Purification and characterization of sulfatases from Haliotis rufescens: evidence for changes in synthesis and heterogeneity during development.
The digestive glands of many marine molluscs are rich sources of arylsulfatase enzymes which may function in the catabolism of sulfated polysaccharides in the diets of herbivorous species. Arylsulfatases, partially purified from the hepatopancreas of the red abalone, Haliotis rufescens, were investigated with respect to heterogeneity, catalytic requirements, and timing of induction during development. Four hepatopancreatic enzymes were purified from adult animals using a combination of hydrophobic interaction and anion-exchange chromatography. Zymograms of the four partially-purified enzymes produced by electrophoresis under nondenaturing conditions revealed a fifth, relatively more basic isozyme. All four partially-purified enzymes appear to be monomeric, with molecular weights of approximately 43,000 Da each, as measured by gel filtration. The affinities for p-nitrocatechol sulfate, pH optima, and strengths of inhibition by anions displayed by these enzymes are similar to the values reported for other molluscan arylsulfatases. Three of the four enzymes have Km values between 0.8 and 2.0 mM for p-nitrocatechol sulfate; the remaining enzyme (A2) has a Km of 6.7 mM. All four enzymes have pH and temperature optima of 5.5 and 45 degrees C, respectively. Three of the four enzymes have-t 1/2 (50 degrees C) values of 3.5 min; the enzyme A4 has a t 1/2 (50 degrees C) of 8.5 min. A monoclonal antibody directed against form A1b does not cross react with any of the other hepatopancreatic arylsulfatases when assayed by Western blot, confirming the structural heterogeneity of the adult enzymes. Total arylsulfatase activity increases in a biphasic manner during early abalone development, with the first increase occurring early in larval maturation.(ABSTRACT TRUNCATED AT 250 WORDS)
Spaulding
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Department of Biological Sciences, University of California, Santa Barbara 93106.
Morse
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D000818:Animals; D000911:Antibodies, Monoclonal; D001192:Arylsulfatases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D015153:Blotting, Western; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D004790:Enzyme Induction / Q000502:physiology; D015971:Gene Expression Regulation, Enzymologic; D008099:Liver / Q000201:enzymology; D008675:Metamorphosis, Biological / Q000502:physiology; D008970:Molecular Weight; D008974:Mollusca / Q000378:metabolism* / Q000502:physiology; D010179:Pancreas / Q000201:enzymology
D000911:Antibodies, Monoclonal; D001192:Arylsulfatases