Preparation of homogeneous human prostatic acid phosphatase using concanavalin A-sepharose 4-B

Etten, Robert L. Van; Saini, Mohan S.

doi:10.1016/0005-2744(77)90104-8

Cited by 28 publications

(9 citation statements)

References 12 publications

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“…Pooled peak activity fractions were concentrated to about 1.5 mL using an Amicon YM-10 ultrafilter. Binding of the enzyme to Con A suggests that, as has been reported for another acid phosphatase (30), PEP phosphatase is a glycoprotein.…”

Section: Con a Chromatographysupporting

confidence: 64%

Purification and Characterization of a Phosphoenolpyruvate Phosphatase from Brassica nigra Suspension Cells

Duff¹,

Lefebvre²,

Plaxton³

1989

Plant Physiol.

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Phosphoenolpyruvate phosphatase from Brassica nigra leaf petiole suspension cells has been purified 1700-fold to apparent homogeneity and a final specific activity of 380 micromole pyruvate produced per minute per milligram protein. Purification steps included: ammonium sulfate fractionation, S-Sepharose, chelating Sepharose, concanavalin A Sepharose, and Superose 12 chromatography. The native protein was monomeric with a molecular mass of 56 kilodaltons as estimated by analytical gel filtration. The enzyme displayed a broad pH optimum of about pH 5.6 and was relatively heat stable. Western blots of microgram quantities of the final preparation showed no cross-reactivity when probed with rabbit polyclonal antibodies prepared against either castor bean endosperm cytosolic pyruvate kinase, or sorghum leaf phosphoenolpyruvate carboxylase. The final preparation exhibited a broad substrate selectivity, showing high activity toward p-nitrophenyl phosphate, adenosine diphosphate, adenosine triphosphate, gluconate 6-phosphate, and phosphoenolpyruvate, and moderate activity toward several other organic phosphates. Phosphoenolpyruvate phosphatase possessed at least a fivefold and sixfold greater affinity and specificity constant, respectively, for phosphoenolpyruvate (apparent Michaelis constant = 50 micromolar) than for any other nonartificial substrate. The enzyme was activated 1.7-fold by 4 millimolar magnesium, but was strongly inhibited by molybdate, fluoride, zinc, copper, iron, and lead ions, as well as by orthophosphate, ascorbate, glutamate, aspartate, and various organic phosphate compounds. It is postulated that phosphoenolpyruvate phosphatase functions to bypass the adenosine diphosphate dependent pyruvate kinase reaction during extended periods of orthophosphate starvation.

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Section: Con a Chromatographysupporting

confidence: 64%

Purification and Characterization of a Phosphoenolpyruvate Phosphatase from Brassica nigra Suspension Cells

Duff¹,

Lefebvre²,

Plaxton³

1989

Plant Physiol.

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“…Procedures for the purification of HPAP have been reviewed by Bodansky [6] and recent methods have utilized a combination of different techniques including exclusion and ion-exchange chromatography [7,11,18], concanavalin A affinity chromatography [9,28,29], and tartrate affinity methods [13,30,31]. Only Choe et a1 [9,29] purified the two enzyme forms to homogeneity in order to determine their antigenic properties, whereas others have concentrated on isolating only the major enzyme form.…”

Section: Discussionmentioning

confidence: 99%

Isolation, characterization, and spontaneous interconversion of two forms of human prostatic acid phosphatase

McTigue¹,

Etten²

1982

The Prostate

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Starting with human prostatic acid phosphatase (HPAP) purified by affinity chromatography on Sepharose-bound N-(6-aminohexyl)tartramic acid [Van Etten, R. L. and Saini, M. S. (1978) Clin. Chem. 24, 1525], two forms of the enzyme can be separated by gradient elution DEAE-cellulose chromatography. The two enzyme forms are electrophoretically distinguishable on high pH gels. Under some conditions of storage one of these forms undergoes a conversion to the other. Both forms have identical amino acid compositions and Vmax values with typical substrates, but differences are observed in circular dichroism spectra, relative fluorescence intensities, Km values, and carbohydrate composition. It is concluded that the two forms differ only in the structure of their (nonphosphorylated) carbohydrate side chains. In view of the possible interconversion of the two forms upon storage, caution must be observed in any attempt to attach clinical significance to the relative amounts of the two forms.

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“…Lectin affinity chromatography has been widely used for the purification of glycoproteins from many sources (Hayman et al, 1973;Van Etten & Saini, 1977;Mintz & Glaser, 1979;Vretblad & Hjorth, 1977). This paper describes the application of WGA affinity chromatography as a specific means of separating and purifying OMVs from crude extracts of surface antigens of N .…”

Section: Introductionmentioning

confidence: 99%

Purification of Pili and Outer Membrane Vesicles of Neisseria gonorrhoeae by Wheat Germ Agglutinin Affinity Chromatography

1982

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Previous studies indicated that OMVs of Neisseria gonorrhoeae reacted specifically with WGA. A technique was therefore developed for the separation of gonococcal pili and OMVs by WGA affinity chromatography. This method was more convenient and more effective than isopycnic centrifugation for obtaining OMVs free of pili. The antigens were further purified but though homogeneous as judged by in vitro analytical methods, they both elicited an antibody response in animals to minor impurities previously undetected.

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Preparation of homogeneous human prostatic acid phosphatase using concanavalin A-sepharose 4-B

Cited by 28 publications

References 12 publications

Purification and Characterization of a Phosphoenolpyruvate Phosphatase from Brassica nigra Suspension Cells

Purification and Characterization of a Phosphoenolpyruvate Phosphatase from Brassica nigra Suspension Cells

Isolation, characterization, and spontaneous interconversion of two forms of human prostatic acid phosphatase

Purification of Pili and Outer Membrane Vesicles of Neisseria gonorrhoeae by Wheat Germ Agglutinin Affinity Chromatography

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