Purification and some properties of pyruvate kinase from the skeletal muscle of African land tortoise Kinixys erosa (Linn)
Department of Biochemistry, Obafemi Awolowo University, Ile-Ife, NigeriaComparative biochemistry and physiology. B, Comparative biochemistry (Impact Factor: 2.07). 12/1991; 99(3):513-521. DOI: 10.1016/0305-0491(91)90331-7
1.1. Pyruvate kinase from Africa land tortoise (Kinixys erosa) skeletal muscle was isolated and purified to homogeneity.2.2. The mol. wt of the enzyme was estimated to be 212,333 ± 2887 with four subunits of 49,680 ± 526.3.3. The enzyme, denatured by 4M guanidine-HCl, regained a maximum of 80–87% of its original activity upon dilution at 20°C and at a protein concentration of 80 μg/ml in appropriate buffer containing 10 mM PEP and 1 mM l-valine. The kinetics of renaturation was first order.4.4. The catalytically active renatured enzyme was a dimer even though it was kinetically similar to the tetrameric native enzyme.
- [Show abstract] [Hide abstract]
ABSTRACT: 1. Glutathione-S-transferase has been purified from the hepatopancreas of Archachatina marginata to homogeneity. 2. The enzyme was found to be a dimer with a molecular weight of 44,000. The subunits sizes were 22,500 and 23,500 respectively. The isoelectric points of the enzyme were 8.35, 7.95 and 4. The enzyme was most stable at temperature below 40 degrees C. Upon denaturation by 4 M urea, only 56% of the activity could be recovered. 3. The Kms for glutathione and 1-chloro-2,4-dinitrobenze (CDNB) were 0.23 mM and 0.4 mM respectively. The specific activity of the enzyme with CDNB and p-nitrophylacetate as substrates were 47 mumol/mg and 38 mumol/mg respectively. 4. Inhibition studies showed that S-hexylglutathione, Rose Bengal, iodoacetamide, sodium azide and Procion Blue H-B were good inhibitors with I50 values ranging from 18.5 microM to 299 mM. 5. The amino acid composition showed that the enzyme had a relatively high content of hydrophobic and acidic amino acid residues. The peptide maps of the tryptic digests of the native and performic acid-oxidised enzyme indicated that there might be about two disulphide bridges per molecule of the enzyme.
- [Show abstract] [Hide abstract]
ABSTRACT: Glutathione transferase from the hepatopancreas of fresh water crayfish Macrobrachium vollenhovenii was purified to apparent homogeneity by ion-exchange chromatography on DEAE-cellulose and by gel filtration on Sephadex G-100. The enzyme appeared to be a homodimer with molecular weight (Mr) of 46.0 +/- 1.4 kDa and a subunit Mr of 24.1 +/- 0.35 kDa. Chromatofocusing of the apparently pure enzyme revealed microheterogeneity and resolved it into two isozymic peaks, which were eluted at pH 8.36 and 8.22 respectively. Inhibition studies showed that the I50 value for cibacron blue, S-hexylglutathione, hematin, and N-ethylmaleimide (NEM) were 0.01 microM, 340 microM, 5 microM and 33 mM respectively. Out of the several substrates tested, only 1-chloro-2,4-dinitrobenzene (CDNB) and 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole could be conjugated with glutathione. Chemical modification studies with DTNB revealed that two sulphydryl groups per dimer were essential to the activity of the enzymes. On the basis of structural and catalytic characteristics, M. vollenhovenii GST seems close, tentatively, to the omega and zeta classes of GST. Initial-velocity studies of the enzyme are consistent with a steady-state random kinetic mechanism. Denaturation and renaturation studies with guanidine HCl (Gdn-HCl) revealed that though low Gdn-HCl concentrations (less than 0.5 M) denatured the enzyme, the enzyme was able to renature completely (100%). At higher concentration of the denaturant (0.5-4 M), refolding studies indicated that complete renaturation was not achieved. The extent of renaturation was however a function of protein concentration. Our results are consistent with a three-state unfolding process.
Data provided are for informational purposes only. Although carefully collected, accuracy cannot be guaranteed. The impact factor represents a rough estimation of the journal's impact factor and does not reflect the actual current impact factor. Publisher conditions are provided by RoMEO. Differing provisions from the publisher's actual policy or licence agreement may be applicable.