Purification and Characterisation of isozymes 1 and 2 of Acid phosphatase from Cicer arietinum

Purification & Characterisation of isozyme-1 and 2 of Acid Phosphatase from Cicer arietinum

Antik K Bose

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Human Biology Division, Fred Hutchinson Cancer Research Center, Seattle, USA.

Antik K Bose

Biochemical properties of acid phosphatase isoforms obtained from germinating seeds of Cicer arietinum have been described. The

isoforms were purified 3604.88 and 2390 fold with 1.4% recovery by CM-Cellulose, DEAE- Cellulose, (L)-tartarate-AH-Sepharose-4B affinity

chromatography and Isoelectric focusing. The molar mass of both isoforms was estimated by Sephadex G-200 to be 100KD and by 6% SDS-

PAGE a band of 55KD was obtained suggesting a homodimer. Temperature optima for both isoforms is 32 C and pH optima are 4.5 and 5.5

for isoforms 1 and 2. The isoforms were moderately glycosylated , 40% and 35% saccharide content for isoforms 1 and 2. Isoform-1

dephosphorylated Phosphotyrosine, PNPP, o-phospho-L- serine, Phosphoenolpyruvate, PPi and ATP. Both isoforms showed product

inhibition by phosphate. The activity was enhanced by Mg2+ ( 2.2% for isoform-1 and 4.9% for isoform-2 ). 4-nitrophenyl phosphate was

hydrolysed with KM and Vmax 2,27 mM and 238.095 µM PNPP consumed/min for isoform-2. Vanadium tetrachloride showed uncompetitive

inhibition on isoform-1 and competitive inhibition on isoform-2.

Nature Methods

ISSN: 1548-7091

EISSN: 1548-7105

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