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.     

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 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.     

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.     

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.     

An extracellular β-galactosidase secreted from cell suspension cultures of carrot. Its purification and involvement in cell wall-polysaccharide hydrolysis

β-Galactosidase (β-Galase, EC 3.2.1.23) activity has been detected in a culture medium of cell suspension cultures of carrot ( Daucus carota L. cv. Kintoki). The extracellular β-Galase (β-Galase-II) was purified to electrophoretic homogeneity from the concentrated medium using ammonium sulfate precipitation, chromatography on CM-Sephadex C-50. DEAE-Sepharose CL-6B and Sephacryl S-200HR, and preparative PAGE. The molecular mass of the purified enzyme was estimated to be 65 kDa by Sephacryl S-200HR gel-permeation, and 60 kDa by SDS-PAGE after treatment with SDS and 2-mercaptoethanol. The pI was 6.5. The K_m and V_max values for p -nitrophenyl (PNP)-β-D-galactopyranoside were 0.17 m M and 31.9 μmol (mg protein)^-1, h^-1, respectively. The optimal activity in McIlvaine's buffer occurred at pH 4.0–4.4. The enzyme activity was inhibited by Co²⁴, Cu²⁺, Hg^2-, p -chloromercuribenzoate (PCMB) and D-galactono-1,4-lactone. The enzyme acted on citrus galactan and larchwood arabinogalactan in an exo-fashion, and was slightly involved in the hydrolysis of an acidic pectic polymer containing arabinosyl and galactosyl residues and in the breakdown of cell walls isolated from carrot cell cultures.     

Purification and characterization of adenine phosphoribosyltransferase from Arabidopsis thaliana

Adenine phosphoribosyltransferase (APRT; EC 2. 4,2. 7) from Arabidopsis thaliana was purified approximately 3800-fold, to apparent homogeneity. The purification procedure involved subjecting a leaf extract to heat denaturation, (NH₄)₂SO₄ precipitation, Sephadex G-25 salt separation, ultracentrifugation and liquid chromatography on Diethylaminoethyl Sephacel, Phenyl Sepharose CL-4B, Blue Sepharose CL-6B and adenosine 5'-monophosphate-Agarose. The purified APRT was a homodimer of approximately 54 kDa and it had a specific activity of approximately 300 μmol (mg total protein)^-1 min^-1. Under standard assay conditions, the temperature optimum for APRT activity was 65°C and the pH optimum was temperature dependent. High enzyme activity was dependent upon the presence of divalent cations (Mn²⁺ or Mg²⁺). In the presence of MnCl₂₊ other divalent cations (Mg²⁺, Ca²⁺, Ba²⁺, Hg²⁺ and Cd²⁺) inhibited the APRT reaction. Kinetic studies indicated that 5-phosphoribose-1-pyrophosphate (PRPP) caused substrate inhibition whereas adenine did not. The K_m for adenine was 4.5±1.5 μ M , the K_m for PRPP was 0.29±0.06 m M and the K_i for PRPP was 1.96±0.45 m M . Assays using radiolabelled cytokinins showed that purified APRT can also catalyze the phosphoribosylation of isopentenyladenine and benzyladenine. The K_m for benzyladenine was approximately 0.73±0.06 m M     

Two forms of fructose 1,6-bisphosphatase from immature wheat endosperm

Fructose 1,6-bisphosphatase (α-D-fructose 1,6-bisphosphate 1-phosphohydrolase, EC 3.1.3.11; FBPase) from immature wheat endosperm has been resolved into two forms, FBPase-I and FBPase-II. Their specific activities over crude homogenate increased 47- and 77-fold, respectively, by using ammonium sulfate fractionation, DEAE-cellulose chromatography and gel filtration through Sephadex G-200. The pH optimum was 7.6 for FBPase-I and 8.4 for FBPase-II. The two forms were highly specific for the substrate FBP with K_m values of 0.17 and 0.08 m M , respectively, for FBPase-I and FBPase-II at their respective pH optimum and saturating Mg²⁺ concentration. pH had no effect on the K_m value for FBPase-I, but that for FBPase-II increased below optimum pH. Neither of the forms had an absolute requirement for Mg²⁺, although it was essential for maximum activity. Mg²⁺ could not be replaced by Cu²⁺, Ca²⁺, Ba²⁺, Co²⁺ or Ni²⁺. Sulfhydryl reagents inactivated both FBPase-I and FBPase-II. Of the metabolites, only 6-phosphogluconate was inhibitory with 50% inhibition at 2 and 4 m M for FBPase-I and FBPase-II, respectively.     

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 .     

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.     

