Purification and characterization of 3-isopropylmalate dehydrogenase from a thermoacidophilic archaebacterium Sulfolobus sp. strain 7

Abstract 3-Isopropylmalate dehydrogenase was purified (about 2000-fold) to homogeneity for the first time from an archaebacterium, Sulfolobus sp. strain 7. The enzyme showed an apparent molecular mass of about 110 kDa by gel filtration and a single 36-kDa polypeptide band on SDS-PAGE, suggesting tri- or tetrameric structure. The p I value was 6.9. The N-terminal amino acid sequence was similar to enzymes from other sources. The enzyme activity was greatly stimulated by the presence of Mn²⁺, Cd²⁺, Mg²⁺, or Co²⁺. In contrast to 3-isopropylmalate dehydrogenase from other sources, monovalent cations such as K²⁺ and Na²⁺ were neither essential for activity nor stability of the protein. The enzyme was extraordinarily thermostable.     

Partial purification and some biochemical properties of acid phosphatase in germinating chickpea (Cicer arietinum) seeds

An acid phosphatase (EC 3.1.3.2.) from the embryonic axes of chickpea seeds ( Cicer arietinum L. cv. Castellana) was purified by ammonium sulphate precipitation, chromatography on Sephacryl S-200 and polyacrylamide gel electrophoresis. The preparation has an apparent molecular weight of 39 kDa, pH optimum for p -nitrophenylphosphate hydrolysis of 5.25, and K _m of 0.57 m M . The enzyme hydrolyzed all the mono- and di-phosphorylated sugars tested, but had no effect on ATP, ADP, AMP and phosphoenolpyruvate. Phosphate was a competitive inhibitor. Mg²⁺. Ca²⁺, Hg²⁺, Fe³⁺, arsenate, K⁺ and Zn²⁺ were inhibitory. Mn²⁺, dithiothreitol and EDTA had no effect, and polyamines were activators.     

Extracellular exo-polygalacturonase secreted from carrot cell cultures. Its purification and involvement in pectic polymer degradation

Exo-polygalacturonase (exo-PGase, EC 3.2.1.67) activity has been detected in a culture filtrate of cell suspension cultures of carrot ( Daucus carota L. cv. Kintoki). The extracellular exo-PGase was purified to electrophoretic homogeneity using DEAE-Sephadex A-50 ion-exchange chromatography, Sephadex G-150 gel filtration, and preparative polyacrylamide gel electrophoresis (PAGE). The molecular mass of the purified enzyme was calculated to be 48 kDa from Sephadex G-200 gel filtration, and 50 kDa from sodium dodecyl sulfate (SDS)-PAGE after treatment with SDS and 2-mercaptoethanol. The isoelectric point was at pH 6.2. The K_m and V_max values for polygalacturonate (degree of polymerization: 52) were 14.4 μ M and 25.6 μmol (mg protein)⁻¹ h⁻¹, respectively. The optimal activity in McIlvaine's buffer occurred at pH 4.6. The enzyme activity was inhibited by Ba²⁺, Cu²⁺, Mn²⁺ and Hg²⁺. The enzyme was involved in ca 15% hydrolysis of the acidic polymer purified from carrot pectic polysaccharides, and connected with the release of galacturonic acid. Even after an exhaustive reaction the enzyme had, however, little or no effect on cell walls from carrot cell cultures.     

A calcium and calmodulin-dependent protein kinase present in differentiating Dictyostelium discoideum

Abstract A protein kinase from Dictyostelium discoideum which phosphorylates the synthetic peptide, calmodulin-dependent protein kinase substrate (CDPKS, amino acid sequence: PLRRTLSVAA) and is stimulated by Ca²⁺/calmodulin is described. This is the first report of a protein kinase with these characteristics in D. discoideum . The enzyme was partially purified by Q-Sepharose chromatography. The protein kinase is very labile, and rapidly loses Ca²⁺/calmodulin-dependence upon standing at 4°C, even in the presence of protease inhibitors, making further purification and characterisation difficult. In the active fractions, a 55 kDa polypeptide is labelled with [γ-³² P]ATP in vitro under conditions in which intramolecular rather than intermolecular reactions are favoured. The phosphorylation of this peptide is stimulated in the presence of Ca²⁺ and calmodulin but not Ca²⁺ alone. Ca²⁺/calmodulin-dependent stimulation is inhibited in the presence of the calmodulin antagonist, trifluoperazine (TFP). It is proposed that the 55 kDa polypeptide may represent the autophosphorylated form of the enzyme.     

