Phosphorylase purification

Miller, R. L., Sabourin, C. L. and Krenitsky, T. A. (1987) Trypanosoma cruzi adenine nucleoside phosphorylase. Purification and substrate specificity. Biochem. Pharmacol. 36 553 560. 

His first work in the Laboratory of Biological Chemistry was on the purification and properties of potato phosphorylase, but he soon changed his subject to the study of amino-containing sugars. It was not well understood at that time whether there were impurities in the products of separation and purification of proteins. Consequently, Onodera made up his mind to devote his whole life to identifying the chemical nature of amino-containing sugars. 

Koga, T., Nakamura, K., Shirokane, Y., Mizusawa, K., Kitao, S., and Kikuchi, M. 1991. Purification and some properities of sucrose phosphorylase from Leuconostoc mes-enteroides. Agric. Biol. Chem.,55,1805-1810. 

Takata, H., Takaha, T., Okada, S., Takagi, M., and Imanaka, T. 1998. Purification and characterization of a-glucan phosphorylase from Bacillus stearothermophilus. J. Ferment. Bioeng., 85,156-161. 

Fifty milliliters of the cell-free extract described above was fractionated by ammonium sulfate precipitation. The fraction precipitating between 30 and 50% saturation was redissolved in a total volume of 10 ml and dialyzed. The solution after dialysis occupied 12 ml and contained 30 mg protein/ml. Twenty microliters of the purified fraction catalyzed the phosphorylase reaction at a rate of 5.9 nmoles/min under the standard assay conditions. Calculate (a) the recovery of the enzyme and (b) the degree of purification obtained in the ammonium sulfate step. 

The XLL cross-axis CPC, with a 250-mL capacity column, was used for the purification of recombinant enzymes such as purine nucleoside phosphorylase (PNP) and uridine phosphorylase (UrdPase) from a crude Escherichia coli lysate. The polymer-phase system used in these separations was 16% (w/w) PEG 1000-12.5% (w/w) potassium phosphate at pH 6.8. The separation was performed at 750 rpm at a flow rate of 0.5 mL/min using the upper phase as a mobile phase. About 1.0 mL of crude lysate, containing PNP in 10 mL of the above solvent system, was loaded into the multilayer coil. Purified PNP was harvested in 45-mL fractions. The SDS-PAGE analysis clearly demonstrated that PNP was highly purified in a one-step elution with the XLL cross-axis CPC. 

Huwel S, Haalck L, Coniath N et al (1997) Maltose phosphorylase firom Lactobacillus brevis purification, characterization, and application in a biosensor for ortho-phosphate. Enzyme Microb Technol 21 413-420... 

Waelkens E, Goris J, Merlevede W (1987) Purification and properties of polycation-stimulated phosphorylase phosphatases from rabbit skeletal muscle. J Biol Chem 262 1049-1059... 

Large scale purification of enzymes in PEG/dextran and PEG/salt have been reported and reviewed by Kula et. al.(18. 19. 20). Examples include pullulanase and 1,4-j-glucan phosphorylase <21 , a-amylase (22), b-galactosidase (jg3), and the general economics of extractive enzyme recovery (24). Process studies have covered the use of continuous centrifuges (18. 19) and countercurrent distribution trains (2j 25). 

Brishammar, S. and N. Juntti RNA-synthesizing enzymes in health and TMV-infected tobacco leaves. Partial purification and characterization of tobacco polynucleotide phosphorylase Arch. Biochem. Biophys. 164 (1974) 224-232. 

Derensy-Dron, D., Krzewinski, R, Brassart, C., and Bouquelet, S. 1999. Beta-1,3-galactosyl-A -acetylhexosamine phosphorylase from Bifidobacterium bifidum DSM 20082 characterization, partial purification and relation to mucin degradation. Biotechnol. Appl. Biochem. 29 3-10. 

Sakai, K. Matsumura, S. Okimura, Y. Yamamura, H. Nishizuka, Y. Liver glycogen phosphorylase kinase. Partial purification and characterization. J. Biol. Chem., 254, 6631-6637 (1979)... 

Pocinwong, S. Blum, H. Malencik, D. Fisher, E.H. Phosphorylase kinase from dogfish skeletal muscle. Purification and properties. Biochemistry, 20, 7219-7226 (1981)... 

Killilea, S.D. Ky, N.M. Purification and partial characterization of bovine heart phosphorylase kinase. Arch. Biochem. Biophys., 221, 333-342 (1983)... 

Crabb, J.W. Heilmeyer, L.M.G. High performance liquid chromatography purification and structural characterization of the subunits of rabbit muscle phosphorylase kinase. J. Biol. Chem., 259, 6346-6350 (1984)... 

Nikolaropoulos, S. Sotiroudis, T.G. Phosphorylase kinase from chicken gizzard. Partial purification and characterization. Eur. J. Biochem., 151, 467-473 (1985)... 

Beleta, J. Benedicto, P. Aymerich, P. GeUa, F.J. Purification and characterization of native and proteolytic forms of rabbit liver phosphorylase kinase. Int. J. Biochem., 22, 443-451 (1990)... 

Lanciotti, R.A. Bender, P.K. Baculovirus-directed expression of the y-sub-unit of phosphorylase kinase purification and calmodulin dependence. Biochem. J., 299, 183-189 (1994)... 

Phosphorylase can be easily isolated from potatoes and, after purification, used to catalyze the polymerization of glucose-1-phosphate in order to obtain linear polysaccharide chains with a-(l— 4) glycosidic linkages, as can be seen in figure 5. 

The enzyme from rabbit muscle may be extracted and crystallized readily, but many recrystallizations may be necessary to ensure freedom from such contaminants as alpha-amylase, debranching enzyme, and branching enzyme. Purification may be facilitated by the use of column chromatography on 0-(2-diethylaminoethyl) cellulose. The enzyme exists in two forms (a and h) that differ in their requirement for adenosine 5 -monophosphate as cofactor. The b form is inactive in the absence of cofactor, and may be converted into the active (adenosine 5 -monophos-phate-independent) a form by phosphorylation of a specific serine residue under the action of (contaminant-free) phosphorylase kinase with adenosine 5 -triphosphate as the phosphate donor. 

Purification of glycogen phosphorylase b Purification of UDP-A -acetylenolpyruvoyl-glucosamine reductase Affinity chromatography of dihydropterin reductase... 

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