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)

A high molecular weight protein phosphatase (phosphatase H-II) was isolated from rabbit skeletal muscle. The enzyme had a Mr = 260,000 as determined by gel filtration and possessed two types of subunit, of Mr = 70,000 and 35,000, respectively, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. On ethanol treatment, the enzyme was dissociated to an active species of Mr = 35,000. The purified phosphatase dephosphorylated lysine-rich histone, phosphorylase a, glycogen synthase, and phosphorylase kinase. It dephosphorylated both the alpha- and beta-subunit phosphates of phosphorylase kinase, with a preference for the dephosphorylation of the alpha-subunit phosphate over the beta-subunit phosphate of phosphorylase kinase. The enzyme also dephosphorylated p-nitrophenyl phosphate at alkaline pH. Phosphatase H-II is distinct from the major phosphorylase phosphatase activities in the muscle extracts. Its enzymatic properties closely resemble that of a Mr = 33,500 protein phosphatase (protein phosphatase C-II) isolated from the same tissue. However, despite their similarity of enzymatic properties, the Mr = 35,000 subunit of phosphatase H-II is physically different from phosphatase C-II as revealed by their different sizes on sodium dodecyl sulfate-gel electrophoresis. On trypsin treatment of the enzyme, this subunit is converted to a form which is a similar size to phosphatase C-II.
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PMID:Isolation and characterization of a high molecular weight protein phosphatase from rabbit skeletal muscle. 633 61

Glycogen synthase I in a homogenate of human polymorphonuclear leukocytes was phosphorylated under imitated physiological conditions utilizing the endogenous protein kinases. At subsequent steps of phosphorylation the 32P-labelled synthase was purified and characterized. Limited tryptic hydrolysis of the 32P-labelled synthase released four phosphopeptides (t-A, t-B, t-C, t-D) and subsequent chymotrypsinization of the trypsin resistant core released three phosphopeptides (c-A, c-B, c-C). One Pi/subunit was incorporated within 8-10 min and 2.2 Pi/subunit within 60 min increasing the Kc for Glc-6-P to 4-6 mM. The initial phosphorylation up to 0.8 Pi/subunit occurred mainly in peptide c-A and a linear relation between ratio of independence (RI) of glycogen synthase in the interval RI 0.85 to RI 0.05 and phosphorylation of this peptide of 0.5 Pi was observed. Phosphorylation of this peptide is responsible for the decrease in ratio of independence. From experiments with inhibitors and activators, the initial phosphorylation was found predominantly catalysed by the endogenous cAMP independent synthase kinase, however, the endogenous cAMP dependent protein kinase and phosphorylase kinase also phosphorylate endogenous glycogen synthase I to a minor degree. Circumstantial evidence for a Ca-dependent synthase kinase different from phosphorylase kinase is presented. The endogenous Glc-6-P dependent glycogen synthase occurring in a homogenate of leukocytes disrupted in the presence of NaF incorporated 1.07 Pi/subunit and Kc for Glc-6 was increased from 6-8 mM to 20 mM. From the present and previous experiments [7] a total of 8 major phosphorylatable sites have been defined, one on each of the peptides t-A, t-B, c-B, c-C and two on peptide c-A, which in addition may contain a third site for phosphorylase kinase. Assuming identical subunits, only 13 out of 32 sites are thus covalently modified at maximum phosphorylation. The operational defined synthase R (Kc for Glc-6-P 0.5 mM) and D (Kc for Glc-6-P 2-8 mM) activities correspond to synthase with about 0.8 Pi and 1.8-2.3 Pi/subunit, respectively.
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PMID:Phosphorylation of glycogen synthase in a homogenate of human polymorphonuclear leukocytes. 678 73

Glycogen synthase is a substrate for five distinct protein kinases in skeletal muscle which phosphorylate seven different serine residues on the enzyme. Cyclic-AMP-dependent protein kinase phosphorylates sites 1a, 1b and 2, phosphorylase kinase, site 2, glycogen synthase kinase 3, sites 3a, 3b and 3c, glycogen synthase kinase 4, site 2 and glycogen synthase kinase 5 site 5. Site 2 is seven residues from the N-terminus of glycogen synthase and is located in a cyanogen bromide peptide termed CB1 (apparent Mr = 9000). The other six phosphorylation sites are located in a cyanogen bromide peptide termed CB2 (apparent Mr = 24 000) at the C-terminal end of the molecule. The sequence of the N-terminal 123 residues of peptide CB2, has been completed. Sites 3a, 3b, 3c, 5, 1a and 1b are located at residues 30, 34, 38, 46, 87 and 100 from the N-terminus of CB2 respectively. Site 1a is the next serine residue after site 5. The region surrounding sites 3a, 3b and 3c is very rich in proline residues while that surrounding sites 1a and 1b contains many serine and threonine residues. The 23 residues following site 5 contain 15 aspartic acid and glutamic acid residues, while the region immediately N-terminal to site 1a is very basic. The whole region is remarkably hydrophilic and is the region at which the native enzyme is attacked by proteinases. The sites at which glycogen synthase is cleaved by trypsin, chymotrypsin and thermolysin have been identified. The finding that trypsin cleaves the enzyme C-terminal to site 3c while chymotrypsin cleaves N-terminal to site 3a has formed the basis of a simple procedure for determining the state of phosphorylation of the seven serine residues in vivo [Parker, P.J., Embi, N., Caudwell, F.B., and Cohen, P. (1982) Eur. J. Biochem. 124, 47-55].
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PMID:Multisite phosphorylation of glycogen synthase from rabbit skeletal muscle. Organisation of the seven sites in the polypeptide chain. 680 97

