Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.21.1 (chymotrypsin)
 10,938 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Several lines of evidence have demonstrated conclusively the presence of cAMP-dependent protein kinase (ecto-RC) activity on the external surface of goat cauda-epididymal intact spermatozoa. The intact-cell ecto-kinase that caused transfer of the terminal phosphate of exogenous ATP to the serine and threonine residues of exogenous histone was specifically activated by cAMP. As well, the ecto-kinase caused phosphorylation of the synthetic peptide Kemptide. The isolated spermatozoa, before or after incubation with reaction mixture for ecto-kinase assays, were approximately 99.5% viable, as shown by the analyses of ethidium bromide fluorescence and the cytosolic marker enzymes lactic dehydrogenase and 3-phosphoglycerate kinase. The ecto-kinase activity was not due to contamination of epididymal plasma and damaged cells or to protein kinase that may have leaked from the cells. There was little uptake of ATP and histone by the cells. The intact-cell kinase activity was strongly (80-90%) inhibited by treatment with membrane nonpenetrating surface probes: p-chloromercuriphenylsulfonic acid (2 microM), diazonium salt of sulfanilic acid (DSS, 0.5 mM), and proteases such as trypsin, chymotrypsin, and pronase (each 125 micrograms/mL). Disruption of sperm plasma membrane by sonication or Triton X-100 (0.2%) caused about a fivefold increase of the intact sperm kinase activity. Highly purified sperm plasma membrane (PM) possessed ecto-kinase activity that was resolved into type I and II kinases by DEAE-cellulose chromatography, the type I isoenzyme being the major (approximately 70%) enzymic species. Treatment of the intact spermatozoa with DSS prior to isolation of PM caused a marked loss of the activities of both the isoenzymes, indicating thereby the "ecto" nature of the PM-bound type I and II kinases. Preparations of vigorously forward-motile spermatozoa with 100% intactness had approximately fourfold higher specific activity of the ecto-kinase than the "composite" cells from which the former cells were isolated. However, the profiles of the type I and II ecto-kinases of the composite, as well as forward-motile spermatozoa, were nearly identical. The data are consistent with the view that ecto-kinases may have role in the regulation of flagellar motility.
 ...
PMID:Type I and II cAMP-dependent ecto-protein kinases in goat epididymal spermatozoa and their enriched activities in forward-motile spermatozoa. 216 Aug 33

ARPP-21 (cAMP-regulated phosphoprotein, Mr = 21,000 as determined by SDS-PAGE) is a major cytosolic substrate for cAMP-stimulated protein phosphorylation in dopamine-innervated regions of the rat CNS. It has recently been purified to homogeneity from bovine caudate nucleus and characterized (Hemmings and Greengard, 1989). ARPP-21 is isolated as 2 isoforms, ARPP-21A and ARPP-21B. The amino acid sequence of purified bovine ARPP-21B has now been determined by gas-phase sequencing. The S-14C-carboxymethylated protein was subjected to enzymatic cleavage with trypsin, chymotrypsin, subtilisin, and endoproteinase Lys-C. The resulting peptides were purified by high-performance liquid chromatography, and selected peptides were subjected to amino acid analysis and/or amino acid sequencing by automated Edman degradation. ARPP-21B consists of a single NH2-terminal blocked polypeptide chain of 88 residues, with a calculated molecular mass of 9561 Da, including an NH2-terminal acetyl group inferred by deblocking with an acylaminopeptidase. This molecular mass is significantly lower than earlier estimates based on SDS-PAGE or hydrodynamic measurements. The seryl residue phosphorylated by cAMP-dependent protein kinase (Hemmings et al., 1989) is located at position 55. The molecule contains 1 cysteinyl residue, at position 71, and contains no methionyl, tyrosyl, phenylalanyl, tryptophanyl, or histidinyl residues. Determination of the primary structure of ARPP-21, one of several phosphoproteins localized to dopaminoceptive neurons in the basal ganglia, provides a framework for further investigations into the molecular mechanisms involved in dopaminergic neurotransmission.
 ...
PMID:ARPP-21, a cyclic AMP-regulated phosphoprotein enriched in dopamine-innervated brain regions. I. Amino acid sequence of ARPP-21B from bovine caudate nucleus. 255 36

L-Thyroxine selectively inhibited Ca2+-calmodulin-activated myosin light chain kinases (MLC kinase) purified from rabbit skeletal muscle, chicken gizzard smooth muscle, bovine thyroid gland, and human platelet with similar Ki values (Ki = 2.5 microM). A detailed analysis of L-thyroxine inhibition of smooth muscle myosin light chain kinase activation was undertaken in order to determine the effect of L-thyroxine on the stoichiometries of Ca2+, calmodulin, and the enzyme in the activation process. The kinetic data indicated that L-thyroxine does not interact with calmodulin but, instead, through direct association with the enzyme, inhibits the binding of the Ca2+-calmodulin complex to MLC kinase. L-[125I]Thyroxine gel overlay revealed that the 95-kDa fragment of chicken gizzard MLC kinase digested by chymotrypsin and all the fragments of 110, 94, 70, and 43 kDa produced by Staphylococcus aureus V8 protease digestion which contain the calmodulin binding domain retain L-[125I]thyroxine binding activity, whereas smaller peptides were not radioactive. Since MLC kinase is phosphorylated by cAMP-dependent protein kinase (2 mol of phosphate/mol of MLC kinase), the effect of L-thyroxine on the phosphorylation of MLC kinase also was examined. L-Thyroxine binding did not inhibit the phosphorylation of MLC kinase and, moreover, reversed the inhibition of phosphorylation obtained with the calmodulin-enzyme complex. These observations support the suggestion that L-thyroxine binds at or near the calmodulin-binding site of MLC kinase. L-Thyroxine may serve as a different type of pharmacological tool for elucidating the biological significance of MLC kinase-mediated reactions.
 ...
PMID:Selective binding of L-thyroxine by myosin light chain kinase. 290 27

