Competition by 4-chloronitrosobenzene for the active glycolaldehyde intermediate of transketolase

The incubation of 4-chloronitrosobenzene with yeast transketolase, Mg²⁺, and thiamime pyrophosphate in the presence of excess xylulose-5-phosphate resulted in the formation of N-(4-chlorophenyl)glycolhydroxamic acid. This enzyme-catalyzed C₂ transfer displayed a K_m of 0.92 mM and a V_max of 5.2 × 10^?2 μmol min^?1 unit enzyme^?1. Conversion was inhibited by the normal acceptor sugar, ribose-5-phosphate, with a K_i of 0.35 mM. Kinetic analysis showed inhibition was competitive in nature, reinforcing the proposed theory for similarity in catalytic formation of both the hydroxamic acid and sedoheptulose-7-phosphate. Most interesting about the conversion of this alternative substrate is that even at high concentrations of ribose-5-phosphate, a significant amount of the nitroso compound was converted to the hydroxamic acid, implying that 4-chloronitrosobenzene can successfully compete for active glycoaldehyde. Using the yeast enzyme as a model for transketolase in higher organisms, the adventitious conversion of such xenobiotics in vivo is proposed.     

Regulation of resynthesis of the CO2-acceptor in photosynthesis. Feedback inhibition of transketolase

The presence of ribulose-5-phosphate epimerase (EC 5.1.3.1, epimerase) in samples of ribose-5-phosphate isomerase (EC 5.3.1.6, isomerase) obtained from spinach ( Spinacea aleracea L. cv. Bloomsdale Long Standing) was determined using (i) a sampling procedure which measured the quantity of xylulose-5-phosphate formed in the reaction mixture and (ii) a coupled enzyme assay in which the rate of oxidation of NADH was measured after establishing steady-state concentrations of xylulose-5-phosphate, dihydroxacetonephosphate and glyceraldehyde-3-phosphate by the action of epimerase, transketolase (EC 2.2.1.1), triosephosphate isomerase (EC 5.3.1.1) and glycerol-3-phosphate dehydrogenase (EC 1.1.1.8). In preparations where the ratio of isomerase to epimerase activities was less than 100, both assay procedures yielded valid indications of epimerase activity. The steady-state assay system was found, however, to seriously underestimate epimerase activity in enzyme preparations which were enriched in isomerase. Cross plots of epimerase activity determined by the sampling and steady-state procedures demonstrated that an inhibitor of the coupling enzyme mixture was formed in the presence of high relative concentrations of the isomerase. The inhibited coupling enzyme mixture was fully active with glycer-aldehyde-3-phosphate. Inhibition of the coupling enzyme mixture was attributed to transketolase. Feedback inhibition of transketolase is proposed to be of physiological significance in the photosynthesis cycle, operating to restrict resynthesis of CO₂-acceptor under conditions where high steady-state concentrations of the intermediates of the photosynthesis cycle are maintained.     

Effects of Free Ca2+ on Kinetic Characteristics of Holotransketolase

Catalytic activity has been demonstrated for holotransketolase in the absence of free bivalent cations in the medium. The two active centers of the enzyme are equivalent in both the catalytic activity and the affinity for the substrates. In the presence of free Ca²⁺ (added to the medium from an external source), this equivalence is lost: negative cooperativity is induced on binding of either xylulose 5-phosphate (donor substrate) or ribose 5-phosphate (acceptor substrate), whereupon the catalytic conversion of the bound substrates causes the interaction between the centers to become positively cooperative. Moreover, the enzyme total activity increase is observed.     

