Studies on acid phosphatases

Campbell, Hugh Douglas (1978). Studies on acid phosphatases PhD Thesis, School of Molecular and Microbial Sciences, The University of Queensland. doi:10.14264/uql.2014.438

       
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Author Campbell, Hugh Douglas
Thesis Title Studies on acid phosphatases
School, Centre or Institute School of Molecular and Microbial Sciences
Institution The University of Queensland
DOI 10.14264/uql.2014.438
Publication date 1978-01-01
Thesis type PhD Thesis
Supervisor Burt Zerner
Total pages 273
Language eng
Subjects 0601 Biochemistry and Cell Biology
Formatted abstract
Gel isoelectric focussing of pig liver extracts revealed multiple bands of acid phosphatase activity. The three major forms were separated by ion-exchange chromatography, and appear to be of lysosomal origin. Neuraminidase treatment converted one fraction to a form resembling one of the two remaining fractions; these differ in other significant ways. The electrophoretic pattern of mouse liver catalase was also simplified by neuraminidase treatment; glucose and galactose were detected in the purified enzyme.

A low molecular weight acid phosphatase also occurs in pig liver, and its properties were correlated with those of various acid phosphatase fractions studied previously.

The major pig liver lysosomal acid phosphatase was purified from chloroform/acetone powder to the highest specific activity observed with any liver acid phosphatase, by ammonium sulphate and acetone fractionations, CM-Sephadex chromatography, and Sephadex G-100 chromatography. No inactive bands were detected on gel isoelectric focussing. The sedimentation coefficient was~5.4 S, and the pI was 6.4.

A beef spleen acid phosphatase containing an unidentified violet chromophore (λmax ~550 nm) was purified by acid extraction, ammonium sulphate fractionation, CM-cellulose chromatography, and Sephadex G-75 chromatography. Spleen activity levels vary widely, and a modified procedure was developed to purify enzyme from extracts of low activity. Enzyme with A280/A550 = 15.1 appears homogeneous by several criteria.

A variety of evidence strongly supports the hypothesis that the enzyme contains tightly-bound iron. The enzyme was the first known example of a metallo-acid-phosphatase, and only the second known hydrolytic enzyme containing iron. It is proposed that the enzyme is a representative of a widely-distributed class of violet, iron-containing acid phosphatases.

Studies on the purified enzyme have included, amongst others, determination of A1%1 cm at 280 nm (15.9), the iron content (1 g atom/19,400 g protein), and the amino acid composition. Gel filtration indicated a MW of ~38,000. The concentration dependence of the sedimentation coefficient was studied (s°20,buffer = 2.85 S); the MW determined via the Svedberg equation was ~37,000.

No sulphydryl groups were detected in the "native" enzyme in the presence or absence of ascorbate, but in the presence of sodium dodecyl sulphate, one sulphydryl group per ~38,000 daltons reacted with 5,5'-dithiobis(2-nitrobenzoic acid).

Sodium dodecyl sulphate gel electrophoresis revealed two bands with MWs of 14-17,000 and 21-25,000; the sum of the apparent MWs was ~35-39,000. The pig uterus violet acid phosphatase gave a single band with a MW of 38-40,000.

The evidence shows that the MW of the spleen enzyme is ~38,000, with two iron atoms and two polypeptide chains per molecule. Data on the pig uterus enzyme, and reported data on other apparently related enzymes, provides evidence in some of these cases also for a species of MW ~40,000, amongst other similarities; it is predicted that the pig uterus enzyme contains two iron atoms per molecule.

Theories for the reductive activation of these enzymes are considered: it is concluded that sulphydryl groups are probably not involved, and that reduction of one or both of the iron atoms is responsible.

In the light of these studies, an extended classification scheme for the mammalian acid phosphatases is proposed.

The problem of the determination of protein MWs from amino acid compositional data was investigated, Only one previously reported function satisfies certain desirable requirements, and other functions in accord with these requirements, but embodying different approaches,are proposed. The application of such functions to a range of data on well-characterized proteins, and a variety of other proteins, including acid phosphatases, is reported.

LIST OF PUBLICATIONS
Campbell, H.D., Dudman, N.P.B., and Zerner, B. (1973), FEBS Lett. 31, 123. doi:10.1016/0014-5793(73)80088-2
Campbell, H.D., and Zerner, B. (1973), Biochem. Biophys Res. Comm. 54, 1498. doi:10.1016/0006-291X(73)91155-8
Hamilton, S.E., Campbell, H.D., de Jersey, J., and Zerner, B. (1975), Biochem. Biophys. Res. Comm. 63, 1146. doi:10.1016/0006-291X(75)90688-9
Keyword Phosphatases

Document type: Thesis
Collection: UQ Theses (RHD) - UQ staff and students only
 
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