Chandra B. Sharma

Myoinositol hexaphosphate as a potential inhibitor of α-amylases

Abstract Myoinositol hexaphosphate (MHP) strongly inhibited α-amylases of different origins. The inhibition of wheat α-amylase is noncompetitive with an apparent Ki value of 1 mM, pH dependent and markedly increased by the preincubation of enzyme with MHP before the addition of substrate. Addition of Ca2+ did not reverse the inhibition of α-amylase indicating that its inhibition was not due to the binding of Ca2+ by MHP.

Multiple forms of phytase in germinating cotyledons of Cucurbita maxima

Abstract Multiple forms of phytase (myoinositol hexaphosphate phosphohydrolase, EC 3.1.3.8) have been isolated in highly purified forms from germinating Cucurbita maxima cotyledons using acetone and ammonium sulphate fractionation, Sephadex gel filtration and ion exchange chromatography on DEAE- and CM-cellulose. Gel filtration produced two peaks of phytase activity; phytase I (high MW) and phytase II (low MW). Phytase I was further resolved into 4 distinct species on CM-cellulose and these were designated phytase IA, IB, IC and ID, according to their elution order. On the other hand, phytase II remained as a single species with a purification of 35-fold. The MWs of each phytase I species were identical (MW 66 500 ± 4000) and they were twice the MW of phytase II (MW 32 400 ± 4000) indicating that I and II may be structurally related. The properties of various molecular forms were compared. The difference in properties between phytase II and phytase I isoenzymes (IA, IB, IC and ID) was more pronounced than that observed among the isoenzymes of phytase I alone.

Purification, by high-performance liquid chromatography, and characterization, by high-field 1H-n.m.r. spectroscopy, of two fucose-containing pentasaccharides of goat's milk.

Two fucose-containing pentasaccharides were isolated from goats milk using a Bio-Gel P-4 column, followed by reverse-phase C-18 high-performance liquid chromatography. The structures of the pentasaccharides as characterized by high-field 1H-n.m.r. spectroscopy and enzymatic digestion were found to be [see text] and [see text].

Purification and properties of an α-amylase protein-inhibitor from Arachis hypogaea seeds

A protein showing highly specific inhibitory activity towards hog pancreatic and human salivary alpha-amylases (1,4-alpha-D-glucan glucanohydrolase, EC 3.2.1.1), but not towards plant and bacterial alpha-amylases, has been purified 197-fold from an aqueous extract of peanut cotyledons using heat treatment, (NH4)2SO4 precipitation and ion-exchange chromatography on DEAE- and CM-cellulose. The purified inhibitor was homogeneous by polyacrylamide gel electrophoresis. Its molecular weight, as determined by Sephadex G-100 gel-filtration, and its electrophoretic mobility at pH 8 relative to bromophenol blue, were 25 000 and 0.14, respectively. The inhibitory activity was relatively resistant to thermal treatment and markedly increased when the inhibitor was preincubated with the enzyme before the addition of starch. Further, the inhibition was found to be pH-dependent and non-competitive in nature.

Goat milk oligosaccharides: purification and characterization by HPLC and high-field 1H-NMR spectroscopy

Three oligosaccharides were isolated from goat milk using Bio-Gel P-4 and reverse-phase C-18 HPLC and were characterized by high-field 1H-NMR spectroscopy as a trisaccharide, GlcNAc(beta 1-6)Gal(beta 1-4)Glc, a tetrasaccharide, Gal(beta 1-4)GlcNAc(beta 1-6)Gal(beta 1-4)Glc, and a pentasaccharide.

Effect of Zn2+ on plant α-amylases in vitro

Abstract A detailed study of the in vitro interaction between Zn 2+ and α-amylases from different origins has shown that under physiological conditions

Purification and characterization of 5′-nucleotidase from the Golgi apparatus of peanut cotyledons

Abstract An adenosine 5′-monophosphatese (AMPase) has been purified from the Golgi-apparatus (GA) of peanut cotyledons by first selective solubilizing the enzyme with 0.5% non-specific detergent, n-octyl-β-D-glucopyranoside, at a protein-to-detergent ratio of 2:3 in the presence of 20 mM MgCl2 and 5 mM EDTA, and then by ion-exchange chromatography on DEAE-cellulose. The GA-AMPase is a glycoprotein having 38.5% carbohydrate and apparent Mr = 53.7 dkDa. It was inhibited competitively by ADP (Ki = 3.4 mM) and non-competitively by NaF (Ki = 41 mM). The values of optimum pH, Km and Vmax for the hydrolysis of AMP were 5.0–5.5, 0.9 mM and 0.4 mM/min per mg, respectively. The enzyme is highly specific for AMP with no hydrolysis of ATP, ADP, GTP, GDP, GMP, UTP, UDP, UMP, CTP and CMP. The purified AMPase from GA resembled the plasma membrane (PM) enzyme closely, except for a slightly lower carbohydrate content. It is suggested that GA-AMPase may be a precursor of the PM-AMPase in peanut cotyledon cells.

Purification and properties of a glycoprotein adenosine 5′-monophosphatase from the plasma membrane fraction of Arachis hypogaea cotyledons

Abstract An adenosine 5′-monophosphatase (AMPase) has been purified from the plasma membrane fraction of germinating cotyledons of peanut (Arachis hypogaea L.) by selective solubilization of the membrane-bound enzyme with 0.5% n-octyl β-glucoside at a protein-to-detergent ratio of 2:3 in the presence of Mg2+ and EDTA, followed by ion exchange chromatography on DEAE-cellulose. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis it showed a single protein band with a molecular weight of 55 000. The enzyme is a glycoprotein with 42.7% carbohydrate content. It had a broad pH optimum of 5.0–6.0. The Km and Vmax values were 1.08·10−3 M and 8.5 μmol/min per mg protein, respectively, with 5′-AMP as substrate. The enzyme is specific for 5′-AMP. Other nucleotides (GMP, UMP, CMP, ADP, GDP, ATP, GTP and UTP) as well as phosphorylated sugars were not hydrolyzed. p-Nitrophenyl phosphate was hydrolyzed at a relatively much lower rate (15%) and the substrate affinity (1/Km was only one-tenth that of AMP. The purified enzyme is competitively inhibited by ADP (Ki = 2.4 mM) and is also inhibited by NaF in a non-competitive manner with a Ki value of 35 mM. Divalent cations, Ca2+, Mg2+, Hg2+, Zn2+, Mn2+, Ni2+ and monovalent cations, K+, Li+ and Na+ had no effect on the enzyme activity. The purified enzyme was highly unstable, losing its total activity within 24 h at −20°C, or 0°C, while under these conditions the unpurified solubilized enzyme (octyl glucoside extract) was stable for several days, indicating that some stabilizing factors, most likely phospholipids, were lost during the enzyme purification.

Multiple forms of α-galatosidase in chick pea seedlings

Abstract Two molecular species of α-galactosidase with apparent MWs of 134 000 and 43 000 were separated and partially purified from germinating Cicer arietinum seeds. The two forms of the enzyme showed different kinetic and thermodynamic properties, and responses towards the specific inhibitors, but their pH optima and pH stabilities were almost identical.

Sensitivity of plant α-amylases to calcium ions in Vitro

Abstract Plant α-amylases were found to be sensitive to high calcium ion concentration in vitro . At 250 mM nearly 70 % of the activity of maize, wheat, an