Phytase activity of Selenomonas ruminantium: a preliminary characterization

Obligately anaerobic ruminal bacteria have been found to possess phytase activity, in particular, Selenomonas ruminantium . The phytase activity of S. ruminantium JY35 was produced late in growth and required neither phytate for induction nor phosphate limitation for derepression. The activity was completely cell-associated with a significant fraction extractable by a magnesium chloride solution. Zymogram analysis suggested that the activity was the result of a single gene product of a monomeric nature and approximately 46 kDa in size. The phytase had a temperature optimum of 50–55 °C, but activity dropped off sharply at 60 °C. Phytase activity was optimal over the pH range of 4·0–5·5, and dependent on the nature of the buffer used. Activity was inhibited by citric acid buffer and by the addition of 5 mmol l⁻¹ Fe²⁺, Fe³⁺, Cu²⁺, Zn²⁺ and Hg²⁺. The addition of 5 mmol l^–1 Pb²⁺ to the enzyme assay appeared to enhance activity of the enzyme.     

Purification and biochemical characterization of a membrane-bound [NiFe]-hydrogenase from a hydrogen-oxidizing, lithotrophic bacterium, Hydrogenophaga sp. AH-24

Membrane-bound [NiFe]-hydrogenase from Hydrogenophaga sp. AH-24 was purified to homogeneity. The molecular weight was estimated as 100±10 kDa, consisting of two different subunits (62 and 37 kDa). The optimal pH values for H₂ oxidation and evolution were 8.0 and 4.0, respectively, and the activity ratio (H₂ oxidation/H₂ evolution) was 1.61 × 10² at pH 7.0. The optimal temperature was 75 °C. The enzyme was quite stable under air atmosphere (the half-life of activity was c . 48 h at 4 °C), which should be important to function in the aerobic habitat of the strain. The enzyme showed high thermal stability under anaerobic conditions, which retained full activity for over 5 h at 50 °C. The activity increased up to 2.5-fold during incubation at 50 °C under H₂. Using methylene blue as an electron acceptor, the kinetic constants of the purified membrane-bound homogenase (MBH) were V _max=336 U mg⁻¹, k _cat=560 s⁻¹, and k _cat/ K _m=2.24 × 10⁷ M⁻¹ s⁻¹. The MBH exhibited prominent electron paramagnetic resonance signals originating from [3Fe–4S]⁺ and [4Fe–4S]⁺ clusters. On the other hand, signals originating from Ni of the active center were very weak, as observed in other oxygen-stable hydrogenases from aerobic H₂-oxidizing bacteria. This is the first report of catalytic and biochemical characterization of the respiratory MBH from Hydrogenophaga .     

Note: Purification of amylase secreted from Bifidobacterium adolescentis

Bifidobacterium adolescentis Int-57 isolated from human faeces produced extracellular amylase. The enzyme was purified from the culture supernatant fluids by ammonium sulphate precipitation, gel-filtration chromatography (Sephadex-G-75), ion-exchange chromatography (CM-cellulose) and FPLC. SDS-PAGE of the purified enzyme revealed a major band with an apparent molecular weight of 66 kDa. The pI was 5·2. Enzyme activity was optimal at 50°C, and at pH 5·5. The enzyme was stable at 20–40°C, and at pH 5–6 with a K _m value of 2·4 g l⁻¹ soluble starch. The activation energy was 42·3 kJ mol⁻¹. The enzyme was significantly inhibited by maltose (10%), glucose (10%), Cu²+ (5 mmol l⁻¹), Zn²+ (5 mmol l⁻¹), N- bromosuccinimide (5 mmol l⁻¹), EDTA (5 mmol l⁻¹), I₂ (1 mmol l⁻¹) and activated by β-mercaptoethanol (10 mmol l⁻¹).     