Regulation by ca of a cytosolic fructose-1,6-bisphosphatase from spinach leaves

Cytosolic fructose-1,6-bisphosphatase from spinach (Spinacia oleracea L.) leaves was purified over 1700-fold. The final preparation was specific for fructose-1,6-bisphosphate in the presence of either Mg²⁺ or Mn²⁺, and was free of interfering enzyme activities. Ca²⁺ was an effector of fructose-1,6-bisphosphatase activity, and showed different kinetics, depending on whether Mg²⁺ or Mn²⁺ was used as cofactor. In the presence of 5 millimolar Mg²⁺, Ca²⁺ appeared as activator or as inhibitor of the enzyme at low or high levels of substrate, respectively. In both cases, a rise in affinity for fructose-1,6-bisphosphate was observed. A model is proposed to describe the complex interaction of fructose-1,6-bisphosphatase with its substrate and Ca²⁺. However, with Mn²⁺ (60 micromolar) as cofactor, Ca²⁺ exhibited the Michaelis-Menten kinetics of a noncompetitive inhibitor. When assayed at constant substrate concentration, Ca²⁺ behaves as a competitive or noncompetitive inhibitor, depending on the use of Mg²⁺ or Mn²⁺ as cofactor, respectively, with a positive cooperativity in both cases. Fructose-2,6-bisphosphate showed a classic competitive allosteric inhibition in the presence of Mg²⁺ as cofactor, but this effect was low with Mn²⁺. From these results we suggest that Ca²⁺ plays a role in the in vivo regulation of cytosolic fructose-1,6-bisphosphatase.     

Cloning, expression, purification, cofactor requirements, and steady state kinetics of phosphoketolase-2 from Lactobacillus plantarum

The genes xpk1 and xpk2(Δ1–21) encoding phosphoketolase-1 and (Δ1–7)-truncated phosphoketolase-2 have been cloned from Lactobacillus plantarum and expressed in Escherichia coli. Both gene-products display phosphoketolase activity on fructose-6-phosphate in extracts. A N-terminal His-tag construct of xpk2(Δ1–21) was also expressed in E. coli and produced active His-tagged (Δ1–7)-truncated phosphoketolase-2 (hereafter phosphoketolase-2). Phosphoketolase-2 is activated by thiamine pyrophosphate (TPP) and the divalent metal ions Mg²⁺, Mn²⁺, or Ca²⁺. Kinetic analysis and data from the literature indicate the activators are MgTPP, MnTPP, or CaTPP, and these species activate by an ordered equilibrium binding pathway, with Me²⁺TPP binding first and then fructose-6-phosphate. Phosphoketolase-2 accepts either fructose-6-phosphate or xylulose-5-phosphate as substrates, together with inorganic phosphate, to produce acetyl phosphate and either erythrose-4-phosphate or glyceraldehyde-3-phosphate, respectively. Steady state kinetic analysis of acetyl phosphate formation with either substrate indicates a ping pong kinetic mechanism. Product inhibition patterns with erythrose-4-phosphate indicate that an intermediate in the ping pong mechanism is formed irreversibly. Background mechanistic information indicates that this intermediate is 2-acetyl-TPP. The irreversibility of 2-acetyl-TPP formation might explain the overall irreversibility of the reaction of phosphoketolase-2.     

Acceptor substrate inhibits transketolase competitively with respect to donor substrate

Two substrates of the transketolase reaction are known to bind with the enzyme according to a ping-pong mechanism [1]. It is shown in this work that high concentrations of ribose-5-phosphate (acceptor substrate) compete with xylulose-5-phosphate (donor substrate), suppressing the transketolase activity (Ki = 3.8 mM). However, interacting with the donor-substrate binding site on the protein molecule, the acceptor substrate, unlike the donor substrate, does not cause any change in the active site of the enzyme. The data are interesting in terms of studying the regulatory mechanism of the transketolase activity and the structure of the enzyme-substrate complex.     