In ultrafiltrated plasma (molecular weight less than 50,000) obtained from four patients with multiple muscular trauma and acute post-traumatic renal failure, it was possible to verify a subcomponential specific digestion of the subunits alpha and gamma of phosphorylase kinase isolated from rabbit skeletal muscle. The activity of free proteolytic enzymes in ultrafiltrated plasma as well as an increase of plasma alpha 1-antitrypsin values were correlated with the severity and unfavourable course of the illness. In contrast, the plasma levels of alpha 2-macroglobulin were drastically lowered. The mean total protein concentration in the sera of patients with post-traumatic ARF was lowered, whereas the mean ultrafiltrate protein concentration was significantly enhanced. In ultrafiltrated plasma of two patients with hyperuricaemic ARF, three patients with ARF after drug over-dosage, one patient with acute pancreatic necrosis combined with acute renal failure and one patient with chronic pancreatitis, no proteolytic activity could be detected using phosphorylase kinase as substrate. Studies on the trypsin binding capacity of the plasma protease inhibitors revealed a significantly lowered level in patients with post-traumatic acute renal failure as compared to healthy controls, patients with chronic renal insufficiency and patients on regular dialysis treatment Proteolytic activity was found in ca. 100-fold concentrated diafiltrates (molecular weight greater than 10,000) of patients on regular dialysis treatment. Our data suggest a participation of proteases on protein catabolism in hypercatabolic states. Whilst the blood coagulation system can largely be excluded as a source of proteases, it is possible that proteolytic enzymes may be released from muscle lysosomes and/or macrophages after multiple muscular trauma.
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PMID:Evidence for the participation of proteases on protein catabolism during hypercatabolic renal failure. 701 64

Aspects of the molecular interaction and subunit structure of rabbit skeletal muscle phosphorylase kinase, (alpha beta gamma delta)4, were investigated. Exogenous addition of the delta subunit (calmodulin) stimulated the activities of nonactivated phosphorylase kinase and the alpha gamma delta complex, but not the gamma delta complex. This stimulatory effect does not seem to affect the activity-pH profile of the native kinase and is expressed at least partly through an interaction of the calmodulin with the alpha subunit, resulting in an increase in the apparent Vm parameters. Low concentrations of trifluoperazine had only slight effect on the activities of the three forms of kinase, whereas high concentrations caused nonspecific inactivation. The effects of trifluoperazine and EGTA on the activities of the alpha gamma delta and gamma delta complexes were additive; together, they inactivated the two complexes to about 10% of their original activities. The delta subunit remained tightly bound to phosphorylase kinase and the alpha gamma delta complex, even in the presence of 8 M urea, but less so with the gamma delta complex. The nonactivated kinase is more stable towards heat inactivation at 37 degrees C than the alpha gamma delta complex, whereas the gamma delta complex is least stable. Similar to the holoenzyme, limited trypsin digestion activated the alpha gamma delta complex. The pH 6.8/8.2 activity ratio of this complex increased from 0.5 to 0.9, with concomitant degradation of the alpha subunit. In contrast, the gamma delta complex is fairly inert to limited proteolysis. By using sucrose density gradient ultracentrifugation and pretreatment of the gamma delta complex with EGTA, results indicated that the gamma subunit may be a Ca2+-independent kinase, which has about 30% of the original phosphorylase kinase activity.
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PMID:Rabbit skeletal muscle phosphorylase kinase. Interactions between subunits and influence of calmodulin on different complexes. 706 20