Properties and significance of an α-amylase produced by Myxococcus coralloides D

M.E.FÁREZ-VIDAL, A. FERNÁNDEZ-VIVAS, F. GONZÁLEZ AND J.M. ARIAS. 1995. The extracellular amylase activity from Myxococcus coralloides D was purified by Sephacryl S-200 gel filtration and by ion-exchange chromatography on DEAE-Sephadex A-25. The molecular weight was estimated by SDS-PAGE and by gel filtration as 22.5 kDa. The optimum temperature was 45°C. The pH range of high activity was between 6.5 and 8.5, with an optimum at pH 8.0. Activity was strongly inhibited by Hg²⁺, Zn²⁺, Cu²⁺, Ag⁺, Pb²⁺, Fe²⁺ and Fe³⁺, EDTA and glutardialdehyde, but was less affected by Ni²⁺ and Cd²⁺. Li⁺, Mg²⁺, Ba²⁺, Ca²⁺, N -ethylmaleimide, carbodiimide and phenyl methyl sulphonyl fluoride had almost no affect. The K _m (45°C, pH 8) for starch hydrolysis was 2.0 times 10^-3 gl^-1. Comparison of the blue value-reducing curves with the time of appearance of maltose identified the enzyme produced by M. coralloides D as an α-amylase.     

Purification and kinetic properties of a castor bean seed acid phosphatase containing sulfhydryl groups

An acid phosphatase (EC 3.1.3.2) has been identified and purified from castor bean ( Ricinus communis L., IAC-80 ) seed through sulphopropyl (SP)-Sephadex, diethylaminoethyl (DEAE)-Sephadex, Sephacryl S-200, and Concanavalin A-Sepharose chromatography. The enzyme was purified 2 000-fold to homogeneity, with a final specific activity of 3.8 μkat mg⁻¹ protein. The purified enzyme revealed a single diffuse band with phosphatase activity on nondenaturing polyacrylamide gel electrophoresis, at pH 8.3. The relative molecular mass, determined by high-performance liquid chromatography (HPLC), was found to be 60 kDa. The acid phosphatase had a pH optimum of 5.5 and an akpparent K_m value for p -nitrophenylphosphate of 0.52 m M . The enzyme-catalyzed reaction was inhibited by inorganic phosphate, fluoride, vanadate, molybdate, p -chloromercuribenzoate ( p CMB), Cu²⁺ and Zn²⁺. The strong inhibition by p CMB, Cu²⁺ and vanadate suggests the presence of sulfhydryl groups essential for catalysis. The castor bean enzyme also recognized tyrosine-phosphate and inorganic pyrophosphate (KPP_i) as substrate. The highest specificity constant (V_max/K_m) was observed with KPP_i, making it a potential physiological substrate.     

Purification and characterization of an endoamylase from tulip (Tulipa gesneriana) bulbs

Amylase activity extracted from tulip ( Tulipa gesneriana L. cv. Apeldoorn) bulbs that had been stored for 6 weeks at 4°C was resolved to 3 peaks by anion-exchange chromatography on diethylaminoethyl-Sephacel. These 3 amylases exhibited different relative mobilities during non-denaturing polyacrylamide gel electrophoresis (PAGE). The most abundant amylase form (amylase I) was purified to apparent homogeneity using hydrophobic interaction chromatography, gel filtration and chromatofocusing. The apparent molecular mass of the purified amylase was estimated to be 51 kDa by sodium dodecyl sulfate-PAGE and 45 kDa by gel filtration chromatography. The purified amylase was determined to be an endoamylase (EC 3.2.1.1) based on substrate specificity and end-product analysis. The enzyme had a pH optimum of 6.0 and a temperature optimum of 55°C. The apparent K_m value with soluble starch (potato) was 1.28 mg ml⁻¹. The presence of Ca²⁺ increased the activity and thermal stability of the enzyme. The presence of dithiothreitol enhanced the activity, while β -mercaptoethanol and reduced glutathione had no significant effect. When pre-incubated in the absence of the substrate, N-ethylmaleimide and 5,5'-dithiobis-(2-nitrobenzoic acid) partially inhibited the enzyme. α -cyclodextrins or β -cyclodextrins had no effect on enzyme activity up to 10 m M . In addition to CaCl₂, CoCl₂ slightly enhanced activity, while MgCl₂ and MnCl₂ had no significant effect at a concentration of 2 m M . ZnCl₂, CuSO₄, AgNO₃ and EDTA partially inhibited enzyme activity, while AgNO₃ and HgCl₂ completely inhibited it at 2.0 m M .     