Effect of acylphosphates on Ca2+ uptake by sarcoplasmic reticulum vesicles

The data presented in this paper concern a kinetic study of the calcium uptake by sarcoplasmic reticulum vesicles and of the hydrolysis of the substrates which support the process. The results show that substrates which are different from ATP, acetylphosphate, and carbamylphosphate are able to support calcium transport. The technique used to follow the process allows us to detect continuously the changes in the concentration of the calcium present in the external medium. In our experimental conditions the calcium uptake supported by all the high energy substrates tested proceeds for several seconds at a constant rate, presumably corresponding to the “steady state” of the process; furthermore the calcium transport is clearly Ca²⁺ and Mg²⁺ dependent: the lowering of the Ca⁺ concentration in the medium from 10^?4 to 10^?5m causes a remarkable reduction of the V of the calcium transport and an apparent increase of the affinity of the sarcoplasmic reticulum vesicles for the acylphosphates; in the absence of Mg²⁺, none of the substrates is able to support the calcium uptake which increases in the presence of rising amounts of Mg²⁺ in the reaction medium. Furthermore, both the calcium transport and the substrate hydrolysis appear to follow the Michaelis-Menten kinetics in the presence of acylphosphates but not in the presence of ATP. The hydrolytic activity of sarcoplasmic reticulum vesicles on ATP and acylphosphates reveals a clear Mg²⁺ dependence; furthermore, in the absence of free Ca²⁺ and in the presence of 5 mm Mg²⁺, the high energy substrates tested reveal a different susceptibility to the hydrolitic attack by sarcoplasmic reticulum vesicles.     

Mg2+ and Mn2+ modulation of Ca2+ transport and ATPase activity in sarcoplasmic reticulum vesicles

Kinetic experimentation was used to characterize the Mg²⁺ and Mn²⁺ modulation of Ca²⁺ transport and ATPase activity in sarcoplasmic reticulum vesicles. In addition to its participation in the ATP·Mg complex as substrate for the ATPase, Mg²⁺ is an activator of phosphoenzyme progression to hydrolylic cleavage. It is shown that this activation is due to Mg²⁺ occupancy of an allosteric site easily accessible on the outer surface of the vesicles, rather than to participation in an antiport mechanism. The Mg²⁺ site is distinct from the Ca²⁺ binding sites which are involved in activation of enzyme phosphorylation by ATP, and Ca²⁺ translocation. The role of Mg²⁺ is quite specific, inasmuch as phosphoenzyme decay is much slower if the Mg²⁺ allosteric site is occupied by Ca²⁺. Conversely, competive occupancy of the Ca²⁺ sites by Mg²⁺ does not permit enzyme phosphorylation by ATP. Intermediate characteristics between Mg²⁺ and Ca²⁺ are displayed by Mn²⁺ which is well able to stimulate phosphoenzyme cleavage by occupancy of the Mg²⁺ allosteric site, and is also able (although at much slower rates) to activate enzyme phosphorylation, and undergo active transport by occupancy of the Ca²⁺ sites.     

Effect of Mg<Superscript>2+</Superscript> versus Ca<Superscript>2+</Superscript> on the behavior of Annexin A5 in a membrane-bound state

Annexin A5 (AnxA5) binds to negatively charged phospholipid membranes in a Ca²⁺ dependent manner. Several studies already demonstrate that Mg²⁺ ions cannot induce the binding. In this paper, quartz crystal microbalance with dissipation monitoring (QCM-D), Brewster angle microscopy (BAM), polarization modulation infrared reflection absorption spectroscopy (PMIRRAS) and molecular dynamics (MD) were performed to elucidate the high specificity of Ca²⁺ versus Mg²⁺ on AnxA5 binding to membrane models. In the presence of Ca²⁺, AnxA5 showed a strong interaction with lipids, the protein is adsorbed mainly in α-helix under the DMPS monolayer, with an orientation of the α-helices axes slightly tilted with respect to the normal of the phospholipid monolayer as revealed by PMIRRAS. The Ca²⁺ ions interact strongly with the phosphate group of the phospholipid monolayer. In the presence of Mg²⁺, instead of Ca²⁺, no interaction of AnxA5 with lipids was detected. Molecular dynamics simulations allow us to explain the high specificity of calcium. Ca²⁺ ions are well exposed and surrounded by labile water molecules at the surface of the protein, which then favour their binding to the phosphate group of the membrane, explaining their specificity. To the contrary, Mg²⁺ ions are embedded in the protein structure, with a smaller number of water molecules strongly bound. We conclude that the embedded Mg²⁺ ions inside the AnxA5 structure are not able to link the protein to the phosphate group of the phospholipids for this reason.     

