I= ACID PHOSPHATESS IN SHEEP BRAIN AND LIVER: ISOLATION, PURIFICATION AND CHARACTERIZATION Title of Thesis


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Title of Thesis

ACID PHOSPHATES IN SHEEP BRAIN AND LIVER: ISOLATION, PURIFICATION AND CHARACTERIZATION

Author(s)

Abdur Rahim Khan

Institute/University/Department Details

Gomal University, Dera Ismail Khan

Session

1997

Subject

Chemistry

Number of Pages

119

 

Keywords (Extracted from title, table of contents and abstract of thesis)

Acid Phosphatases, Sheep brain, Liver, Mr acid phosphatase, Homogeneity, Chromatography, Filtration, Amino acid, pH optimum, Molecular weight determination

 

Abstract

The low Mr acid phosphatase from sheep brain was purified to homogeneity using cation exchange chromatography and gel filtration. The molecular weight was estimated to be 16,000 D by sodium dodecyl sulphate polyacrylamide gel electrophoresis. The enzyme was characterized with respect to amino acid composition, sequence, pH optimum and temperature optimum. The enzyme activity was decreased in the presence of 25mM Sn++, Ca++ and Hg++ while 5mM flouride and 0.25 % Triton -x -405 had no effect on the enzyme activity.

Subcellular localization of high and low Mr acid phosphatases was reported in the liver of sheep. Both enzyme were isolated by gel filtration on Sephadex G -100. Biochemical properties such as optimal pH, molecular weight determination, effect of inhibitors and some modifier substances on both enzymes were also described. Two isoenzymes of low Mr acid phosphatase namely AcPase A and B were purified to specific activity of 27 and 23 U/mg of protein respectively. The isoenzymes were homogeneous on SDS -PAGE, moved as single bands of Mr 18,000 and showed isoelectric points of 6.0 and 5.8 respectively. Both isoenzymes catalysed the hydrolysis of p -nitrophenyl phosphate and phenyl phosphate, phosphotyrosine and FMN but at different rates. AcPase B showed higher km than AcPase A toward O -phosphotyrosine and FMN. AcPase B was effectively activated by purine compounds whereas AcPase A was not. No differences in sensitivity to inhibitors or modifier substances between AcPase A and AcPase B were observed.

 

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S. No. Chapter Title of the Chapters

Page

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    Table of Contents  

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1 1 INTRODUCTION 1

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2 2 MATERIALS AND METHODS 7

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3 3 PURIFICATION 19

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4 4 ELECTROPHORESIS 48

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5 5 AN APPROACH TO AFFINITY CHROMATOGRAPHY

57

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6 6 CHARACTERIZATION

65

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7 7 ACID PHOSPHATESES OF SHEEP LIVER

74

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8 8 CHARACTERIZATION OF LOW AND HIGH Mr ACID PHOSPHATASES

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9 9 DISCUSSION

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10 10 SUMMARY

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11   REFERENCES

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