Purification and properties of NADP-dependent glutamate dehydrogenase from Sphaerostilbe repens

NADP-dependent glutamate dehydrogenase (EC 1.4.1.4) extracted from Sphaerostilbe repens was purified to homogeneity by using ammonium sullate fractionation hydroxyapatite and DEAE-cellulose column chromatography and, finally, preparative polyacrylamide gel electrophoresis. The turnover number of the enzyme for the amination reaction was about 66000 mol substrate transformed min^-1 (molecule of GDH)^-1. Molecular weight of the native enzyme was estimated to be 280000 dalton by polyacrylamide gradient gel electrophoresis. The same technique in the presence of sodium dodecyl sulfatc gave a single protein band that corresponded to the subunit molecular weight of 48000 dalton. Thus, it is concluded that NADP-GDH is composed of six identical polypeptidic chains.
The pH optimums were 6.9 and 8.4 for the forward and reverse reactions respectively. The NADP-GDH lost practically none of its activity for ten days at 4°C and for 15 h at room temperature, but was inactivated by higher temperatures. Thiol compounds such as 2-mercaptoethanol and dithiolhrcitol protected the enzyme from rapid inactivation. The Michaelis constants for GDH were 0.64, 0.049. 0.043 and 5.5 m M for α-ketoglutaratc. NADPH, NADP and glutamate, respectively. The enzyme had a negative cooperativity for ammonium (Hill number of 0.66), and its K_m value increased from 2.6 to 21.2 m M when the ammonium concentration exceeded 16 m M . The deamination reaction was highly sensitive to inhibition by ammonium, while the amination reaction was only slightly inhibited by glutamate. These results, considered together with the K_m values, indicate that the NADP-GDH in Sphaerostilbe repens is primarily concerned with glutamate biosynthesis.     

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.     

Isolation and characterization of cytoplasmic NADP+-malic enzyme from Lupinus luteus leaves

NADP⁺-dependent malic enzyme (L-malate : NADP⁺ oxidoreductase, decarboxylating, EC 1.1.1.40) was extracted from the leaves of yellow lupine. The purification procedure included fractionation with (NH₄)₂SO₄ and Sephadex G-25 chromatography, followed by purification on DEAE-cellulose and Sephadex G-200 columns. The enzyme was purified 122-fold. The enzyme affinity towards L-malate was found to be significantly higher with Mn²⁺ than with Mg²⁺. The Hill coefficient for Mg²⁺ depended on concentration and was 1.6 for the lower and 3.9 for the higher concentrations. The dependence of the enzyme activity on NADP⁺ followed a hyperbolic curve. K_m values and Hill coefficients for NADP⁺ were similar with both Mn²⁺ and Mg²⁺. The enzyme activity was strictly dependent on divalent cations and followed a sigmoidal curve at least for Mg²⁺. The enzyme had 4-fold higher affinity towards Mn²⁺ than towards Mg²⁺, the K_m values being 0.3 and 1.15 m M respectively. Of several tested organic acids, oxalate was the most effective inhibitor followed by oxaloacetate while succinate was the strongest activator.     

Characterization of ATPase activities in rice root plasmalemma vesicles isolated by two-phase partitioning

Plasma membrane vesicles were isolated from the roots of 7-day-old rice plants ( Oryza sativa L. cv. Bahía) by utilizing an aqueous polymer two-phase system with 6.2%:6.2% (w/w) Dextran T500 and polyethylene glycol 3350 (PEG) at pH 7.6. Plasmalemma vesicles of high purity were obtained as indicated by the vanadate-sensitive K⁺, Mg²⁺-ATPase activity that was 18 times higher in the upper (PEG-rich) phase than in the lower (Dextran-rich) phase and by specific staining with sodium silicotungstate. Two peaks of ATPase activity were found. One showed a pH optimum at 6.0 in the presence of 150 m M KCl and 3 m M ATP with apparent K_m (ATP) and V_max of 0.75 m M and 79 μmol (mg protein)⁻¹ h⁻¹, respectively. With 50 m M KCl and 7 m M ATP a pH optimum of 6.5, an apparent K_m (ATP) of 6.3 m M and V_max of 159 μmol (mg protein)⁻¹ h⁻¹ were determined. Both activities were specific for ATP, unspecific for monovalent cations, sensitive to sodium vanadate and Ca²⁺ but insensitive to azide and nitrate.     

Shikimate dehydrogenase from pepper (Capsicum annuum) seedlings. Purification and properties

Shikimate dehydrogenase (SKDH, EC 1.1.1.25) was extracted from seedlings of pepper ( Capsicum annuum L.) and purified 347-fold. The purification procedure included precipitation with ammonium sulphate and chromatography in columns of Reactive Red-agarose, Q-Sepharose and Sephadex G-100. Pepper SKDH isozymes are separable only using PAGE. The purified enzyme has a relative molecular mass of 67 000 as estimated by gel filtration. The optimum pH of enzyme activity is 10.5 and the optimum temperature is 50°C, but the enzyme is quickly inactivated at temperatures higher than 40°C. The purified enzyme exhibited typical Michaelis-Menten kinetics and K_m values are 0.087 m M for shikimic acid and 0.017 m M for NADP. The mechanism of reaction is sequential considering NADP as a cosubstrate. Ions such as Ca²⁺, Mg²⁺ and Mn²⁺ activate the enzyme, but Zn²⁺ and Cu²⁺ are strong inhibitors. Some phenolic compounds such as guaiacol, protocatechuic acid and 2,4-D are competitive inhibitors of pepper SKDH, showing K_i values of 0.38 m M , 0.27 m M and 0.16 m M , respectively.     