Purification of an α-L-arabinofuranosidase from carrot cell cultures and its involvement in arabinose-rich polymer degradation

Konno, H., Yamasaki, Y. and Katoh, K. 1987. Purification of an α-L-arabinofurano-sidase from carrot cell cultures and its involvement in arabinose-rich polymer degradation.
An α-L-arabinofuranosidase (α-L-arabinofuranoside arabinofuranohydrolase, EC 3.2.1.55) was isolated from a homogenate of cell suspension cultures of carrot ( Daucus carota L. cv. Kintoki). The buffer-soluble enzyme was purified to homogeneity by a procedure involving ammonium sulfate fractionation, chromatography on DEAE-Sephadex A-50, Sephadex G-150, Con A-Sepharose 4B and CM-Sephadex C-50, and preparative polyacrylamide gel electrophoresis. The size of this enzyme as determined by polyacrylamide gel electrophoresis in the presence of sodium laurylsulfate and by Sephadex G-200 gel filtration was 94 and 110 kDa, respectively. The isoelectric point was at pH 4.7. The K_m and V_max values for p-nitrophenyl α-L-arabinofuranoside were 1.33 mM and 20.2 μimol (mg protein)^-1 h^-1, respectively. The optimal activity occurred at pH 4.2 with Mcllvaine buffer. The enzyme was stimulated by Ca²⁺ and Zn²⁺, whereas it was strongly inhibited by Cu²⁺, Ag²⁺, Hg²⁺, p-chloromercuri-benzoate and L-arabono-l,4-lactone. The enzyme acted on beet arabinan in an exo-fashion. Furthermore, the enzyme was partially involved in the hydrolysis of the ara-binogalactan and pectic polymer purified from carrot cell walls.     

Partial purification and some biochemical properties of nonspecific alkaline phosphatase in germinating chick-pea (Cicer arietinum) seeds

A procedure for the partial purification of a non-specific alkaline phosphatase (EC 3.1.3.1.) from the embryonic axes of chick-pea seeds is described. Ammonium sulphate precipitation, DEAE-cellulase chromatography, Sephacryl S-200 chroma-tography and polyacrylamide gel electrophoresis are the most important steps. The molecular weight of this non-specific enzyme, as determined by Sephacryl S–200 gel filtration and SDS–polyacrylamide gel electrophoresis, was estimated as being 68 and 78 kDa respectively; the optimum pH for p-nitrophenylphosphate hydrolysis was 7.5, and the K_m for this artificial substrate was 0.5 mM. The enzyme catalyzes the hydrolysis of a variety of organic phosphate esters. The best substrates are: phos-phoenolpymvate (K_m= 2.4 m M ), NADP⁺ (K_m= 4.0 m M ), 5'-AMP (K_m= 4.5 m M ), 5'-ADP (K_m= 6.1 m M ) and ribose-5P (K_m= 5.8 m M ); but it is unable to hydrolyze 5'-ATP, phosphocreatine and tripolyphosptiate. Phospate was a competitive inhibitor. Zn²⁺, K⁺, Hg²⁺ and Mo⁶⁺ were strong inhibitors, whereas F⁻ and Ca²⁺ inhibited weakly; Co²⁺ and Ni²⁺ were activators.     

