Activation and inhibition of ribulose 1,5-diphosphate carboxylase by 6-phosphogluconate

Ribulose 1,5-diphosphate carboxylase, when activated by preincubation with 1 mm bicarbonate and 10 mm MgCl₂ in the absence of ribulose 1,5-diphosphate, remains activated for 20 minutes or longer after reaction is initiated by addition of ribulose diphosphate. If as little as 50 μm 6-phosphogluconate is added during this preincubation period, 5 minutes before the start of the reaction, a further 188% activation is observed. However, addition of 6-phosphogluconate at the same time or later than addition of ribulose diphosphate, or at any time with 50 mm bicarbonate, gives inhibition of the enzyme activity. Possible relevance of these effects in vivo regulatory effects is discussed.     

Inhibition of ribulose 1,5-diphosphate carboxylase by 6-phosphogluconate

6-Phosphogluconate is a much more effective inhibitor of the photosynthetic carboxylation enzyme, ribulose-1, 5-diphosphate carboxylase, than other sugar phosphates and sugar acids of the reductive and oxidative pentose phosphate cycles. The inhibition appears to be noncompetitive with ribulose 1,5-diphosphate. Since 6-phosphogluconate is unique to the oxidative cycle and inhibits at concentrations comparable to those found in vivo, it is proposed that its inhibition of the carboxylase may be a regulatory factor. If so, it would operate during darkness as a different control factor from those factors postulated to activate the carboxylase during photosynthesis.     

Bicarbonate stabilization of ribulose 1,5-diphosphate carboxylase.

The carboxylase and oxygenase activities of purified soybean ribulose 1,5-di-P carboxylase (EC4.1.1.39) were unstable when reactions were initiated with enzyme. Time courses of carboxylase and oxygenase activities were curvilinear, approximating hyperbolas. Double reciprocal plots of amount of CO2 incorporated and P-glycolate produced vs. time were constructed to determine a constant representing the half-time of initial enzyme activity, K. K increased with increasing bicarbonate concentration but was independent of O2 tensions between 0.21 and 5 atm. When time courses of carboxylase and oxygenase activities were determined simultaneously, K was identical for both activities. Linear time courses were obtained py preincubation of the enzyme for 10 min in the absence of bicarbonate or by adding 46 mM MgCl2 to the reaction mixture. The observed bicarbonate-dependent decline in ribulose 1,5-di-P carboxylase activity with time is the probable cause for the anomalously high Km(CO2) values previously reported for this enzyme. In the experiments reported here, the apparent Km(CO2) at pH 8.5 increased from 6 muM CO2 at zero time to 78 muM CO2 at 10 min. The corresponding bicarbonate Km values ar 1;3 and 17 mM, respectively, The interaction between bicarbonate and enzyme may be important in the light activation of photosynthetic CO2 fixation in vivo.     

Regulation of ribulose 1,5-diphosphate carboxylase by substrates and other metabolites: further evidence for several types of binding sites

Ribulose 1,5-diphosphate carboxylase (RuDPCase, EC 4.1.1.39) isolated from spinach leaves is metabolically regulated at 10 mm Mg(2+) and low CO(2) concentrations by its substrates (RuDP and CO(2)) and by effectors which include 6-phosphogluconate (6-PGluA), NADPH, and fructose 1,6-diphosphate (FDP), but not fructose 6-phosphate. Physiological concentrations of RuDP severely inhibit the enzyme activity when the enzyme has not been preincubated with HCO(3) (-) and Mg(2-), and this inactivity persists for 20 minutes or longer after 1 mm HCO(3) (-) and 10 mm Mg(2+) are added. Maximum activity requires that the preincubation mixture also include either 0.01 mm 6-PGluA or 0.5 mm NADPH.When the enzyme, following preincubation with HCO(3) (-) and Mg(2+), is presented with RuDP plus either 6-PGluA or FDP, competitive inhibition is observed with respect to RuDP. The Ki value for 6-PGluA is 0.02 mm and the Ki value for FDP is 190 mum. NADPH or 3-phosphoglycerate (PGA) at physiological concentrations does not have any effect when presented simultaneously with RuDP. Other studies on the order of addition of substrates and effectors, concentration effects, and kinetics provide additional information that serves as a basis for a proposed model of allosteric regulation combined with competitive inhibition.In this model, there are catalytic sites at which the substrates and 6-PGluA and FDP can bind, and at least four allosteric regulatory sites, which we designate I, A(1), A(2), and A(3). RuDP binds very tightly to site I (in the absence of Mg(2+) or HCO(3) (-)), causing a conformational change in the protein to an inactive form which persists for as long as 20 minutes in the subsequent presence of Mg(2+) and 1 mm HCO(3) (-). Mg(2+) and HCO(3) (-) (or CO(2)) bind to site A(3) (in the absence of RuDP), holding the enzyme in an active form which has a much lower affinity for RuDP at site I, so that when physiological levels of RuDP are then added, only part of the enzyme activity is lost. This active form of the enzyme can bind 6-PGluA or FDP at site A(1) and NADPH at site A(2) during preincubation with Mg(2+) and HCO(3) (-). With optimal levels of bound effectors, 6-PGluA or NADPH, enzyme activity is fully maintained, even when RuDP is subsequently added. Without one of these effectors present, addition of RuDP following preincubation reduces enzyme activity to about 40% at the levels of substrates and effectors studied. FDP is a much poorer effector, and this is ascribed to a possible binding of FDP at site I, as well as at site A(1).The physiological role of this regulation is discussed, particularly with respect to protection of "C-3" plants against oxidation of RuDP to phosphoglycolate.     

