Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.21.4 (trypsin)
 42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Rat liver glycogen synthase was purified to homogeneity by an improved procedure that yielded enzyme almost exclusively as a polypeptide of Mr 85,000. The phosphorylation of this enzyme by eight protein kinases was analyzed by cleavage of the enzyme subunit followed by mapping of the phosphopeptides using polyacrylamide gel electrophoresis in the presence of SDS, reverse-phase high-performance liquid chromatography and thin-layer electrophoresis. Cyclic AMP-dependent protein kinase, phosphorylase kinase, protein kinase C and the calmodulin-dependent protein kinase all phosphorylated the same small peptide (approx. 20 amino acids) located in a 14 kDa CNBr-fragment (CB-1). Calmodulin-dependent protein kinase and protein kinase C also modified second sites in CB-1. A larger CNBr-fragment (CB-2) of approx. 28 kDa was the dominant site of action for casein kinases I and II, FA/GSK-3 and the heparin-activated protein kinase. The sites modified were all localized in a 14 kDa species generated by trypsin digestion. Further proteolysis with V8 proteinase indicated that FA/GSK-3 and the heparin-activated enzyme recognized the same smaller peptide within CB-2, which may also be phosphorylated by casein kinase 1. Casein kinase 1 also modified a distinct peptide, as did casein kinase II. The results lead us to suggest homology to the muscle enzyme with regard to CB-1 phosphorylation and the region recognized by FA/GSK-3, which in rabbit muscle is characterized by a high density of proline and serine residues. A striking difference with the muscle isozyme is the apparent lack of phosphorylations corresponding to the muscle sites 1a and 1b. These results provide further evidence for the presence of liver- and muscle-specific glycogen synthase isozymes in the rat. That the isozymes differ subtly as to phosphorylation sites may provide a clue to the functional differences between the isozymes.
 ...
PMID:Multiple phosphorylation sites of rat liver glycogen synthase. 309 Oct 84

Rat liver glycogen synthase bound to the glycogen particle was partially purified by repeated high-speed centrifugation. This synthase preparation was labeled with 32P by incubations with cAMP-dependent protein kinase and cAMP-independent synthase (casein) kinase-1 in the presence of [gamma-32P]ATP. The phosphorylated synthase was separated from other proteins in the glycogen pellet by immunoprecipitation with rabbit anti-rat liver glycogen synthase serum. Analysis of the immunoprecipitates by sodium dodecyl sulfate-gel electrophoresis showed that synthase subunits of Mr 85,000 and 80,000 were present in varying proportions. The 32P-labeled synthase in the immunoprecipitate was digested with trypsin, and the resulting peptides were analyzed by isoelectric focusing. Synthase bound to the glycogen particle was phosphorylated by cAMP-dependent protein kinase at more sites and by cAMP-independent synthase (casein) kinase-1 at less sites than when the homogeneous synthase was incubated with these kinases. Phosphorylation of synthase in the glycogen pellet by either cAMP-dependent protein kinase or cAMP-independent synthase (casein) kinase-1 did not cause a significant inactivation as has been observed when the synthase was incubated with these kinases. Inactivation of synthase in the glycogen pellet, however, can be achieved by the combination of both kinases. This inactivation appears to result from the phosphorylation of a new site by cAMP-independent synthase (casein) kinase-1 neighboring a site previously phosphorylated by cAMP-dependent protein kinase.
 ...
PMID:Phosphorylation of rat liver glycogen synthase bound to the glycogen particle. 609 7

Methods are described for the purification, close to homogeneity, of rabbit liver glycogen synthase in forms dependent on (D-form) or independent (I-form) of glucose-6-P for activity. In previous studies (Camici, M., DePaoli-Roach, A. A., and Roach, P. J. (1982) J. Biol. Chem. 257, 9898-9901), the D-form enzyme was shown to have apparent subunit molecular weight by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (Mapp) of 90,000 and to be susceptible to partial proteolytic degradation. We report here that the purified I-form consisted of a single polypeptide of Mapp = 85,000, even when protease inhibitors were present during the purification. However, appropriate phosphorylation of the I-form enzyme led to a decrease in the electrophoretic mobility of the subunit to generate a species of Mapp = 90,000, identical to that of the D-form. Exposure of the I-form enzyme (subunit Mapp = 85,000) to trypsin caused degradation in the sequence 85,000----82,000----79,000----72,000; concomitantly, the enzyme underwent partial inactivation whether assayed in the presence or absence of glucose-6-P. As purified, the I-form enzyme had a Vmax, determined from variation of UDP-glucose concentration, some 35 times greater than that of the D-form. The UDP-glucose concentration necessary for half-maximal activity was not greatly different, in the range 1-2 mM, for the two enzyme forms.
 ...
PMID:Rabbit liver glycogen synthase. Purification and comparison of the properties of glucose-6-P-dependent and glucose-6-P-independent forms of the enzyme. 642 31

Rabbit liver glycogen synthase have been purified close to apparent homogeneity in a form dependent on glucose-6-P for full activity. From analyses of the purified enzyme by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, some five polypeptides, apparent molecular weights in the range 79,000 to 90,000, correlated with enzyme activity. The relative abundance of the species varied in different preparations but enzyme could be prepared that was composed almost entirely of a 90,000-dalton polypeptide. Treatment of such enzyme with trypsin generated smaller polypeptides in the sequence 90,000 leads to 85,000 leads to 82,000 leads to 80,000; concomitantly, the enzyme was activated when assayed either in the presence or absence of glucose-6-P. Tryptic proteolysis caused as much as a 16-fold increase in Vmax and a 20-fold increase in the concentration of its substrate, UDP-glucose, necessary for half-maximal activity. The concentration of the activator, glucose-6-P, needed for half-maximal stimulation was decreased 3.5-fold. We propose that rabbit liver glycogen synthase in a glucose-6-P-dependent form has a subunit of apparent molecular weight approximately 90,000, larger than previously reported, and that the enzyme is sensitive to proteolytic degradation.
 ...
PMID:Rabbit liver glycogen synthase. Susceptibility of the enzyme subunit to proteolysis. 680 51