Glucose 6-phosphate dehydrogenase (EC 1. 1.1. 49) has been partially purified from Anacystis nidulans and Anabaena flos-aquae by means of anmnonium sulfate fractionation and exclusion gel chromatography and the kinetic properties de-termined.Glucose 6-phosphate dehydrogenase from these blue-green algae exhibits Michaelis-Menten kinetics at pH 6.7. At this pH, Km values of 0.37 mM for glucose 6-phosphate and 10 zM for NADP were determined. At a pH above 7.4, the enzyme exhibits sigmoidal kinetics with respect to glucose 6-phosphate saturation but the saturation curve for NADP remains hyperbolic.ATP is an inhibitor of the enzyme competitively with NADP with a Ki of 2 to 5 mM. NADPH inhibits the enzyme competitively with glucose 6-phosphate. The inhibition curves for NADPH are hyperbolic at pH 6.7 and sigmoidal at pH 8.6.The significance of these in ritro kinetics are discussed relative to in vivo data on the control of glucose 6-phosphate turnover in blue-green algae.The blue-green algae are procaryotic organisms with a generally obligate photoautotrophic mode of growth (22). Their mechanism for fixing CO, seems to be similar to the mechanism exhibited by higher plants, but To whom correspondence should be addressed.and Bassham (17). The relationship between the degradative shunt and photosynthesis has come under scrutiny only recently. It has been suggested that the pentose phosphate pathway may be regulated by photosynthesis, and that this regulation may be expressed at the first enzyme of the pathway, glucose-6-P dehydrogenase (16).Pelroy and Bassham (17) have found that photosynthesis causes an increase in the glucose-6-P pool size. In view of the finding that the pool size of 6-P-gluconate is very small during photosynthesis, it has been suggested that the blockage occurs at the point of conversion of glucose-6-P to 6-P-gluconate (16,17). Such data indicate that photosynthesis may regulate the flow of carbon through the pentose phosphate pathway by controlling the activity of glucose-6-P dehydrogenase. A variety of factors may contribute to the apparent light-promoted decrease in dehydrogenase activity. The enzyme isolated from the sweet potato tuber displays decreased activity in the presence of either NADPH or ATP (11). During photosynthesis, noncyclic electron transport results in an increased cellular NADPH concentration, whereas cyclic electron flow causes an increase in the cellular ATP concentration. Such increases may reduce the rate of carbon flow through the pentose phosphate pathway via suppression of glucose-6-P dehydrogenase activity. It has recently been reported that ribulose-1, 5-diP, a Calvin cycle intermediate which also shows a marked increase during photosynthesis, causes diminished dehydrogenase activity (16). In addition to the possible enzyme regulation via Calvin cycle intermediates and electron flow products, light-induced pH changes in the extrathylakoidal environment may cause an alteration in enzyme activity. Neumann and Jagendorf (13) have demonstrated that light causes ...

Glyceraldehyde 3-phosphate dehydrogenases (EC 1.2.1.12 and 1.2.1.13) have been purified from the seed, root, etiolated, and green shoot of peas (Pisum sativum). These enzymes are tetramers of 140,000 daltons, with subunits of 35,000 daltons. The enzymes differ in isoelectric point. The seed enzyme has a pl of 5.1, and the root enzyme has a pI of 4.5. The cytoplasmic enzyme from etiolated shoots is slightly acidic with a pI of 5.7 to 6.1 and is found in two separable forms. The chloroplast enzyme (from green shoots) is most basic with a pI of 8.0.In immunodiffusion experiments, the seed, root, and cytoplasmic enzymes of the etiolated shoot share antigenic homology, while the chloroplast enzyme does not cross react antigenically with the extra-chloroplast enzymes. The antiserum to the pea chloroplast enzyme did, however, cross react with glyceraldehyde 3-phosphate dehydrogenase purified from the spinach chloroplast. Therefore, the chloroplast enzyme is significantly different from the extra-chloroplast enzymes with respect to prinary sequence.The NADP analog phosphoadenosine diphosphoribose showed competitive inhibition to the chloroplast enzyme with either pyridine nucleotide. The NAD analog pyridine 3-aldehyde NAD was competitive with respect to the NAD activity but was hyperbolic competitive in the presence of NADP, indicating a complexity in the binding of pyridine nucleotide to the chloroplast enzyme.Five glyceraldehyde-3-P dehydrogenases have been recognized in higher plants. Of these, three are NAD-associated enzymes, located in the seed (14), root (12). and cytoplasm of the leaf (20). A fourth is the chloroplast enzyme linked with either pyridine nucleotide (12). Finally, there is the nonphosphaterequiring, irreversible enzyme located in the cytoplasm of the leaf which utilizes NADP (4, 18).The structural relationship of the cytoplasmic enzyme, which is NAD specific, to the chloroplast enzyme, which uses both pyridine nucleotides, is unknown. Based on their studies in 1This research was supported by Atomic Energy Commission Grant AT (1 1-1 Incorporation of the cytoplasmic enzyme into the chloroplast enzyme would mean that the purified chloroplast and cytoplasmic enzymes should have similar structural properties. In order to evaluate the structural similarity or dissimilarity of the chloroplast and cytoplasmic glyceraldehyde-3-P dehydrogenases, it is necessary to characterize the purified enzymes. Yonuschot et al. (31) have purified the spinach chloroplast enzyme and shown it to be of a higher mol wt (600,000) than any previously studied glyceraldehyde-3-P dehydrogenase, including the various muscle and yeast enzymes (1, 2, 7). Another characteristic of the spinach chloroplast enzyme is a reversible polymerization between a 600,000 and a 140,000 mol wt form affected by NADP (25).These reports clearly indicate the need for a comparative study of the purified chloroplast and cytoplasmic glyceraldehyde-3-P dehydrogenases so that the structural similarity or dissimilarity of the molecules might be inves...