Effects of divalent metal ions on the fluorescence and glucose-quenching of yeast hexokinase isozymes

Titrations of the quenching of the tryptophan fluorescence of yeast hexokinase isozymes P-I and P-II by Mg²⁺, Mn²⁺, Ca²⁺, Cd²⁺, and Zn²⁺ ions and by glucose in the presence of each of these ions (10mM) were performed at pH 5.5 and 6.5 at 20°C. At the higher pH there was a reversal of the type of glucose-binding cooperativity for P-II from negative to positive when either Mn²⁺ or Ca²⁺ was present in the buffered isozyme solution before the glucose titration, whereas Mg²⁺ caused the glucose binding to become noncooperative. Zn²⁺ and Cd²⁺ decreased the glucose quenching of P-II fluorescence drastically at pH 5.5, from a value of 15% in buffer to only 4%. Thus, only these two ions, of the five studied, cause the conformation change that results in quenching of the glucose-quenchable cleft tryptophan of P-II. Glucose binding to the P-I isozyme exhibited positive cooperativity in the presence of either Ca²⁺, Mg²⁺, or Mn²⁺, as well as in buffer alone, at both pH's. At the lower pH, Ca²⁺ enhanced the efficiency of glucose quenching of P-I fluorescence several-fold, while Mn²⁺ increased it only about 40% and Mg²⁺ not at all. Further, Ca²⁺ raised the degree of cooperativity (Hill coefficient) of glucose binding to P-I at this pH from the value of 1.42 in buffer and in the presence of Mg²⁺ and Mn²⁺ to 1.94, i.e., almost up to the highest possible value, 2, for dimeric hexokinase. However, at pH 6.5 the Ca²⁺ effect on the cooperativity was negligible, while Mg²⁺ and Mn²⁺ decreased the coefficient from 1.6 in buffer to about 1.4. The biological implications of these diverse metal ion effects are discussed.     

Metal ion inhibition of corn root plasma membrane atpase

In the presence of K⁺, the hydrolysis of ATP catalysed by the ATPase of corn plasma membrane showed negative cooperative kinetics. When the complexes of ATP and Mg²⁺, Mn²⁺, Ca²⁺ or Cd²⁺ were used as substrates, the catalysed hydrolysis changed to follow simple Michaelis-Menten kinetics. However, this change was not observed with Zn²⁺-ATP as the substrate. A substantial enhancement of the hydrolysis was observed only when the complexes of Mg²⁺ and Mn²⁺ were used. Kinetic parameter determination indicated that the enzyme exhibited a similar binding property but a different catalytic efficiency to Mg²⁺, Mn²⁺ and Ca²⁺-ATP. The enzyme formed a more stable but less reactive complex with Cd²⁺-ATP. The presence of aluminium ions competitively inhibited the membrane-catalysed hydrolysis of Mg²⁺-ATP, but showed no effect when free ATP was the substrate. This finding suggested that aluminium might bind in the vicinity of the Mg²⁺ of Mg²⁺-ATP in the active site of the enzyme. On the basis of these observed inhibitory effects, possible origins of metal ion toxicity to root plasma membrane ATPase activity are discussed.     

Regulation of erythrocyte membrane shape by Ca2+

In the presence of 1.0 mM ATP and MgCl₂, the specific viscosity of suspensions of human erythrocyte ghosts decreases 35% in 20 minutes at 22°C. The changes in viscosity are a sensitive index of Mg-ATP dependent shape changes in these membranes. Low concentrations of Ca²⁺ (1 to 5 μM) inhibit Mg-ATP dependent viscosity changes. If ghosts were preincubated with 1 mM Mg-ATP and 20 μM A23187 to produce a maximal decrease in viscosity, addition of 10 μM Ca²⁺ to the preincubated ghosts increased the viscosity to levels observed in ghosts preincubated without ATP. Ca²⁺ (1 to 5 μM) also inhibited Mg²⁺ dependent phosphorylation 30% and stimulated dephosphorylation 25% in ghost membranes. These effects of Ca²⁺ on viscosity and phosphorylation may be due to a membrane bound Ca²⁺ phosphatase activity which dephosphorylates membranes phosphorylated by a Mg²⁺ dependent kinase activity.     

