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Purification and characterization of sulfatases from Haliotis rufescens: evidence for changes in synthesis and heterogeneity during development

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Summary

The digestive glands of many marine molluscs are rich sources of arylsufatase 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 K m values between 0.8 and 2.0 mM for p-nitrocatechol sulfate; the remaining enzyme (A2) has a K m of 6.7 mM. All four enzymes have pH and temperature optima of 5.5 and 45°C, respectively. Three of the four enzymes have-t1/2(50°C) values of 3.5 min; the enzyme A4 has a t1/2 has a t1/2(50°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. The secoad phase of enzyme expression is dependent upon the induction of settlement and metamorphosis of the competent veliger larvae, strongly suggesting that the expression of arylsulfatase synthesis (and the maturation of the digestive gland, the hepatopancreas) is controlled by genetic events which occur as a result of metamorphosis. Competent veliger larvae express only two arylsulfatase forms, which share many physicochemical and kinetic characteristics with the adult hepatopancreatic enzymes. However, neither of the larval arylsulfatases is recognized by the monoclonal antibody to form A1b from adult hepatopancreas. Endogenous enzyme inhibitor levels in larvae remain constant throughout the period of arylsulfatase induction, and therefore do not contribute to the control of arylsulfatase activity levels during development.

These results are the first documentation of the developmental induction of a specific protein(s) in abalone as a result of metamorphosis. The significance of the timing of arylsulfatase expression is discussed in relation to potential physiological substrates and the dietary switching which occurs at metamorphosis. Possible genetic events which are consistent with the observed patterns of expression of these enzymes also are considered.

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Abbreviations

BSA :

bovine serum albumin

C :

centigrade

Da :

daltons

DEAE :

diethylaminoethyl

ELISA :

enzyme-linked immunosorbent assay

FPLC :

fast protein and polynucleotide liquid chromatography

GABA :

γ-aminobutyric acid

HAT:

hypoxanthine, aminopterin, thymidine

Hepes:

N-(2-Hydroxythyl)piperazine-N'-(2-ethanesulfonic acid)

HPLC :

high pressure liquid chromatography

KBS :

Kantor's balanced salt solution

K m :

Michaelis constant

PBS :

phosphate buffered saline

R m :

relative mobility

RPMI :

Roswell Park Memorial Institute

SDS :

sodium dodecyl sulfate

T :

time

TBS :

TRIS buffered saline

V max :

maximal velocity

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Authors and Affiliations

  1. Department of Biological Sciences, University of California, 93106, Santa Barbara, CA, USA

    D. C. Spaulding & D. E. Morse

  2. the Marine Science Institute, University of California, 93106, Santa Barbara, CA, USA

    D. C. Spaulding & D. E. Morse

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  1. D. C. Spaulding
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Spaulding, D.C., Morse, D.E. Purification and characterization of sulfatases from Haliotis rufescens: evidence for changes in synthesis and heterogeneity during development. J Comp Physiol B 161, 498–515 (1991). https://doi.org/10.1007/BF00257905

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