Phosphorylase isozyme

Structure of Phosphorylase Isozymes from Potato Tuber 109... 

The transit peptide of the type-L phosphorylase isozyme of potato amyloplast has the following amino acid composition and sequence ... 

Another interesting feature of the transit peptide of the type-L phosphorylase isozyme is the high content of histidine the peptide contains five histidyl residues in a total of 50 amino acid residues, in contrast to other transit peptides that contain no or little, if any, histidyl residues. It is not known if these residues have any special role other than increasing the positive charge of the peptide. 

Figure 6.1 also includes the sequence comparison with the rabbit muscle phosphorylase.I6,30) The sequence homologies of the animal enzyme with the potato type-L and type-H phosphorylase isozymes are 38% and 47%, respectively. Further sequence comparison of the plant and animal enzymes with the enzyme from E. co/i82 reveals an overall homology of at least 40%. These high similarities indicate that the phosphorylase family is one of the well-conserved protein groups. 

Previously, it has been shown that most of the residues directly interacting with AMP as well as the phosphorylatable Ser14 and its surroundings in the rabbit muscle enzyme are far less conserved in the potato type-L isozyme sequence.63 Likewise, the amino-terminal region of the potato type-H isozyme is completely different from that of the rabbit muscle enzyme over the first 80 amino acid residues, in which the sites of covalent phosphorylation and of allosteric regulation by AMP are all included. These variances in sequence are compatible with the lack of regulation in the plant phosphorylase isozymes. 

IN-VITRO TRANSLATION AND PROCESSING OF COMPARTMENT-SPECIFIC GLUCAN PHOSPHORYLASE ISOZYMES FROM PISUM SATIVUM L. 

In the present study, biosynthesis of both enzyme forms has been studied using heterotrophic tissues of Pisum sativum L. For this purpose, cotyledons of germinating or developing seeds were chosen. Poly-adenylated RNA was isolated and translated in vitro. Phosphorylase isozymes were identified by immunoprecipitation and SDS-PAGE. Following in-vitro translation, the size of the cytosolic isozyme was indistinguishable from that of the mature protein. In contrast, the plastidic isozyme was synthesized as a precursor which was processed to its final size by a stromal fraction of isolated pea chloroplasts. 

When in-vitro translation was performed using polyadenylated RNA from developing pea seeds the plastidic phosphorylase form (in its high molecular weight state) was the predominantly translated phosphorylase isozyme whereas the cytosolic counterpart was recovered as a very weak band of radioactivity (data not shown). Again, the apparent molecular weight of the labeled plastidic phosphorylase was lager by 11 kD than that of the purified enzyme form (Fig. 2C lane a). A complete conversion of the precursor to the size of the mature isozyme was achieved when the translation mixture (prior to precipitation with anti-plasti-dic phosphorylase IgG) was incubated with a stromal fraction (precipitate between 40-70% saturation of ammonium sulfate) of isolated pea chloroplasts (Fig. 2C lane b 50 pi stromal fraction per translation mixture, lane c 100 pi stromal fraction). The identity of the precursor was further confirmed by competition experiments (data not shown). 

A SOLUBLE POLYSACCHARIDE FRACTION FROM HIGHER PLANTS A POSSIBLE PHYSIOLOGICAL SUBSTRATE OF THE CYTOSOLIC PHOSPHORYLASE ISOZYME... 

Cytosol- and plastid-specific phosphorylase isozymes from Pisum sativum were purified as previously described (4). 

Enzyme activity assays Phosphorolytic activity of phosphorylase isozymes was determined as previously described (3). Glucan synthesizing activity was monitored continuously as orthophosphate liberation using a modification of the procedure of Fossati (5). 

In order to investigate a possible physiological function of the carbohydrate fraction its interaction with compartment-specific phosphorylase isozymes was studied by affinity electrophoresis and kinetic measurements. 

For affinity electrophoresis varying concentrations of a polysaccharide fraction which had been isolated from cotyledons were immobilized in a polyacrylamide gel. Equal volumes of a pea leaflet extract were applied to each tube. Following electrophoresis and phosphorylase activity staining, the migration distance of each phosphorylase form, relative to that of bromophenol blue, was determined (Fig. 1). At increasing polysaccharide concentrations the migration velocity of the cytosolic phosphorylase isozyme decreased markedly whereas the retardation of the two chloroplast isozymes was minor. This indicates that the cytosolic phosphorylase isozyme exhibits a much higher affinity towards the immobilized polysaccharide than the plastidic enzyme forms. 

This conclusion was confirmed by kinetic measurements. Preparations of purified cytosolic and plastidic phosphorylase isozymes from Pisum sativum were adjusted to an approximately equal activity concentration (as determined at saturating levels of soluble starch). Using these isozyme preparations kinetic measurements were performed at varying levels of the polysaccharide fraction. Orthophosphate or glucose 1-phosphate levels were saturating. The initial rates of phosphorolysis (Fig. 2A) or of polysaccharide biosynthesis (Fig. 2B) were determined. 

A Soluble Polysaccharide Fraction from Higher Plants A Possible Physiological Substrate of the Cytosolic Phosphorylase Isozyme 99... 

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