The consistent application of phosphatase inhibitors and a novel final purification step using a connected series of DE-51, DE-52, and DE-53 anion-exchange chromatography columns facilitate the preparation of electrophoretically homogeneous subpopulations of rabbit muscle phosphofructokinase which differ in their catalytic properties and endogenous covalent phosphate content. A band of "high"-phosphate enzyme (fraction II) flanked by regions of "low"-phosphate enzyme (fractions I and III) is an unusual feature of the final purification profile. Fractions I (containing in this case 0.42 mol of P/82 000 g of enzyme) and II (containing 1.26 mol of P/82 000 g of enzyme) exhibit the most pronounced functional differences of the fractions. Following our original report [Liou, R.-S., & Anderson, S. R. (1980) Biochemistry 19, 2684], both are activated by the addition of rabbit skeletal muscle F-actin. Under the assay conditions, half-maximal stimulation of phosphofructokinase activity occurs at 15.4 nM actin (in terms of monomer) for fraction I and 9.7 nM for fraction II. The low-phosphate enzyme is synergistically activated in the presence of 0.12 microM actin plus 3.0 microM fructose 2,6-bisphosphate, with a marked increase in Vmax, while the high-phosphate enzyme is not. Neither fraction is activated appreciably by the addition of G-actin or the chymotrypsin-resistant actin "core". The covalently cross-linked trimer of actin stimulates the activity of both the low- and high-phosphate enzyme fractions. However, the previously mentioned synergistic activation characteristic of fraction I fails to occur in solutions containing the trimer plus fructose 2,6-bisphosphate. Phosphorylation of fraction I in an in vitro reaction catalyzed by the cAMP-dependent protein kinase causes its properties to become more like those of fraction II. The total amount of covalent phosphate present after in vitro phosphorylation approaches 2 mol of P/82 000 g of enzyme for both fractions.
 ...
PMID:Factors affecting the activation of rabbit muscle phosphofructokinase by actin. 293 27

Human sperm-free seminal plasma (HSP) contains inhibitors (I) of the seminal plasma histone kinase activity (HK). One I is dialyzable and the other I is nondialyzable and precipitable by dialysis of HSP against a hypotonic buffer. When the nondialyzable, precipitable I fraction is resolubilized, it inhibits HK in a concentration-dependent manner. Sephadex G-25 column chromatography of whole HSP resolves I in both the void (Vo) and inclusion (Vi) volumes. Rechromatography of the VoI resolves I solely in the Vo. These and other data suggest that the ViI does not originate from the VoI, and that both I activities represent separate molecular entities. VoI was further characterized and found to be heat labile, trypsin and neuraminidase insensitive, and alpha-chymotrypsin sensitive. VoI is not soluble in CHCl3 or CHCl3:CH3OH (2:1) and is not adsorbed by charcoal. Chromatography of VoI on Sephadex G-100 yields a broad peak of I that migrates just past the Vo. VoI has no detectable cyclic AMP (cAMP) binding activity and VoI activity is not affected by coincubation of VoI and HK with cAMP. VoI also does not bind to zinc-chelate or phenothiazine affinity columns. These data suggest that VoI is protein in nature with properties distinct from the class of previously described protein kinase inhibitors. Although the identity of VoI is not known, it does not appear to be the regulatory subunit of a cAMP-dependent protein kinase, calsemin or a zinc binding protein.
 ...
PMID:Characterization of a seminal plasma-associated inhibitor of human seminal plasma protein kinase. 298 35

