Chloroplast Phosphofructokinase: II. Partial Purification, Kinetic and Regulatory Properties

Chloroplast phosphofructokinase from spinach (Spinacia oleracea L.) was purified approximately 40-fold by a combination of fractionations with ammonium sulfate and acetone followed by chromatography on DEAE-Sephadex A-50. Positive cooperative kinetics was observed for the interaction between the enzyme and the substrate fructose 6-phosphate. The optimum pH shifted from 7.7 toward 7.0 as the fructose 6-phosphate concentration was taken below 0.5 mm. The second substrate was MgATP(2-) (Michaelis constant 30 mum). Free ATP inhibited the enzyme. Chloroplast phosphofructokinase was sensitive to inhibition by low concentration of phosphoenolpyruvate and glycolate 2-phosphate (especially at higher pH); these compounds inhibited in a positively cooperative fashion. Inhibitions by glycerate 2-phosphate (and probably glycerate 3-phosphate), citrate, and inorganic phosphate were also recorded; however, inorganic phosphate effectively relieved the inhibitions by phosphoenolpyruvate and glycolate 2-phosphate. These regulatory properties are considered to complement those of ADP-glucose pyrophosphorylase and fructosebisphosphatase in the regulation of chloroplast starch metabolism.     

Kinetic Properties of the ATP-Dependent Phosphofructokinase Isoenzymes from Cucumber Seeds

Two isoenzymes of ATP:D-fructose-6-phosphate 1-phosphotransferase(phosphofructokinase) are present in germinating cucumber seeds,one in the plastids and the other in the cytosol. Both isoenzymeswere purified and some of their kinetic properties studied.These two isoenzymes differ kinetically, the pH optimum of thecytosolic isoenzyme being 7.2 and that of the plastid isoenzymebeing 8.0. Both isoenzymes are activated by phosphate althoughthe concentration required for activation is much lower forthe plastid isoenzyme than cytosolic isoenzyme. Phosphate increasesthe affinity of the isoenzymes for fructose-6-phosphate andalso changes the sigmoidal kinetics of the plastid isoenzymefor this substrate to hyperbolic kinetics at pH 7.2. The fructose-6-phosphatesaturation kinetics of the cytosolic isoenzyme becomes moresigmoidal with an increase in pH while the opposite is truefor the plastid isoenzyme. The cytosolic isoenzyme has a higheraffinity for fructose-6-phosphate at pH 7.2 than pH 8.0 whilethe affinity of the plastid isoenzyme for fructose-6-phosphateis highest at pH 8.0. Both isoenzymes are inhibited by ATP andthe extent of inhibition is pH dependent. The cytosolic isoenzymeis more sensitive to ATP inhibition at pH 8.0 than pH 7.2 whilethe opposite holds for the plastid isoenzyme. Magnesium alleviatesthe ATP inhibition of the plastid isoenzyme suggesting thatfree ATP is the inhibitory form. In contrast the ATP inhibitionof the cytosolic isoenzyme apparently appears to be caused bythe magnesium-ATP complex. (Received May 19, 1987; Accepted January 18, 1988)     

Phosphofructokinase from Ascaris suum. Purification and properties

A rapid and efficient procedure has been developed to purify phosphofructokinase from the muscle of the parasitic roundworm, Ascaris suum. The procedure can be accomplished in 1 day with a 420-fold purification and a 60% yield. The enzyme was shown to be homogeneous by two-dimensional electrophoresis, Sepharose 6B column chromatography, and high performance liquid chromatography utilizing a size exclusion column. The subunit molecular weight of the enzyme was found to be 95,000 by electrophoresis in the presence of sodium dodecyl sulfate. In solutions of low ionic strength, the native enzyme aggregated to species of higher molecular weight than did the rabbit muscle phosphofructokinase. In the presence of 0.2 M (NH4)2SO4, the minimum native molecular weight was determined to be 398,000 by high performance liquid chromatography and Sepharose 6B column chromatography. Therefore, the enzyme appears to be a tetramer with identical or near-identical subunits. The apparent isoelectric point of the enzyme was shown to be 7.3 to 7.4 by both column and gel isoelectric focusing. Amino acid analysis revealed a lower number of the aromatic residues Phe, Tyr, and Trp than in the rabbit muscle enzyme and this is in agreement with the lower extinction coefficient of E1%280 nm = 6.5. Analysis of the purified enzyme revealed 7.4 +/- 0.6 mol of phosphate/mol of enzyme.     

