soils (0–15 cm) including 1 Ferrosol, 2 Tenosols, 2 Kurosols, 1 Sodosol, 1 Chromosol, and 1 Kandosol were collected from mainly pasture sites in New South Wales. The soils had different physico-chemical properties and there were some differences between the sites in climatic conditions. Soil microbial biomass carbon (MBC) was estimated by the chloroform-fumigation extraction method, and substrate utilisation patterns determined by the Biolog method were used to assess the amount, functional diversity, substrate richness and evenness, and community structure of the microorganisms in these soils. The amount of MBC (585 µg C/g) and the microbial diversity (H´ = 3.24) were high in soils that had high clay (33%), organic C (5.96%), total N (0.45%), free iron (7.06%), moisture content (50%), and cation exchange capacitiy (133.5 mmolc/kg). These soil properties, e.g. soil moisture (r2 = 0.72), organic C (r2 = 0.58), total N (r2 = 0.63), free iron (r2 = 0.44), and EC (r2 = 0.53), were positively correlated with MBC and microbial diversity index, whereas pH and sand and silt content showed negative correlations. The climatic factors (temperature and rainfall) had no significant influence on either MBC or diversity.

The measurement and interpretation of isotherms for sorption of water onto, and oxygen permeability of, polymer substrates are important for designing polymers in applications such as agricultural seed coatings. In a preliminary study of water sorption in the commonly used copolymer poly(vinyl alcohol–co-vinyl acetate), moisture sorption isotherms were measured for a series of substrates of different comonomer composition at 25°C. The data were fitted to the D'Arcy–Watt model, which gave physically reasonable values for parameters related to the strength of monolayer adsorption to high affinity sites on the substrate and the number of sites for secondary water adsorption per unit mass. The oxygen permeability of poly(vinyl alcohol) films was found to be a function only of the water content of the films, with no other obvious contributing factors. Marked differences were seen in the populations of 'bound' and 'unbound' water characterized by 1H nuclear magnetic resonance (NMR) spectroscopy and moisture sorption isotherm fitting. There was no correlation of oxygen permeability with 'bound' or 'unbound' water measured by any means, a fact suggesting that there is a continuum of water environments within the materials.

The wheat-grain proteome was investigated, as a basis for devising more efficient methods of cultivar identification or discrimination. Australian wheats (Halberd, Cranbrook, CD87 and Katepwa) were used as the basis of this study. These cultivars were selected on the basis of differences in the quality types represented, in terms of dough-processing attributes that can suit one cultivar better than another for specific types of industrial utilisation. Total wheat endosperm (flour) protein extracts were prepared from mature wheat for two-dimensional electrophoresis, across both acidic (pH 4-7) and basic (pH 6-11) pH ranges. Three particular regions of the proteome maps were chosen for close comparison, involving two sets of gluten proteins and a nongluten protein region (involving small heat shock proteins), based on previous protein characterisation. Differences in the nongluten protein regions (heat shock proteins and other unidentified polypeptides) are of particular interest as being possible targets for use in developing new approaches to cultivar discrimination, such as the development of simple immunoassays.

Acetohydroxy acid reductoisomerase (EC 1.1.1.86, AHAR) was purified to a high degree from green shoots of wheat (Triticum aestivum L. cv. Vulcan). The enzyme was localised in the chloroplasts, and activity was at a maximum approximately 4 d after germination. The subunit molecular mass of wheat AHAR was 57 kD and activity of the native enzyme had an elution volume from size exclusion columns that corresponded to a molecular mass of 47 kD. The enzyme did not require the addition of Mg 2+ ions to reaction mixtures for activity. The Km values for (R,S)-2-acetolactate and (R,S)-2-aceto-2-hydroxybutyrate were 91 and 9 mM, respectively, and the corresponding maximum velocities were 430 and 451 mU mg –1 protein. The Km for NADPH was approximately 10 mM when either of the acetohydroxy acids was the other substrate. Preparation of the acetohydroxy acid substrates by hydrolysis of the parent esters in strong base led to the formation of inhibitory by-products. Racemisation of the acetohydroxy acids was detected in assay mixtures.