Purification and characterization of M3 protein expressed on the surface of group A streptococcal type 3 strain C203

Abstract Monoclonal antibodies (mAbs) have been produced by immunizing BALB/C mice with whole M⁺ bacteria in incomplete Freund adjuvant and the resulting mAbs for M3 protein have been selected by an indirect immuno-fluorescent technique using formaldehyde-fixed M⁺ and M⁻ bacteria. Four mAbs reacted with a 65 kDa protein in an extract obtained from the cell wall of M⁺ bacteria after treatment with N -acetyl muramidase and lysozyme. The purified 65 kDa protein neutralized the phagocytic activity of rabbit anti-M3 antibody. The N-terminal amino acid sequence of the 65 kDa protein was identical with that of protein generated by the M3 gene which has been previously cloned and sequenced. The evidence indicates that the 65 kDa protein is M3 protein. The M3 protein bound not only human fibrinogen but also human serum albumin (HSA). When the M3 protein was purified by gel-filtration and ion-exchange chromatography in the absence of phenylmethyl sulfonyl fluoride (PMSF), four fragments (35 kDa, 32 kDa, 30 kDa, and 25 kDa) in addition to the intact molecule appeared. N-terminal amino acid sequence analysis showed that 35 kDa and 25 kDa fragments were ANAAD and DARSV, respectively, being identical at positions 1–5 and 198–202 to the M3 gene derived protein. Therefore, the 35 kDa and 25 kDa fragments, which were presumed to be cleavage products, may be derived from the C-terminal part and N-terminal part of the intact molecule, respectively. When the effect of purified M3 protein in the bactericidal activity of normal human blood in the presence of M⁻ bacteria was investigated, the M3 protein was responsible for the organism's resistance to attack by phagocytic cells.     

An extracellular α-L-arabinofuranosidase secreted from cell suspension cultures of carrot

Carrot ( Daucus carota L. cv. Kintoki) cell cultures secrete an α-L-arabinofuranosidase (α-L-AFase, EC 3.2.1.55) into their culture medium during growth. The extracellular α-L-AFase (α-L-AFase-II) was purified to electrophoretic homogeneity from the concentrated medium using ammonium sulfate precipitation, chromatography on DEAE-Sepharose CL-6B, CM-Sepharose CL-6B, Sephacryl S-200HR and Concanavalin A-Sepharose, and preparative PAGE. The molecular mass of the purified enzyme was estimated to be 84 kDa by Sephacryl S-200HR gel-permeation, and 80 kDa by SDS-PAGE under denaturing conditions. The enzyme contained carbohydrate and protein in a ratio of 1:5 (w/w), and was analyzed for amino acid composition and the sequence of the first 21 amino acids of the N-terminus. The isoelectric point was pH 5.6, the pH optimum 3.8, and the temperature optimum 55°C. The activity was inhibited by Zn²⁺, Ag²⁺, Cu²⁺, Hg²⁺ and p -chloromercuribenzoate. The K_m and V_max values for p -nitrophenyl-α-L-arabinofuranoside were 0.22 m M and 0.11 mmol (mg protein)⁻¹ h⁻¹, respectively. The enzyme acted on beet arabinan in an exo-fashion, and was capable of hydrolysing arabinose-rich polymers purified from pectic polysaccha-rides of carrot cell cultures. However, even after an exhaustive reaction, the enzyme had little or no effect on cell walls from carrot cell cultures.     

Interaction of Guanine Nucleotides with [3H]Kainate and 6-[3H]Cyano-7-Nitroquinoxaline-2,3-dione Binding in Goldfish Brain