Properties and localization of the homoglutathione synthetase from Phaseolus coccineus leaves

The synthesis of homoglutathione (hGSH) by several plants of the tribe Phaseoleae is shown to be catalysed by a β-alanine-specific hGSH synthetase, Properties of the enzyme from Phaseolus coccineus L. cv. Preisgewinner were studied, using ammonium sulfate precipitates of primary leaf extracts. The hGSH synthetase showed a broad pH optimum at pH 8–9, an absolute requirement for Mg²⁺, a stimulation by K⁺, and a high affinity for γ-glutamylcysteine [K_m(app.) 73 μ M ]. The enzyme exhibited a high specificity for β-alanine [K_m(app.) 1.34 m M ] compared to glycine [K_m(app.) 98 m M ]. Chloroplasts, isolated from the leaves of Phaseolus coccineus , contained about 17% of the hGSH synthetase activity in the leaf cells.     

Characteristics of β-galactosidase purified from cell suspension cultures of carrot

Five glycosidase activities from cell homogenate of carrot ( Daucus carota L. cv. Kintoki) cell cultures were assayed after extraction successively by phosphate buffer (pH 7.0) and the buffer plus 2 M NaCl. A β-galactosidase (EC 3.2.1.23) was isolated in a highly purified state from the buffer-soluble protein fraction by ammonium sulfate fractionation and chromatography on CM-Sephadex C-50, DEAE-Sephadex A-50 and Sephadex G-200. The molecular weight of this enzyme was ca 104 000 and the isoelectric point was pH 7.8. The optimal activity occurred at pH 4.4 with McIlvaine buffer. The K_m and V_max values were 1.67 m M and 201 units (mg protein)⁻¹, respectively, for p -nitrophenyl β- d -galactopyranoside. The enzyme activity was strongly inhibited by Zn²⁺, Cu²⁺, Hg²⁺ and d -galactono-1,4-lactone. The enzyme acted on the β-1,4-linked galactan prepared from citrus pectin in an exo-fashion. Furthermore, the enzyme was slightly involved in the hydrolysis of the pectic polymer and cell walls purified from carrot cell cultures.     

Purification and characterization of type I DNA topoisomerase from Lentinus edodes

Abstract Type I DNA topoisomerase was purified from the lower eukaryote Lentinus edodes . Like the topoisomerase I from other eukaryotic cells, the L. edodes enzyme removed both positive and negative superhelical turns. The M _r of the enzyme was determined to be 71,500 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). On gel filtration by Sephacryl S-200, the enzyme appeared to be an aggregate with a native M _r of about 235 000 daltons. No energy cofactor was required and ATP did not affect the enzyme. Activity was enhanced about 10-fold by Mg²⁺ (10 mM) and about 8-fold by KCl (100 mM).     

Indole-3-acetaldehyde reductase in Phycomyces blakesleeanus. Characterization of the enzyme

lndole-3-acetaldehyde reductase (lAAld reductase EC 1.2.3.1) from Phycomyces blakesleeanus Bgff., a 38 kDa polypeptide as determined by gel filtration, is probably localized in the cytoplasm. The formation of indole-3-ethanol (lEt) is dependent on the presence of NAD(P)H. The enzymatic reduction of IAAId shows a pH optimum between 6 and 8 and a temperature optimum at 30°C. Enzyme activity follows Michaelis Menten kinetic (K_m= 200 μ M for IAAId; K_m= 24 μ M for NADPH). The isoelectric point of the IAAId reductase is at pH 5.4. Phenylacetaldehyde and benzaldehyde are competitive substrates. Hydroxymeihylindole promotes the reductive IEt formation, whereas NADP⁺ is a non-competitive inhibitor. Changes in lAAJd reductase activity correlate with certain developmental stages of the fungus.     

GTP-specific pyrophosphorylation of thiamin in dark-grown soybean (Glycine max) seedling axes

ATP:thiamin pyrophosphotransferase (TPT: EC 2.7.6.2) was purified 5 900-fold from 48 h dark-grown soybean [ Glycine max (L.), Merr. cv. Ransom II] seedling axes. TPT activity was monitored during purification by measuring the formation of thiamin pyrophosphate (TPP) from [2-¹⁴C]-thiamin at optimal pH (7.3). Although other nucleoside triophosphates were active as pyrophosphate donors (apparent K_ms from 21 to 138 m M ), GTP was the preferred nucleotide with an apparent K_m of 0.021 m M . TPT activity was extremely sensitive to TPP formation, suggesting product feedback inhibition of TPT activity in vivo. Sulfhydryl, H⁺ and Mg²⁺ concentrations, either independently or in concert, were found to affect TPT activity.