The distribution of the nodule-specific form of sucrose synthase (nodulin-100) in soybean nodules has been studied by immunocytochemical methods. Ultrathin sections were labelled with immunogold, and semi-thin sections with immunogold followed by staining with silver. The enzyme was located in the host cytosol, and was present to a much greater extent in uninfected inner cortical cells and interstitial cells than in infected cells. This adds further evidence that conversion of sucrose to dicarboxylic acids takes place to a greater extent in uninfected inner cortical cells and interstitial cells, than in infected cells.

Steady state velocity studies and inhibition studies with products and substrate analogs have been conducted on the amination reaction of glutamate dehydrogenase ( l -glutamate: NAD+ oxidoreductase (deaminating) EC 1.4.1.2) from lupin nodules. The results are qualitatively and quantitatively consistent with a fully ordered mechanism in which NADH binds to the enzyme first followed by 2-oxoglutarate and then NH4+, and in which the product l -glutamate is released before NAD+. This mechanism is the only plausible mechanism consistent with the results of studies on the reaction in both directions.

The subcellular location and properties of enzymes concerned with the phosphorylation of glucose and fructose in avocado (Persea americana Mill. cv. Hass) have been studied. A substantial amount of glucose-phosphorylating activity was particulate and fractionation of extracts by sucrose density gradient centrifugation indicated that most of this activity was associated with the mitochondria. Three hexose-phosphorylating enzymes were resolved by DEAE-cellulose chromatography of the cytosolic fraction. These were a hexokinase (EC 2.7.1.1), which had strong preference for glucose as substrate, and two specific fructokinases (EC 2.7.1.4). ATP was the preferred phosphoryl donor for the hexose kinases of avocado.

At low uncoupler concentrations the binding of carbonyl-cyanide-m-chlorophenyl-hydrazone to mitochondria was found to depend sensitively on the metabolic state of mitochondria. The binding data are consistent with the assumption that at low concentrations and pH 7.4 the uncoupler is bound mainly in anionic form to the inner mitochondrial membrane and that upon energization the inner membrane undergoes conformation change, exposes buried ionizable groups and hence acquires a negative net membrane charge. Deenergization of the inner membrane by a small amount of uncoupler removes the negative net membrane charge and consequently increases the apparent binding constants. Based upon the present results on uncoupler binding and previous observations on the physiological properties of alkylating uncouplers, a possible molecular mechanism involving electron carriers and coupling factors is suggested for coupling electron transport to phosphorylation.

A fructokinase (EC 2.7.1.4) was obtained from pea (Pisum sativum L.) seeds. This enzyme, termed fructokinase (fraction IV), was specific for fructose as substrate and had little activity with glucose or mannose. Excess fructose inhibited the enzyme at the optimum pH (8.2) but not at pH 6.6. MgATP was inhibitory at pH 6.6. The apparent Michaelis-Menten constants at pH 8.2 were 0.057 mm for fructose and 0.10 mm for MgATP. Mg2+ ions were essential for activity; Mn2+ could partially replace Mg2+. Fructokinase (fraction IV) had a requirement for K+ ions which could be substantially replaced by Rb+ or NH4+ but not by Na+. The enzyme was inhibited by MgADP. The possible significance of fructokinase (fraction IV) in plant carbohydrate metabolism is discussed.

SUCROSE SYNTHASE (UDPGLUCOSE: d-fructose 2-alpha-d-glucosyl transferase, EC 2.4.1.13) has been purified from the plant cytosolic fraction of soybean (Glycine max L. Merr cv Williams) nodules. The native enzyme had a molecular weight of 400,000. The subunit molecular weight was 90,000 and a tetrameric structure is proposed for soybean nodule sucrose synthase. Optimum activity in the sucrose cleavage and synthesis directions was at pH 6 and pH 9.5 respectively, and the enzyme displayed typical Michaelis-Menten kinetics. Soybean nodule sucrose synthase had a high affinity for UDP (K(m), 5 micromolar) and a relatively low affinity for ADP (apparent K(m), 0.13 millimolar) and CDP (apparent K(m), 1.1 millimolar). The K(m) for sucrose was 31 millimolar. In the synthesis direction, UDPglucose (K(m), 0.012 millimolar) was a more effective glucosyl donor than ADPglucose (K(m), 1.6 millimolar) and the K(m) for fructose was 3.7 millimolar. Divalent cations stimulated activity in both the cleavage and synthesis directions and the enzyme was very sensitive to inhibition by heavy metals.