Cooperative binding of calcium to glycerinated skeletal muscle fibers

The binding of ⁴⁵Ca²⁺ to glycerinated rabbit psoas fibers was measured by means of a double isotope technique. With 5 mM Mg²⁺ (no ATP) binding was half-maximal at 1.4 · 10^?6M Ca²⁺ and the maximal amount bound was 1.6 μmol/g protein. At < 50% saturation, the Scatchard plot had a positive slope and the Hill coefficient was 2.2. At greater than 50% saturation, the Scatchard plot was linear with a negative slope (K′ = 0.8 · 10⁶ M^?1) and the Hill coefficient was 1.0. In the absence of Mg²⁺, binding was half-maximal at 3 · 10^?7 M Ca²⁺ and the maximal amount bound was 2.9 μmol/g protein. The Scatchard plot indicated two classes of sites with K′ values of about 2 · 10⁷ and 2 · 10⁶ M^?1. The Hill coefficient in the mid-saturation range was approx. 0.6. The data indicate that in the presence of Mg²⁺ binding to about half of the total Ca²⁺ binding sites is suppressed and there is a strong positive cooperativity involving half of the remaining sites.     

Purification and characterization of a Ca2+/Mg2+ ecto-ATPase from rat heart sarcolemma

The Ca²⁺/Mg²⁺ ATPase of the rat heart sarcolemmal particles was solublized with Triton X-100 after treating the membranes with trypsin and purified by high speed centrifugation, ammonium sulfate fractionation, hydrophobic chromatography and gel filtration. The purified enzyme was seen as a single protein band in nondenaturing polyacrylamide gel electrophoresis and its molecular weight by gel filtration was found to be about 240000. The enzyme utilized Ca-ATP or Mg-ATP as a substrate with high affinity sites (Km = 0.12 – 0.16 mM) and low affinity sites (Km = 1 mM). The enzyme also utilized CTP, GTP, ITP, UTP and ADP as substrates but at a lower rate in comparison to ATP. The enzyme was activated by Ca²⁺ (Ka = 0.4 mM) and Mg²⁺ (Ka = 0.2 mM) as well as by other cations in the order Ca^2– > Mg²⁺ > Mn²⁺ > Sr²⁺ > Ba²⁺ > Ni²⁺ > Cu²⁺. The ATPase activity in the presence of Ca²⁺ was markedly inhibited by Mg²⁺, Mn²⁺, Ni²⁺ and Cu²⁺ whereas the monovalent cations such as Na⁺ and K⁺ were without effect. The enzyme did not exhibit Ca²⁺ stimulated Mg²⁺ dependent ATPase activity and was insensitive to calmodulin, ouabain, verapamil, D-600, oligomycin, azide and vanadate. Optimum pH for Ca²⁺ or Mg²⁺ ATPase activity was 8.5 – 9.0. In view of the possible ectoenzyme nature of the ATPase, its role in adenine nucleotide and Ca²⁺ metabolism in the myocardium is discussed.     

Effect of inhibitors on the sigmoidicity of the calcium ion transport kinetics in rat liver mitochondria

