Purification and characterization of acid-type phosphatases from a heavy-metal-accumulating Citrobacter sp.
An acid phosphatase from a heavy-metal-accumulating strain of a Citrobacter sp. was resolved into two forms on the basis of their nonbinding (phosphatase I) or binding (phosphatase II) behaviour on the cation-exchange resin SP-Sephadex C50. Both holoenzymes had a molecular mass of 103-108 kDa as determined by Superose Q-6 column chromatography in the presence of 150 mM KCl and a subunit molecular mass of 27 kDa as determined by SDS-PAGE; the enzyme was tetrameric. Both enzymes had a pI approximately 9.0 and were immunologically cross-reactive. There were minor differences in amino acid composition and in peptide maps following tryptic digest. The pH optimum for phosphatases I and II was 5.5 and 6.25, respectively; phosphatase II alone retained activity at pH values up to 9.0. Phosphatase I was more resistant to mechanical shear, gamma-irradiation, high temperature, and toxins (F- and formaldehyde). Glycerol increased the thermostability of both enzymes, particularly the more thermosensitive phosphatase II. Phosphatase II had a lower Km and a lower Vmax for glycerol 2-phosphate hydrolysis. The production of enzyme isoforms is a phenomenon similar to that described previously for the alkaline phosphatase of Escherichia coli, where the isoforms relate to precursive and final processed forms of the enzyme. Acid phosphatase is physiologically distinct, with a role that is still obscure but that may relate to cellular stress responses.
Jeong
B C
BC
Department of Biological Science, College of Science, Myong Ji University, 38-2 San, Nam-ri, Yongin-eup, Yongin-gun, Kyunggi-do, 449-728, South Korea.
Poole
P S
PS
Willis
A C
AC
Macaskie
L E
LE
D000135:Acid Phosphatase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D002954:Citrobacter / Q000201:enzymology*; D004795:Enzyme Stability; D005990:Glycerol / Q000494:pharmacology; D007526:Isoelectric Point; D008970:Molecular Weight; D013379:Substrate Specificity
D000135:Acid Phosphatase; D005990:Glycerol