The activities of enzymes of carbohydrate metabolism have been measured in the plant and bacteroid fractions of soybean ( Glycine max ) nodules and in roots of non-nodulated soybeans. The plant fraction of the nodules contained the enzymes required to convert sucrose to pyruvate and to oxaloacetate as well as enzymes of starch synthesis and degradation. In contrast, the bacteroids had only limited capacity for carbohydrate metabolism and appeared to lack the complete glycolytic pathway. The distribution of enzymes of carbohydrate metabolism between the cortical tissue and central infected zone of the nodules was investigated. The conversion of sucrose to organic acids may take place to a large extent in the cortical tissue of the nodules.

SUMMARY: Soluble extracts of Bradyrhizobium japonicum bacteroids from soybean root nodules showed substantial rates of NAD+ and NADP+ reduction which were malate and MnCl2 dependent. Pyruvate was formed stoichiometrically and the NAD- and NADP-dependent rates were additive, indicating the presence of two malic enzymes. The NADP-dependent malic enzyme had a high affinity for malate (apparent K m = 0·1 mM) and was stimulated by ammonium. The NAD-dependent malic enzyme had a lower affinity for malate (apparent K m = 1·9 mM) and was stimulated by potassium and ammonium salts. The maximum velocities of the two enzymes were similar and of comparable magnitude to the activities of tricarboxylic acid cycle enzymes in the extracts. Possible roles of the malic enzymes in the metabolism of malate and succinate in bacteroids are discussed.

A second fructokinase (EC 2.7.1.4) was obtained from pea seed (Pisum sativum L. var. Progress No. 9) extracts. The enzyme, termed fructokinase (fraction III), was specific for fructose and had little activity with glucose. With fructose concentrations above 0.25 millimolar, there was strong substrate inhibition at the optimum pH (8.0) and also at pH 6.6. The apparent K(m) values at pH 8.0 for fructose and glucose were 0.06 millimolar and 0.14 millimolar, respectively. The apparent K(m) for Mg adenosine 5'-triphosphate (MgATP) was 0.06 millimolar and excess MgATP was inhibitory. Mg(2+) was essential for activity but the enzyme was inhibited by excess Mg(2+) or ATP. Mg adenosine 5'-pyrophosphate was also inhibitory. Activity was stimulated by the addition of monovalent cations: of those tested K(+), Rb(+), and NH(4) (+) were the most effective. The possible role of fructokinase (fraction III) is discussed.

Glutamate dehydrogenase, GDH (l-glutamate: NAD+ oxidoreductase (deaminating) EC 1.4.1.2) was purified from the plant fraction of lupin nodules and the purity of the preparation established by gel electrophoresis and electrofocusing. The purified enzyme existed as 4 charge isozymes with a MW of 270000. The subunit MW, as determined by dodecyl sulphate electrophoresis, was 45 000. On the basis of the results of the MW determinations a hexameric structure is proposed for lupin-nodule GDH. The pH optima for the enzyme were pH 8.2 for the amination reaction and pH 8.8 for the deamination reaction. GDH from lupin nodules showed a marked preference for NADH over NADPH in the amination reaction and used only NAD+ for the deamination reaction. Pyridoxal-5′-P and EDTA inhibited activity. The enzyme displayed Michaelis-Menten kinetics with respect to all substrates except NAD+. When NAD+ was the varied substrate, there was a deviation from Michaelis-Menten behaviour towards higher activity at high concentrations of NAD+.

The enzymes responsible for the phosphorylation of hexoses in the plant cytosolic fraction of soybean (Glycine max L. Merr cv Williams) nodules have been studied and a hexokinase (ATP:d-hexose 6-phosphotransferase EC 2.7.1.1) and fructokinase (ATP:d-fructose 6-phosphotransferase EC 2.7.1.4) shown to be involved. The plant cytosolic hexokinase had optimum activity from pH 8.2 to 8.9 and the enzyme displayed typical Michaelis-Menten kinetics. Hexokinase had a higher affinity for glucose (K(m) 0.075 millimolar) than fructose (K(m) 2.5 millimolar) and is likely to phosphorylate mainly glucose in vivo. The plant cytosolic fructokinase had a pH optimum of 8.2 and required K(+) ions for maximum activity. The enzyme was specific for fructose (apparent K(m) 0.077 millimolar) but concentrations of fructose greater than 0.4 millimolar were inhibitory. The native molecular weight of fructokinase was 84,000 +/- 5,000. The roles of these enzymes in the metabolism of glucose and fructose in the host cytoplasm of soybean nodules are discussed.