Abstract— Recent reports have suggested that a major proportion of [³H]kainate binding in goldfish brain is to a novel form of G-protein-linked glutamate receptor. Here we confirm that guanine nucleotides decrease [³H]kainate binding in goldfish brain membranes, but that binding is also reduced to a similar extent under conditions where G-protein modulation should be minimised. Inclusion of GTPγS resulted in an approximately twofold decrease in the affinity of [³H]kainate binding and a 50% reduction in the apparent B _max values in both Mg²⁺/Na⁺ and Mg²⁺/Na⁺-free buffer when assayed at 0°c. The pharmacology of [³H]kainate binding is similar to that of well-characterised ionotropic kainate receptors but unlike that of known me-tabotropic glutamate receptors, with neither 1 S ,3 R -amino-1,3-cyclopentanedicarboxylic acid (1 S ,3 R -ACPD) nor ibo-tenic acid being effective competitors. The molecular mass of the [³H]kainate binding protein, as determined by radiation inactivation, was 40 kDa, similar to the subunit sizes of other lower vertebrate kainate binding proteins that are believed to comprise ligand-gated ion channels. Furthermore, GTP-γS also inhibited the binding of the non-NMDA receptor-selective antagonist 6-[³H]cyano-7-ni-troquinoxaline-2,3-dione. These data strongly suggest that the regulatory interaction between guanine nucleotides and [³H]kainate and 6-[³H]cyano-7-nitroquinoxaline-2,3-dione binding is complex and involves competition at the agonist/antagonist binding site in addition to any G-protein-mediated modulation.     

Purification of a β-galactosidase from rice shoots and its involvement in hydrolysis of the natural substrate in cell walls

Several glycosidase and glycanase activities have been detected in homogenates of rice ( Oryza sativa L. cv. Nipponbare) shoots after successive extraction with K-phosphate (pH 7. 0) and buffer containing 3 M LiCl. The major β-D-galactosidase (EC 3. 2. 1. 23) present in the buffer-soluble protein fraction was purified to electrophoretic homogeneity by a combination of chromatographic techniques including DEAE-Sepharose CL-6B, Sephacryl S-200HR and p -aminophcnyl-β-D-thiogalactopyranoside–Sepharose 4B. Analysis by denaturing gel electrophoresis revealed a single polypeptide chain with an apparent molecular mass of 42 kDa. Similar to the value of 40 kDa estimated for the native protein by gel-permeation. The isoelectric point was pH 6. 0. The K_m and V_max values for p -nitrophenyl (PNP)-β-D-galactopyra-noside were 0. 63 m M and 0. 32 mmol (mg protein)⁻¹ h⁻¹, respectively. Maximum activity in McIlvaine buffer occurred at pH 3. 4, and the activity was inhibited by Ag²⁺, Cu²⁺. Hg²⁺, p -chloromercuribenzoate (PCMB) and D-galactono-l,4-lactone. The enzyme hydrolyzed larchwood arabinogalactan in an exo-fashion, and acted weakly on arabinosyl and galactosyl residue-rich polymer of pectic polysaccharides and cell walls from rice shoots.     

Purification and some properties of (1R,2S)-1,2-dihydroxy-3,5-cyclohexadiene-1,4-dicarboxylate dehydrogenase from Comamonas testosteroni T-2

Abstract Inducible (1 R ,2 S )-1,2-dihydroxy-3,5-cyclohexadiene-l,4-dicarboxylate (diene-diol) dehydrogenase was found in extracts of Comamonas testosteroni T-2 grown in p -toluate-or terephthalate-salts medium and it was purified using anion exchange, hydrophobic interaction and gel filtration chromatography. The enzyme is a homodimer with subunit M r 39000. It had a specific activity of 500 mkat/kg of protein and was activated by the addition of Fe²⁺. The dehydrogenase converted 1 mol diene-diol and 1 mol NAD⁺ to 1 mol protocatechuic acid, 1 mol NADH and 1 mol CO₂. Apparent K _m-values of 43 μM (NAD⁺) and about 90 μM (diene-diol) were determined. The hydride ion was transferred to the si face of NAD⁺.     