Purification and Kinetic Properties of Betaine-Homocysteine Methyltransferase from Aphanothece halophytica

Betaine-homocysteine methyl transferase (BHMT) from Aphanothece halophytica was purified to homogeneity by hydroxyapatite, DEAE-Sepharose CL-6B and Sephadex G-200 column chromatography. A 24-fold purification and 11% overall yield were achieved with a specific activity of 595 nmol h⁻¹ mg⁻¹. The subunit molecular weight was determined to be 45 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the native enzyme was found to have a molecular weight of 350 kDa, suggesting an octameric structure of the enzyme. The enzyme shows optimum activity at 37°C, pH 7.5. The apparent Km values for glycinebetaine and L-homocysteine were 4.3 mM and 1.3 mM, respectively. The enzyme was 70% inactivated by 5 mM dimethylglycine whereas the same concentration of sarcosine slightly inactivated the enzyme. Two analogs of glycinebetaine were also tested for enzyme inactivation and it was found that 5 mM choline inactivated 60% of the enzyme activity and 2.5 mM betaine aldehyde completely abolished the enzyme activity. NaCl at 200 mM or higher also completely inactivated the enzyme. Received: 6 December 2000 / Accepted: 10 January 2001     

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.     

Purification and characterization of a novel haemagglutinin from Chlorella pyrenoidosa

We previously identified a strong haemagglutination activity in the freshwater unicellular green alga, Chlorella pyrenoidosa. Here, we sought to purify and characterize the haemagglutinin associated with this activity. Ammonium sulfate precipitation, gel filtration on sephacryl S-200 and DEAE-Sepharose ion-exchange chromatography were used to purify the haemagglutinin, which was designated CPH (Chlorella pyrenoidosa haemagglutinin). The molecular weight of CPH was estimated as 58 kDa by SDS-PAGE and 60 kDa by gel filtration of the native protein, indicating that this haemagglutinin exists as a monomer. The haemagglutinin activity of CPH was inhibited by glycoproteins, especially yeast mannan, but not by monosaccharides or disaccharides, indicating that CPH is carbohydrate-specific. In addition to the composition of CPH shown to be rich in glycine and acidic amino acids, heamagglutinating activity of CPH was insensitive to variations in pH or the presence of divalent cations, and atomic force microscopy revealed that the protein is rod-shaped. These results indicate that the characteristics of CPH are consistent with its identification as a haemagglutinin, and suggest that CPH may be a viable candidate for applications in a variety of biomedical fields.     

Purification and Kinetic Properties of 6-Phosphogluconate Dehydrogenase from Rat Small Intestine

6-Phosphogluconate dehydrogenase (6PG) was purified from rat small intestine with 36% yield and a specific activity of 15 U/mg. On SDS/PAGE, one band with a mass of 52 kDa was found. On native PAGE three protein and two activity bands were observed. The pH optimum was 7.35. Using Arrhenius plots, Ea, ΔH, Q₁₀ and Tm for 6PGD were found to be 7.52 kcal/mol, 6.90 kcal/mol, 1.49 and 49.4°C, respectively. The enzyme obeyed “Rapid Equilibrium Random Bi Bi” kinetic model with K_m values of 595 ± 213 μM for 6PG and 53.03±1.99 μM for NADP. 1/V_m versus 1/6PG and 1/NADP plots gave a V_m value of 8.91±1.92 U/mg protein. NADPH is the competitive inhibitor with a K_i of 31.91±1.31 μM. The relatively small K_i for the 6PGD:NADPH complex indicates the importance of NADPH in the regulation of the pentose phosphate pathway through G6PD and 6PGD.     

Partial Purification of the NADH-Nitrate Reductase Complex from Chlorella pyrenoidosa

The nitrate reductase complex from Chlorella pyrenoidosa has been purified by a procedure which includes as main steps, ammonium sulfate fractionation, polyethylene glycol treatment, and DEAE-cellulose chromatography. The Michaelis constants for NADH, FAD, and NO₃⁻ in the NADH-nitrate reductase assay are 10 μm, 2.6 μm, and 0.23 mm, respectively. Heat treatment exerts varying effects on the enzymatic activities associated with the nitrate reductase complex.     