The subcellular localization of hexose phosphorylating activity in extracts of pea stems has been studied by differential centrifugation and sucrose density gradient centrifugation. The hexokinase (EC 2.7.1.1) was associated with the mitochondria, whereas fructokinase (EC 2.7.1.4) was in the cytosolic fraction. Some properties of the mitochondrial hexokinase were studied. The enzyme had a high affinity for glucose (K(m) 76 micromolar) and mannose (K(m) 71 micromolar) and a relatively low affinity for fructose (K(m) 15.7 millimolar). The K(m) for MgATP was 180 micromolar. The addition of salts stimulated the activity of the hexokinase. Al(3+) was a strong inhibitor at pH 7 but not at the optimum pH (8.2). The enzyme was not readily solubilized but, in experiments with intact mitochondria, was susceptible to proteolysis. A location on the outer mitochondrial membrane is suggested for the hexokinase of pea stems.

The uncoupler dinitrobromoacetoxyethoxyphenol (DNBP) has been synthesized and found to label rapidly and specifically a small number of cysteine residues in rat liver mitochondria. The labeling reaction was essentially complete in a few minutes. Only eight of the mitochondrial polypeptide bands, of MW 97, 58, 52, 43, 30, 26, 22, 13 × 10 3 , respectively, as separated by SDS gel electrophoresis, were found to carry the radioactive label. In each case, the label which survived acid hydrolysis was covalently bound to cysteine residues through alkylation reaction. Under the present experimental conditions, only 0.45 mole of the label was covalently bound to mitochondrial protein per mole of cytochrome aa 3 and only 1 out of about 650 sulfhydryl groups was so labeled. Consideration of the specificity of the labeling and the observed time-dependence of DNBP-stimulated respiration suggests that the labeled protein molecules are either at or very close to the mitochondrial coupling sites and probably play an important role in the primary energy transduction process.

The labeling of rat liver mitochondria (RLM) by the uncoupler 2,4-dinitro-5-(bromoacetoxyethoxy)phenol (DNBP) was studied and related to the effect of this molecule on oxidative phosphorylation. Alkylation of the cysteine residues was measured both with respect to incubation time of RLM with DNBP and with increasing DNBP concentration. At 3.3 × 10−5 m DNBP, the amount of S-carboxymethyl-cysteine formed was found to level off after about 3 min. The rate of ATP synthesis in RLM is reduced by increasing concentrations of DNBP and falls to zero, with either hydroxybutyrate or succinate as substrate, at 2 × 10−4 m DNBP. To characterize the effect of labeling on oxidative phosphorylation, the P O ratio were measured after incubating RLM with DNBP for various times between 10 and 300 sec. The P O ratio increases and tends to level off as the incubation time increases. No increase in P O ratio was noted when RLM were similarly incubated with the nonlabeling uncoupler 2,4-dinitro-5-(acetoxyethoxy) phenol. Further, the effect of labeling on oxidative phosphorylation was determined with RLM which had been treated with DNBP and then washed free of the excess unreacted uncoupler. DNBP produces specific labeling in RLM which, when related to the effects of this uncoupler on oxidative phosphorylation, suggests that the labeled proteins may be involved in the primary energy transduction process.

By feeding (methylene-14C)-L-tryptophan, a biosynthetic precursor of the alkaloid harman, to Passiflora edulis it can be shown that the concentration of (3-14C)-harman initially increases and then decreases. By feeding (3-14C)-harman, the decrease has been further investigated and found to be due to excretion and not catabolic reactions.

Extraction and fractionation of the beta-D-mannanases in germinating lucerne produced four fractions if extracts were stored prior to chromatography. However, when extracts were applied rapidly to DEAE-cellulose, one of the fractions of high molecular weight (separated subsequently by gel chromatography) was not detected, indicating that it was formed after maceration. On storage, the amount of the lower-molecular-weight, unbound fraction from chromatography on DEAE-cellulose did not increase relative to that bound. During germination, the bound and unbound fractions both increased initially and then decreased, with only small changes in the ratio of each. The results suggest that three of the beta-D-mannanase fractions are formed in vivo prior to maceration.