Activation of the neutrophil nicotinamide adenine dinucleotide phosphate oxidase by galectin-1

Galectins are a group of lactose-binding proteins widely distributed in nature. Twelve mammalian galectins have so far been identified, but their functions are to a large extent unknown. In this work we study galectin-1 in its interaction with human neutrophils, with regard to both cell surface binding and activation of the superoxide-producing NADPH-oxidase. We show that galectin-1 is able to activate the neutrophil NADPH-oxidase, provided that the cells have been primed by extravasation from the blood into the tissue, an activation pattern that is similar to that of galectin-3. Using in vitro priming protocols, the galectin-1 responsiveness was found to correlate to granule mobilization and galectin-1 binding to the cells, suggesting the presence of granule-localized receptors that are up-regulated to the cell surface upon priming. By galectin-1 overlay of fractionated neutrophils we identified potential galectin-1 receptor candidates localized in the membranes of the secretory vesicle and gelatinase granules. The binding of galectin-1 and galectin-3 to neutrophil proteins was compared, as were the dose dependencies for activation by the two lectins. The results suggest that, although similarities are found between the two galectins, they appear to activate the NADPH-oxidase using different receptors. In conclusion, galectin-1 appears to have proinflammatory functions, mediated through activation of the neutrophil respiratory burst.     

Activation of ribulose-1,5-bisphosphate carboxylase by chloroplast metabolites in a reconstituted spinach chloroplast system

In a preparation of soluble components from isolated spinach (Spinecia oleracea L.) chloroplasts, the activity of ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39) is strongly increased by 6-phosphogluconate or by NADPH at pH 8.0. When the thylakoid system is added to these soluble components (reconstituted chloroplast system) plus ferredoxin, the carboxylase is even more strongly activated in the light. This light activation appears to be due to reduction of endogenous NADP⁺ by electrons from the light reactions transferred via ferredoxin, since NADPH alone can activate the purified enzyme in the dark while reduced ferredoxin does not. The regulatory properties of the enzyme in the reconstituted chloroplast system are compared with those of the isolated enzyme, and their possible physiologic significance is discussed.Abbreviations Fd ferredoxin - PPC pentose phosphate cycle - 6-PGluA 6-phosphogluconate - Rib-5-P ribose-5-phosphate - RuBP ribulose-1,5-bisphosphate     

Light-induced conversion of nicotinamide adenine dinucleotide to nicotinamide adenine dinucleotide phosphate in higher plant leaves

Light-induced conversion of NAD to NADP was investigated in higher plants. Upon illumination, conversion of NAD to NADP was observed in intact leaves of wheat and pea following incubation in the dark. This conversion was also observed in mesophyll protoplasts of wheat leaves when they were isolated in the dark or isolated in light and then preincubated in the dark. Chloroplasts isolated from wheat protoplasts prepared in the dark carried out the conversion. The conversion in the mechanically isolated spinach chloroplasts was observed only when they were isolated in the dark from leaves preincubated in darkness.     

A nicotinamide adenine dinucleotide phosphate phosphatase fromChlorella

A phosphatase enzyme hydrolysing NADP⁺ and NADPH to NAD⁺ and NADH was found to be present in extracts ofChlorella pyrenoidosa     

Polyhedral bodies and ribulose 1,5-diphosphate carboxylase of the blue-green alga Anabaena cylindrica

Summary The ribulose 1,5-diphosphate carboxylase (RUDP Case E.C. 4.1.1.39) activity of late log phase Anabaena cylindrica Lemm. was measured in vitro in fractions obtained by sucrose density gradient centrifugation. Two peaks of enzymic activity were obtained. One, accounting for about 80% of the total measurable activity occurred at the top of the gradient and appeared to be soluble activity; the second showed maximum activity in the 55% (w/w) sucrose fraction and represented 20% of the total activity. When the distribution of RUDP Case was assayed by immunoprecipitation using antiserum to RUDP Case from Euglena gracilis, the corresponding values were 59% and 41%. Electron microscopy of the various fractions showed that polyhedral bodies, which are sites of RUDP Case activity in other autotrophic prokaryotes, were also most abundant in the 55% (w/w) sucrose fraction.     

Activation and inhibition of spinach ribulose 1,5-bisphosphate carboxylase by 2-phosphoglycolate

Ribulose 1,5-bisphosphate carboxylase when activated by preincubation with 1 mM bicarbonate and 10 mM magnesium chloride can be further activated ca 20–500% by incubating with 2.5 mM phosphoglycolate depending upon the pH of the preincubation medium. The activation effects were seen only under specific preincubation conditions. The activation by phosphoglycolate was a slow reaction requiring ca 15 min for maximal effect. Even though magnesium was essential for phosphoglycolate activation, concentrations higher than 15 mM progressively inhibited the activation of the enzyme by phosphoglycolate. When added directly to the reaction mixture, phosphoglycolate was a potent inhibitor of the carboxylase activity. Even under preincubating conditions, phosphoglycolate showed slight inhibitory effect at 0.1 mM and activation was observed at concentrations higher than 0.5 mM. The K_A value for phosphoglycolate was 2.8 mM.     

A simplified purification and some properties of ribulose 1,5-diphosphate carboxylase from barley

A rapid procedure was developed for purifying ribulose 1,5-diphosphate carboxylase from barley leaves. After (NH₄)₂SO₄ fractionation, the unique sedimentation properties of the enzyme were exploited to effect a single step purification to 90% homogeneity. High speed centrifugation pelleted the enzyme with complete recovery of activity. Residual impurities were then removed by diethylaminoethyl cellulose chromatography and density gradient centrifugation. The purified protein exhibited size heterogeneity due to polymerization. The polymerization products were enzymatically active aggregates of ribulose 1,5-diphosphate carboxylase and were precipitated by an antibody specific for the enzyme.