Identification and Characterization of Two DNA Polymerase Activities Present in Trypanosoma brucei Mitochondria

We have identified and partially purified two DNA polymerase activities from purified Trypanosoma brucei mitochondrial extracts. The DNA polymerase activity eluted from the single-stranded DNA agarose column at 0.15 M KCI (polymerase MI) was significantly inhibited by salt concentrations greater than 100 mM, utilized Mg²⁺ in preference to Mn²⁺ as a cofactor on deoxyribonucleotide templates with deoxyribose primers, and in the presence of Mn²⁺ favored a ribonucleotide template with a deoxyribose primer. A 44 kDa peptide in this fraction crossreacted with antisera against the Crithidia fasciculata β-like mitochondrial polymerase. In activity gels the catalytic peptide migrated at an apparent molecular weight of 35 kDa. The DNA polymerase activity present in the 0.3 M KCI DNA agarose fraction (polymerase M2) exhibited optimum activity at 120-180 mM KCI, used both Mg²⁺ and Mn²⁺ as cofactors, and used deoxyribonucleotide templates primed with either deoxyribose or ribose oligomers. Activity gel assays indicate that the native catalytic peptide(s) is ˜ 80 kDa in size. The two polymerases showed different sensitivities to several inhibitors: polymerase MI shows similarities to the Crithidia fasciculata β-like mitochondrial polymerase while polymerase M2 is a novel, salt-activated enzyme of higher molecular weight.     

Reconstitution and rapid partial purification of the red beet plasma membrane ATPase

Red beet ( Beta vulgaris L., cv. Detroit Dark Red) plasma membrane ATPase solubilized from a deoxycholate-extracted plasma membrane fraction with Zwittergent 3–14 was reconstituted into liposomes. Detergent removal and reconstitution was carried out by column chromatography on Sephadex G-200 followed by centrifugation at 100 000 g for I h. Prior to reconstitution, optimal activity in the solubilized preparation was observed when dormant red beet tissue was used in the extraction/solubilization procedure. Following reconstitution into liposomes, ATP-dependent proton transport could be demonstrated by measuring the quenching of acridine orange fluorescence. Proton transport and ATPase activity in the reconstituted enzyme preparation were inhibited by orthovandate but stimulated by KNO₃. This stimulation most likely results from a reduction in the membrane potential generated during electrogenic proton transport by the reconstituted ATPase. The ATPase activity of the reconstituted ATPase was further characterized and found to have a pH optimum of 6.5 in the presence of both Mg²⁺ and K⁺. The activity was specific for ATP, insensitive to ouabain and azide but inhibited by N;N-dicyclohexylcarbodiimide and diethylstilbestrol. Stimulation of ATP hydrolytic activity occurred in the sequence: K⁺ Rb⁺ Na⁺ Cs⁺ Li⁺ and the kinetics of K⁺ stimulation of ATPase activity followed non-Michaelis-Menten kinetics as observed for both the membrane-bound and solubilized forms of the enzyme. Reconstitution of the plasma membrane ATPase from red beet allowed a substantial purification of the enzyme and resulted in the enrichment of a 100 kDa polypeptide representing the ATPase catalytic subunit.     

Properties of NADP+-malic enzyme from pod walls of chickpea (Cicer arietinum)

NADP⁺-malic enzyme ( l -malate: NADP⁺ oxidoreductase, decarboxylating EC 1.1.1.40) from pod walls of chickpea was purified 51-fold by ammonium sulphate fractionation, DEAE- cellulose chromatography and gel filtration through Sepharose 4B. The purified enzyme required a divalent cation, either Mn²⁺ or Mg²⁺, for its activity. K_m values at pH 7.8 for malate, NADP⁺ and Mn²⁺ were 4.0, 0.031 and 0.71 m M , respectively. Mn²⁺-dependent activity was inhibited by heavy metal ions such as Cd²⁺, Zn²⁺, Hg²⁺, and to a lesser extent by Pb²⁺ and Al³⁺. Among the organic acids examined, sodium salts of oxalate and oxaloacetate were inhibitory. Kinetics of the reaction mechanism showed sequential binding of malate and NADP⁺ to the enzyme. Products of reaction, viz. pyruvate, bicarbonate and NADPH, inhibited the enzyme activity. At limiting concentrations of NADP⁺, pyruvate and bicarbonate induced a positive cooperative effect by malate. It is proposed that the activity of NADP⁺-malic enzyme is controlled by intracellular concentrations of substrates and products.