The kinetic parameters of three forms of glutamate dehydrogenase from developing soybean seed have been determined. For all enzymes double reciprocal plots were intersecting in animating and deaminating directions when each substrate was held at a fixed concentration in turn. In amination all plots were linear and the KNH4+ and KNAD(P)H values determined using (NH4)2SO4 were about one-fifth of values obtained with NH4CL. In deamination, plots were linear when l -glutamate and NADP+ were varied and biphasic with varied NAD+. It was not possible to determine the mechanisms of the enzymes but the data are consistent with nucleotide binding to the free enzyme forms. The role of the enzymes in the tissue is discussed. They could be active in deamination in the later stages of seed development when the supply of carbohydrate could be limiting. They could also be important in ammonia assimilation and their kinetic properties are discussed in relation to known properties of glutamine synthetase and glutamate synthase and cellular levels of enzyme substrates.

Fructokinase I of pea seeds has been purified to homogeneity and the enzyme shown to be monomeric, with a molecular weight of 72,000 ± 4000. The reaction mechanism was investigated by means of initial velocity studies. Both substrates inhibited the enzyme; the inhibition caused by MgATP was linear-uncompetitive with respect to fructose whereas that caused by d -fructose was hyperbolic-noncompetitive against MgATP. The product d -fructose 6-phosphate caused hyperbolic-noncompetitive inhibition with respect to both substrates. MgADP caused noncompetitive inhibition, which gave intercept and slope replots that were linear with d -fructose but hyperbolic with MgATP. Free Mg 2+ caused linear-uncompetitive inhibition when either substrate was varied. l -Sorbose and β,γ-methyleneadenosine 5′-triphosphate were used as analogs of d -fructose and MgATP, respectively. Inhibition experiments using these compounds indicated that substrate addition was steady-state ordered, with MgATP adding first. The product inhibition experiments were found to be consistent with a steady-state random release of products. The substrate inhibition caused by MgATP was most likely due to the formation of an enzyme-MgATP-product dead-end complex, whereas that caused by d -fructose was due to alternative pathways in the reaction mechanism. The inhibition caused by Mg 2+ can be explained in terms of a dead-end complex with either a central complex or an enzyme-product complex.

The specific activities of acid and alkaline invertases (beta-d-fructofuranoside fructohydrolase, EC 3.2.1.26), sucrose synthase (UDPglucose: d-fructose 2-alpha-d-glucosyltransferase, EC 2.4.1.13), hexokinase (ATP: d-hexose 6-phosphotransferase, EC 2.7.1.1), and fructokinase (ATP: d-fructose 6-phosphotransferase, EC 2.7.1.4) were determined in soybean (Glycine max L. Merr cv Williams) nodules at different stages of development and, for comparison, in roots of nonnodulated soybeans. Alkaline invertase and sucrose synthase were both involved in sucrose metabolism in the nodules, but there was only a small amount of acid invertase present. The nodules contained more phosphorylating activity with fructose than glucose. Essentially all of the alkaline invertase, sucrose synthase, and fructokinase were in the soluble fraction of nodule extracts whereas hexokinase was in the bacteroid, plant particulate, and soluble fractions.Soybean nodule alkaline invertase was partially purified and shown to be a beta-d-fructofuranosidase which was specific for sucrose. The pH optimum was 7.6 and the K(m) for sucrose was 10 millimolar. Fructose was a competitive inhibitor. Tris was a noncompetitive inhibitor and the enzyme was very sensitive to inhibition by heavy metals.