The interaction of muscle and liver phosphorylase kinase with some proteins has been studied. It was shown that muscle G-actin has a visible stimulating effect on the dephosphorylated form of muscle phosphorylase kinase. The effect of F-actin on this enzyme is very low. The interaction of phosphorylase kinase with G-actin probably is one of the additional links between glycogenolysis and muscle contraction. To answer the question what subunit(s) of phosphorylase kinase is involved in the interaction with G-actin we studied the influence of actin on the kinase preparations previously activated to a different degree by partial proteolysis with endogenous protease(s) or with trypsin. G-actin has almost no stimulating effect on the preparations of phosphorylase kinase deeply activated by proteolysis (pH 6.8-8.2 activity ratio more than 0.2). The experiments with partial proteolysis allow us to suppose that alpha-subunit is involved in the interaction of phosphorylase kinase with G-actin. Skeletal muscle G-actin activates purified preparations of liver phosphorylase kinase but to a lower degree than muscle enzyme. Brain and liver calmodulin has a low activating effect on liver phosphorylase kinase in the presence of calcium. Calcium-independent action of calmodulin on the preparations of liver phosphorylase kinase is stronger; probably it is connected with a nonspecific effect of this small acidic protein on the liver enzyme. The basic protein protamine has a strong inhibitory effect on liver phosphorylase kinase.
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PMID:Some comparative aspects of regulation of muscle and liver phosphorylase kinase. 711

In this study, we examined protein phosphatase 1 (PP-1) and protein phosphatase 2A (PP-2A) activities during various stages of myogenesis and their regulation by insulin in rat skeletal muscle cells. Protein phosphatase activities were measured using 32P-labeled phosphorylase a, glycogen synthase, and phosphorylase kinase as substrates. Spontaneous PP-1 activity increased progressively in cultures from 2 to 5 days, PP-2A activities remained constant in days 2-4 cultures and increased sharply on day 5. Most of the times in culture, a significant proportion (approximately 65%) of PP-1 was in a form that could be activated by trypsin. Insulin stimulated PP-1 activity (40-80% increase over basal) in a time (t1/2 approximately 5 min)- and dose (EC50 approximately 0.1 nM)-dependent manner. Insulin activation of PP-1 was accompanied by a corresponding inhibition in PP-2A activity. The effects of insulin on PP-1 and PP-2A were differentiation dependent and were observed only in cells at fusion (day 5) and post-fusion. The insulin's effect on PP-1 correlated with the gradual appearance of PP-1 G subunit in cells at fusion. Immunoprecipitation of PP-1 from 32P-labeled cells with an antibody directed against the site 1 sequence of rabbit skeletal muscle PP-1G detected a 160-kDa protein, phosphorylation of which was significantly increased by insulin. This correlated well with the increase observed in immunoprecipitated PP-1G activity. Treatment of cells with a cAMP agonist (SpcAMP) completely blocked activation of PP-1 by insulin and diminished insulin-stimulated phosphorylation of the 160-kDa protein. The likely identity of the 160-kDa band as the regulatory subunit of PP-1 was confirmed by assay of PP-1 activity in the immunoprecipitates and by competition studies with the site 1 peptide against which the antibody was made. From these studies, we conclude that insulin activates PP-1 in L6 cells by increasing the phosphorylation of its regulatory subunit.
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PMID:Regulation of protein phosphatase 1 and 2A activities by insulin during myogenesis in rat skeletal muscle cells in culture. 817 60

Various proteins/enzymes obtained commercially were tested for the presence of endogenously nitrated tyrosine by Western blot analysis omitting reducing agent in the step of SDS-PAGE. Histones II-S and VIII-S, IgG, cAMP-dependent protein kinase (PKA), phosphorylase b, and phosphorylase kinase exhibited strong immunoreactive bands. Histone VI-S, glycogen synthase, lactate dehydrogenase, actin, thyroglobulin, and macroglobulin exhibited moderate immunoreactivity. Histone III-S, casein, acetyl cholinesterase, DNase I, and lipase had only traceable immunoreactivity. Whereas histone VII-S, pyruvate kinase, trypsin, pepsin, chymotrypsin, protease IV, and protease XIII, and glutathione S-transferase lacked immunoreactivity. A variation of immunoreactivity between hypertensive and normaltensive rat hearts was found in the histone-agarose fractions of crude extracts. Additionally, nitrotyrosine immunoreactivity was observed in non-mammalian organisms including Eschericia coli, Saccharomyces cerevisiae and Triticum vulgaris. Upon the treatment of 15 microM peroxynitrite (PN), strong oxidant derived from nitric oxide (NO), the apparent Km of PKA for cAMP increased from approximately 10(-8) to 10(-6) M. The results imply that the varied nitration of tyrosine residues in proteins/enzymes may occur as a post-translational modification in vivo, and such discriminative nitration may be vital in PN/NO-regulated signal transduction cascade.
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PMID:Protein nitration. 1119 83


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