The kinetic plot (initial rate of Ca²⁺ transport versus concentration) of mitochondrial Ca²⁺ transport is hyperbolic in a sucrose medium. The plot becomes sigmoidal in the presence of competitive inhibitors of Ca²⁺ binding to low affinity sites of the membrane surface such as Mg²⁺ and K⁺. The plot also becomes sigmoidal in the presence of Ba²⁺. Ba²⁺ is a competitive inhibitor of both Ca²⁺ transport and Ca²⁺ binding to the low affinity sites. The K_i for the inhibition of Ca²⁺ transport by Ba²⁺ increases in the presence of K⁺ and Mg²⁺, which suggests a competition for the low affinity sites between the cations. The plot is still hyperbolic in the presence of La³⁺, which inhibits Ca²⁺ transport competitively. Ruthenium red which is a pure non-competitive inhibitor of mitochondrial Ca²⁺ transport, does not affect the shape of the kinetic plot. These results indicate that the surface potential, which depends on the ions bound to the low affinity sites, determines whether the kinetics of Ca²⁺ uptake in mitochondria is sigmoidal or hyperbolic.     

兔肌3-磷酸甘油脱氢酶的提纯及性质研究

目的:研究兔肌3-磷酸甘油脱氢酶的分离纯化方法及其酶学性质,为测定血清甘油三酯所用酶联试剂的开发提供试验基础和理论依据。方法:通过硫酸铵分级沉淀、DEAE-Sepharose、Blue-Sepharose和羟磷灰石纯化兔肌3-磷酸甘油脱氢酶,利用凝胶过滤和梯度PAGE(5%～15%)法测定酶分子量,采用常规酶学动力学分析方法,考察pH、温度、底物浓度以及部分金属离子与有机化合物对酶促反应的影响。结果 纯化后的兔肌3-磷酸甘油脱氢酶经PAGE(12%)分析为单一条带;酶分子量为115～122 kDa;酶最适温度45℃,最适pH 9;酸碱稳定范围pH6～9,低于45℃时热稳定性好;最适条件下,以3-磷酸甘油和NAD⁺为底物,测得酶的K_m分别为7.4×10^-3mol/L和1.47×10^-4mol/L;Ba²⁺、Mn²⁺、Fe²⁺、Al³⁺、Cu²⁺、Ni²⁺、Ag⁺、Hg²⁺、NaN₃、EDTA对酶有不同程度的抑制作用,Mg²⁺、Ca²⁺、Co²⁺、Zn²⁺有一定程度的激活作用,其中Co²⁺和Zn²⁺对酶的激活作用能达到200%以上,有机化合物NaF对酶的活性没有影响。     

Purification and Regulatory Properties of Fructose 1,6-Diphosphatase from Hydrogenomonas eutropha

Fructose diphosphatase of Hydrogenomonas eutropha H 16, produced during autotrophic growth, was purified 247-fold from extracts of cells. The molecular weight of the enzyme was estimated to be 170,000. The enzyme showed a pH optimum of 8.5 in both crude extracts and purified preparation. The shape of the pH curve was not changed in the presence of ethylenediaminetetraacetic acid. The enzyme required Mg²⁺ for activity. The MgCl₂ saturation curve was sigmoidal and the degree of positive cooperativity increased at lower fructose diphosphate concentrations. Mn²⁺ can replace Mg²⁺, but maximal activity was lower than that observed with Mg²⁺ and the optimal concentration range was narrow. The fructose diphosphate curve was also sigmoidal. The purified enzyme also hydrolyzed sedoheptulose diphosphate but at a much lower rate than fructose diphosphate. The enzyme was not inhibited by adenosine 5′-monophosphate but was inhibited by ribulose 5-phosphate and adenosine 5′-triphosphate. Adenosine 5′-triphosphate did not affect the degree of cooperativity among the sites for fructose diphosphate. The inhibition by adenosine 5′-triphosphate was mixed and by ribulose 5-phosphate was noncompetitive. An attempt was made to correlate the properties of fructose diphosphatase from H. eutropha with its physiological role during autotrophic growth.     

Calcium binding to rabbit skeletal myosin under physiological conditions

At a free Mg²⁺ concentration of 1.0 mM, myosin binds one Ca²⁺ per molecule when the Ca²⁺ concentration is 20 μM, a value in the concentration range expected during contraction of skeletal muscle. Mg²⁺ alters Ca²⁺ binding in a complex manner, not by simple competition. In the range from 20 to 100 μM Mg²⁺ it produces positive cooperativity between the high-affinity Ca²⁺ binding sites, in addition to shifting binding to higher Ca²⁺ concentrations. High-affinity Ca²⁺ binding is not significantly affected by the addition of ATP, increase in ionic strength to 0.1 and changes in temperature. Ca²⁺ binding did not increase actin-activated ATPase activity in the absence of regulatory proteins, but rather inhibited it.     