A procedure has been developed for the purification of amine oxidase (E.C. 1.4.3.4) from etiolated pea epicotyls (Pisum sativum cv. Little Marvel). The enzyme is sensitive to copper chelating reagents and carbonyl reagents, but is not inhibited by sulfhydryl reagents. The purified enzyme has a molecular weight of 1.85 X 105, as determined by sedimentation equilibrium centrifugation, and has been shown to be specifically stimulated by phosphate.enzyme. In addition, the enzymes from bovine and porcine plasma have been found to contain pyridoxal phosphate (4, 23). The amine oxidase from porcine plasma has a molecular weight of 1.95 X 105 (5), and the amine oxidase from bovine plasma has a molecular weight of 1.7 x 10' (1). The enzyme from bovine plasma has a subunit molecular weight of 8.05 X 104(1).The study reported here describes a purification procedure for the amine oxidase from etiolated pea epicotyls, confirmation of copper and R-COR as functional groups in catalysis, the molecular weight as determined by ultracentrifugation, and the requirement of phosphate for maximal catalysis.Recent studies in this laboratory have indicated the possibility that IAA synthesis in Pisum could proceed through tryptamine and that the enzyme responsible for catalyzing this reaction could be purified (10, 14). As part of an investigation of IAA biosynthesis in Pisum, an enzyme which forms IAA from tryptamine was purified and found to be an amine oxidase. In view of the wide interest in amine oxidases from mammals and the one amine oxidase purified from a higher plant, it was decided to characterize this enzyme for a further understanding of the plant enzyme and for a comparison with the mammalian enzymes.Amine oxidase from plants was first purified and studied by Mann, who found that the enzyme was a pink, copper-containing protein which was sensitive to carbonyl reagents (12). The visible absorption spectrum of this enzyme had an absorbance maximum at 500 nm (13). This absorbance maximum could be shifted to lower wavelengths by incubation of the enzyme with substrate under anaerobic conditions or by incubation of the enzyme with copper-chelating reagents. These spectral shifts could be reversed by incubation under aerobic conditions in the first case and cupric ions in the second (9). The enzyme was found to be active in catalyzing the oxidative deamination of a wide variety of substrates (9). The purified enzyme had an s5,,0 of 7.7S, measured from sedimentation velocity centrifugation, and a molecular weight of 9.6 X 104, calculated from electron micrographs (9). Recently, Yamasaki et al. (24) have shown that the binding of oxygen to amine oxidase from peas is dependent upon amine concentration.Amine oxidase from bovine plasma has been studied extensively by Yasunobu and co-workers (21-23) who have found that the visible absorbance spectrum, copper content, and sensitivity to carbonyl reagents are similar to the plant

The effect of various sugar phosphates on CO2 fixation in Anabacna flos-aquae was investigated and found to be very smilar to that found for isolated spinch chloroplasts. One exception, glucose 6-phosphate, has a stimulatory effect on CO2 fixation in Anabena but not in isolated chloroplast.Further examination of the role of glucose 6-phosphate metabolsm in The generally obligate nature of photoautotrophic growth in the blue-green algae has prompted the investigation of the biochemical basis for this growth habit. Although their mode of photosynthesis seems to be essentially identical to that of eucaryotic organisms (9), a blocked tricarboxylic acid cycle and a limited glycolytic cycle, due to the absence or low levels of phosphofructokinase (11,12,16,18), suggest that carbon flow in these organisms is either by a catabolic pentose phosphate shunt, as shown by Cheung and Gibbs (5), or by direct utilization of triose produced during photosynthetic carbon reduction. The questionable functioning of glycolysis and the tricarboxylic acid cycle, and the concomitant lack of ATP synthesis in the dark, might be the reason for limited growth on glucose or acetate (11,12,16).Recently, Pelroy and , have shown that turnover of glucose-6-P is restricted in the light and that rapid turnover occurs in the dark. Grossman and McGowan (6) have investigated the kinetics of glucose-6-P dehydrogenase in Anacystis and Anabaena and have determined regulation of the enzyme's catalytic activity by pH, NADPH, and ATP. These data suggest that if glucose-6-P could be assimilated in the dark, its turnover via the pentose phosphate pathway should go unhindered. However, it is generally believed that sugar phosphates are not assimilated by whole algal cells, or that if they are, they are first dephosphorylated at the cell membrane.In this study we present data which suggest that some sugar