鸡蛋果叶片细胞质丙酮酸激酶的部分纯化及其调节性质

鸡蛋果叶片细胞质丙酮酸激酶(PK_c)纯化92.6倍.其最适pH为7.2,对热较稳定。PEP的K_m为0.037 mmol/L,ADP的K_m为0.05 mmol/L。ASP、Asn、Cys、α—酮戊二酸和苹果酸均对PK?有轻微的激活作用,但草酸、ATP、CaCl_2则具强烈的抑制作用。     

Cloning, Sequencing, Expression, and Purification of the C Isozyme of Mouse Phosphofructokinase

The cDNA of mouse phosphofructo-1-kinase isozyme C was cloned and sequenced. The coding region translates into a protein of 85,473 Da containing 785 amino acids. The cDNA includes 57 base pairs of a 5'-untranslated region and a 3' untranslated region of 284 base pairs containing a polyadenylation signal, AUUAAA, located 17 bases upstream from the poly(A) tail. The cDNA was ligated into a pET vector and transformed into a pfk(-) strain of Escherichia coli (DF1020) that contained the pLysS plasmid and an integrated lambda DE3 prophage that includes a single copy of the gene for T7 RNA polymerase under control of the inducible LacUV5 promoter. Conditions for maximum induction of soluble enzyme activity was developed to produce up to 2400 units of soluble enzyme activity per liter of growth medium. The enzyme could be purified to homogeneity with a yield of approximately 60% by a single purification step on ATP-Sepharose.     

Fructose-1,6-Diphosphate-Dependent Lactate Dehydrogenase from a Cariogenic Streptococcus: Purification and Regulatory Properties

The lactate dehydrogenase (LDH) from Streptococcus mutans NCTC 10449 is under stringent metabolic control. The partially purified enzyme was specifically activated by high concentrations of fructose-1,6-diphosphate (FDP) and was inhibited by adenosine triphosphate. There appeared to be at least two binding sites for the activator which interacted in a cooperative manner. The interaction between the FDP sites was independent of the pH of the assay system, although the relative affinity of the enzyme for the activator was influenced by pH. There also appeared to be at least two pyruvate binding sites on the S. mutans LDH with some cooperative interaction between them, and the interaction between these sites was also independent of the hydrogen ion concentration. Two pyruvate analogues had different effects on the interaction of pyruvate with the LDH. One of the analogues, alpha-ketobutyrate, stimulated enzyme activity at limiting pyruvate concentrations, but had no significant effect at saturating concentrations of the substrate. The net effect of alpha-ketobutyrate was to shift the pyruvate saturation curve from sigmoidal to hyperbolic and to decrease the Hill coefficient from about 2.0 to 1.0. The other pyruvate analogue, oxamate, inhibited enzyme activity at all pyruvate concentrations but had no effect on the sigmoidal nature of the pyruvate saturation curve or on the apparent kinetic order of the reaction with respect to substrate. These results suggested that there may be two types of pyruvate binding sites on the LDH from S. mutans. Other kinetic properties of the S. mutans NCTC 10449 enzyme were studied and compared with those exhibited by the LDH from several other strains of the organism.     

Phosphofructokinase from lycopersicon esculentum fruits-I. Kinetic properties in relation to its substrates

The kinetic properties of phosphofructokinase with regard to its substrates are discussed. Free ATP is inhibitory to the enzyme while the Mg-ATP complex at a concentration up to 5 mM is not. The kinetics with respect to Mg-ATP follow simple Michaelis-Menten kinetics and this pattern is not affected by changes in concentration of the second substrate fructose-6-phosphate (F6P). The kinetics with respect to F6P showed apparent negative co-operative interactions in the presence of saturating levels of Mg²⁺ relative to ATP. In the presence of inhibitory levels of free ATP, the kinetics showed positive co-operative interactions. The relationship between the nature of the kinetics of the enzyme with F6P and the various molecular forms of PFK are discussed.     