14CO2 fixation and glycolate metabolism in the dark in isolated maize (Zea mays L.) bundle sheath strands

Bundle sheath strands capable of assimilating up to 68 μmoles CO₂ per mg chlorophyll per hr in the dark have been isolated from fully expanded leaves of Zea mays L. This dark CO₂-fixing system is dependent on exogenous ribose-5-phosphate, ADP or ATP, and Mg²⁺ for maximum activity. The principal product of dark fixation in this system is 3-phosphoglycerate, indicating that the CO₂-fixing reaction is mediated by ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39). The rate of dark CO₂ uptake in the strands in the presence of saturating levels of ribose-5-phosphate plus ADP is inhibited by oxygen. The inhibitory effect of oxygen is rapidly and completely reversible, and is relieved by increased levels of CO₂. Glycolate is synthesized in this dark system in the presence of [U-¹⁴C]ribose-5-phosphate, ADP, oxygen, and an inhibitor of glycolate oxidase (EC 1.1.3.1). Glycolate formation is completely abolished by heating the strands, and the rate of glycolate synthesis is markedly reduced by either lowering the oxygen tension or increasing the level of CO₂.These results, obtained with intact cells in the absence of light, indicate that the direct inhibitory effect of oxygen on photosynthesis is associated with photosynthetic carbon metabolism, probably at the level of ribulose-1,5-bisphosphate carboxylase, and not with photophosphorylation or photosynthetic electron transport. Furthermore, the findings indicate that the synthesis of glycolate from exogenous substrate can readily occur in the absence of photosynthetic electron transport, an observation consistent with the ribulose-1, 5-bisphosphate “oxygenase” scheme for glycolate formation during photosynthesis.     

The regulatory control of β-receptor dependent adenylate cyclase

The characteristics of the β-receptor in turkey erythrocyte adenylate cyclase were studied using both kinetics of enzyme activation and direct binding measurement of the β-agonists and antagonists to the β-receptor. The regulatory ligands Gpp(NH)p and Ca²⁺ do not have any direct effect on the β-receptor, but modulate the enzyme activity through the interaction with specific regulatory sites. It was found that the role of the catecholamine hormone is to facilitate the activation of the enzyme by the guanyl nucleotide. The regulatory guanyl nucleotide binds to its allosteric site in the absence of hormone, but the activation of the enzyme is slow in the absence of hormone. This role of the hormone can be described by the scheme: Where R is the receptor, E the enzyme, G the guanyl nucleotide, H the hormone, and E′ the activated form of the enzyme. The binding steps are fast and reversible but the conversion of the inactive enzyme E to its active form occurs with a k～1.0 min^?1 In the absence of the β-agonist (l-catecholamine) at the β-receptor and at physiological free Mg²⁺ concentrations, the activation of the enzyme is insignificant. Thus the presence of a guanyl nucleotide at the allosteric site is obligatory but not sufficient to induce the conversion of the inactive enzyme to its active form. At high (nonphysiological) Mg²⁺ concentration the conversion of E to E′ occurs slowly in the absence of hormone probably by another pathway. There are two classes of Gpp(NH)p regulatory sites: tight sites and loose sites, both of which can be identified kinetically. We have also identified the tight sites by direct binding studies using ³H-Gpp(NH)p. It is not clear, however, whether these are two distinct classes of sites or whether their existence reflects the presence of negative cooperativity among the guanyl nucleotide regulatory sites. Calcium was found to be a negative allosteric inhibitor of adenylate cyclase. The inhibitory effect of Ca²⁺ is exerted on the nonactivated enzyme as well as on the Gpp(NH)p preactivated enzyme.