The kinetics of the reductive amination reaction of lupine-nodule glutamate dehydrogenase ( l -glutamate:NAD oxidoreductase (deaminating), EC 1.4.1.2) were found to vary with the identity of the ammonium salt which was used as a substrate. Normal Michaelis-Menten kinetics were obtained with (NH 4 ) 2 SO 4 but when NH 4 Cl or NH 4 -acetate was varied apparent substrate inhibition was observed. Linear double-reciprocal plots were obtained with NH 4 Cl and NH 4 -acetate, however, if the concentration of Cl − or acetate was maintained constant by adding KCl or K-acetate. Chloride and acetate were subsequently found to cause linear noncompetitive inhibition with respect to NH 4 + and the apparent substrate inhibition by NH 4 Cl and NH 4 -acetate can be explained as the result varying a substrate and a noncompetitive inhibitor in constant ratio. Other anions were also found to be inhibitors of the glutamate dehydrogenase reaction; I − caused parabolic noncompetitive inhibition with respect to NH 4 + and NO 3 − caused slope-parabolic noncompetitive inhibition with respect to all three substrates of the reductive amination reaction. For the oxidation deamination reaction, Cl − was a linear competitive inhibitor with respect to both NAD and l -glutamate whereas NO 3 − caused parabolic competitive inhibition with respect to these reactants. To explain the results, it is proposed that anions bind to an allosteric site and cause a change in some of the rate constants of the reaction. Specifically, the results are consistent with anions causing decreases in the rates of association of NADH and 2-oxoglutarate with the enzyme and an increase in the rate of dissociation of NAD.

ADPglucose pyrophosphorylase from spinach leaves has been purified to homogeneity by hydrophobic chromatography carried out in 1 molar phosphate buffer. After polyacrylamide gel electrophoresis, the preparation showed only one protein staining band that coincided with a single activity stain. The enzyme appears to be composed of two subunits with molecular weights of 44,000 and 48,000, respectively, as determined by SDS polyacrylamide gel electrophoresis. Thus ADPglucose pyrophosphorylase of spinach appears to be comprised of subunits which are similar in size to the subunits of ADPglucose pyrophosphorylase isolated from bacterial sources. In contrast, a subunit molecular weight of 96,000 has been reported for the maize endosperm ADPglucose pyrophosphorylase (Fuchs RL and JO Smith 1979 Biochim Biophys Acta 556: 40-48). The purified enzyme retains similar allosteric and catalytic properties as reported previously and is more sensitive to phosphate inhibition under “dark”-simulated conditions than under “light”-simulated conditions.

A second hexokinase (EC 2.7.1.1) was obtained from pea seed (Pisum sativum L. var. Progress No. 9) extracts. The enzyme, termed hexokinase II, had a high affinity (K(m), 48 micromolar) for glucose and a relatively low affinity (K(m), 10 millimolar) for fructose. The K(m) for MgATP was 86 micromolar. Mg(2+) was required for activity, but excess Mg(2+) was inhibitory. MgADP inhibited hexokinase II. The addition of salts of monovalent cations increased hexokinase II activity. Al(3+) was a strong inhibitor of the enzyme at pH 6.6 but not at the optimum pH (8.2). Citrate and 3-phosphoglycerate activated pea seed hexokinase II at pH 6.6, probably by coordinating with aluminum present as a contaminant in commercial ATP. The properties of hexokinase II are compared with those of the other three hexose kinases obtained from pea seed extracts. The possible role of these enzymes in plant carbohydrate metabolism is discussed.

We propose that plant foods containing high quantities of starch were essential for the evolution of the human phenotype during the Pleistocene. Although previous studies have highlighted a stone tool-mediated shift from primarily plant-based to primarily meat-based diets as critical in the development of the brain and other human traits, we argue that digestible carbohydrates were also necessary to accommodate the increased metabolic demands of a growing brain. Furthermore, we acknowledge the adaptive role cooking played in improving the digestibility and palatability of key carbohydrates. We provide evidence that cooked starch, a source of preformed glucose, greatly increased energy availability to human tissues with high glucose demands, such as the brain, red blood cells, and the developing fetus. We also highlight the auxiliary role copy number variation in the salivary amylase genes may have played in increasing the importance of starch in human evolution following the origins of cooking. Salivary amylases are largely ineffective on raw crystalline starch, but cooking substantially increases both their energy-yielding potential and glycemia. Although uncertainties remain regarding the antiquity of cooking and the origins of salivary amylase gene copy number variation, the hypothesis we present makes a testable prediction that these events are correlated.

Tryptophan-1- 14 C was shown to be incorporated into 6-hydroxykynurenic acid in Nicotiana tabacum and decarboxylation showed that virtually all the label was incorporated into the carboxyl group.