Preparation of Protoplasts from Chiorella ellipsoidea C-27

Protoplasts from Chlorella ellipsoidea IAM C-27 were rapidlyobtained by enzymatic digestion with a mixture of ChitosanaseKI, mixed glycosidases, and a cell wall-lytic enzyme found inthe cell homogenate of C-27 cells. The formation of naked protoplastswas demonstrated by electron microscopy. (Received December 18, 1991; Accepted May 12, 1992)     

Partial Purification of Isocitrate Lyase from Candida tropicalis and Some Kinetic Properties of the Enzyme

Isocitrate lyase was purified partially from n-alkane-grown cells and glucose-grown cells of Candida tropicalis by means of ammonium sulfate fractionation and DEAE-cellulose column chromatography. The preparation from alkane-grown cells showed one peak of the enzyme activity, while that from glucose-grown cells showed two distinct peaks of the activity, on DEAE-cellulose column chromatography. These enzymes, having the similar pH optima (around 7.0) and Km values with dl-isocitrate (1.2 ~ 1.7 mm), were inhibited by various metabolic intermediates, such as 6-phosphogluconate and phosphoenolpyruvate.

Time-course changes in the activities of isocitrate lyase and isocitrate dehydrogenases of C. tropicalis during the growth indicated that the lyase would participate preferentially in alkane assimilation and NAD-linked isocitrate dehydrogenase in glucose utilization of the yeast.

Regulation of isocitrate metabolism in C. tropicalis through glyoxylate cycle and tricarboxylic acid cycle is discussed based on the kinetic properties, cellular localization and time- course changes in the levels of isocitrate lyase and NAD-linked and NADP-linked isocitrate dehydrogenases.     

Human Brain 6-Phosphogluconate Dehydrogenase: Purification and Kinetic Properties

6-Phosphogluconate dehydrogenase has been purified from human brain to a specific activity of 22.8 U/mg protein. The molecular weight was 90,000. At low ionic strengths enzyme activity increased, due to an increase in Vmax and a decrease in Km for 6-phosphogluconate, and activity subsequently decreased as the ionic strength was increased (above 0.12). Both 6-phosphogluconate and NADP+ provided good protection against thermal inactivation, with 6-phosphogluconate also providing considerable protection against loss of activity caused by p-chloromercuribenzoate and iodoacetamide. Initial velocity studies indicated the enzyme mechanism was sequential. NADPH was a competitive inhibitor with respect to NADP+, and the Ki values for this inhibition were dependent on the concentration of 6-phosphogluconate. Product inhibition by NADPH was noncompetitive when 6-phosphogluconate was the variable substrate, whereas inhibition by the products CO2 and ribulose 5-phosphogluconate and NADP+ were varied. In totality these data suggest that binding of substrates to the enzyme is random. CO2 and ribulose 5-phosphate are released from the enzyme in random order with NADPH as the last product released.     

Phosphofructokinase from porcine heart, liver and erythrocytes

1. Phosphofructokinase from porcine heart, liver and erythrocytes were purified by affinity chromatographies on Cibacron Blue Sepharose and N6-ATP agarose. 2. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate revealed that the heart and liver enzymes consist of only one kind of subunit, namely, M and L type subunits, respectively, whereas the erythrocyte enzyme comprises of three kinds of subunits, M, L and C types. 3. Some kinetic and regulatory properties of the enzymes were also measured.     

菠菜叶片中两种酶型的磷酸果糖激酶性质的比较

菠菜叶片提取液经ＰＥＧ－６０００沉淀、ＤＥ－５２离子交换柱层析及分子筛ＳｅｐｈｅｃｔｙｌＳ－３００凝胶过滤得到两种分子量不同的依赖ＡＴＰ的磷酸果糖激酶（ＰＦＫ）。一为大分子酸型,分子量大于２０００ｋＤ,其活力可被Ｐｉ、３－ＰＧＡ、柠檬酸激活,被ＰＥＰ强烈抑制,Ｐｉ能减缓此抑制作用,Ｍｇ２＋为必需金属离子,但其浓度高于０．５ｍｍｏｌ／Ｌ时酶活力降低;一为小分子酸型,分子量为３００ｋＤ,其活性受Ｐｉ、３－ＰＧＡ、柠檬酸和ＰＥＰ抑制,Ｍｇ２＋亦为必需金属离子,Ｈｉｌｌ系数为０．６７,表现负协同效应。实验证明小分子酸型可能存在叶绿体中,大分子酸型属于胞质酶。