cAMP-dependent protein kinase, derived from either calf lens or bovine heart, promotes the phosphorylation of three lens plasma membrane proteins of molecular mass 28 kDa, 26 kDa and 18 kDa. Correlation of the maximal level of phosphorylation of these components with the Coomassie blue staining intensity of fractionated lens membranes suggests that the phosphorylation of the 28 kDa and 18 kDa components may be approximately stoichiometric. The protein kinase substrates could be dephosphorylated by a cardiac sarcoplasmic-reticulum-bound protein phosphatase activity. The 26 k Da component comigrated with MP26, the major lens membrane component that has been localized to the lens fiber cell junction. Treatment of phosphorylated lens membranes with chymotrypsin did not suggest that any of the three major phosphorylated components was derived from the partial proteolysis of a larger phosphoprotein. After electrophoretic separation of phosphorylated proteins, treatment with N-chlorosuccinimide confirmed that there was little similarity in the structure of the three phosphoproteins. Chymotrypsin did, however, reveal a cryptic phosphorylation site in a 22 kDa fragment that appeared to be derived from MP26. Treatment of phosphorylated membranes with reducing agents resulted in the disappearance of the 28 kDa phosphorylated component and the appearance of a new phosphorylated component of 18 kDa; neither MP26 nor the original 18 kDa component was affected by such treatment. It is not clear whether the original 18 kDa phosphoprotein, present in unreduced samples, is the same as that generated with reducing agents from the 28 kDa phosphorylated lens membrane component.
 ...
PMID:Characterization of the bovine lens plasma membrane substrates for cAMP-dependent protein kinase. 299 Sep 30

Purified phospholamban isolated from canine cardiac sarcoplasmic reticulum vesicles was subjected to proteolysis and peptide mapping to localize the different sites of phosphorylation on the protein and to gain further information on its subunit structure. Five different proteases (trypsin, papain, chymotrypsin, elastase, and Pronase) degraded the oligomeric 27-kDa phosphoprotein into a major 21-22-kDa protease-resistant fragment. No 32P was retained by this protease-resistant fragment, regardless of whether phospholamban had been phosphorylated by cAMP-dependent protein kinase, Ca2+/calmodulin-dependent protein kinase, or protein kinase C. Phosphoamino acid analysis and thin-layer electrophoresis of liberated phosphopeptides revealed that 1 threonine and 2 serine residues were phosphorylated in phospholamban and that 1 of these serine residues and the threonine residue were in close proximity. Only serine was phosphorylated by cAMP-dependent protein kinase, whereas Ca2+-calmodulin-dependent protein kinase phosphorylated exclusively threonine. The results demonstrate that phospholamban has a large protease-resistant domain and a smaller protease-sensitive domain, the latter of which contains all of the sites of phosphorylation. The 21-22-kDa protease-resistant domain, although devoid of incorporated 32P, was completely dissociated into identical lower molecular weight subunits by boiling in sodium dodecyl sulfate, suggesting that this region of the molecule promotes the relatively strong interactions that hold the subunits together. The data presented lend further support for a model of phospholamban structure in which several identical low molecular weight subunits are noncovalently bound to one another, each containing one site of phosphorylation for cAMP-dependent protein kinase and another site of phosphorylation for Ca2+/calmodulin-dependent protein kinase.
 ...
PMID:Proteolytic cleavage of phospholamban purified from canine cardiac sarcoplasmic reticulum vesicles. Generation of a low resolution model of phospholamban structure. 300 93

Calmodulin-dependent protein phosphatase purified from bovine cardiac muscle catalyzed the rapid dephosphorylation of Ser-95 of bovine cardiac cAMP-dependent protein kinase regulatory subunit (RII). The kinetic constants determined for the reaction (Km = 20 microM; Vmax = 2 mumol min-1 mg-1) are comparable to those determined for other good substrates of this phosphatase. Because little is known about the determinants of substrate specificity for the calmodulin-dependent phosphatase, various phosphopeptides were used to investigate the structural features important for substrate recognition. Limited proteolysis of phospho-RII with trypsin and chymotrypsin yielded fragments (residues 93-400 and 91-400, respectively) that were poor substrates, whereas digestion with Staphylococcal aureus V8 protease produced three phosphopeptides that were all dephosphorylated as rapidly as intact RII. The sequence of the shortest phosphopeptide produced by S. aureus V8 protease was determined by sequence analysis to be Asp-Leu-Asp-Val-Pro-Ile-Pro-Gly-Arg-Phe-Asp-Arg-Arg-Val-Ser-Val-Cys-Ala-Glu, corresponding to residues 81-99 of RII. Synthetic phosphopeptides corresponding to residues 81-99, 85-99, 90-99, and 91-99 were prepared to determine the minimum sequence necessary for substrate recognition. Only the 19-residue peptide (81-99) was dephosphorylated with kinetics comparable to RII (Km = 26 microM, Vmax = 1.7 mumol min-1 mg-1). Structural analysis of this peptide indicates that an amphipathic beta-sheet structure may be an important structural determinant for some substrates of the calmodulin-dependent phosphatase.
 ...
PMID:Dephosphorylation of cAMP-dependent protein kinase regulatory subunit (type II) by calmodulin-dependent protein phosphatase. Determinants of substrate specificity. 301 43

The nature of cytosolic factors which modulate the activity of rat liver phosphatidylethanolamine (PE) methyltransferase was investigated. The combined additions of cytosol, Mg X ATP, and NaF to incubations with rat liver microsomes produced a 1.6-fold activation of the methyltransferase at pH 9.2 and a 1.3-fold stimulation at pH 7.0. Nonhydrolyzable 5'-adenylylimidodiphosphate could not substitute for ATP, although GTP could. The activation was time dependent, stable to reisolation of the microsomes by ultracentrifugation, and partially preventable by other cytosolic components. Despite these indications that PE methyltransferase might be a substrate for cytosolic protein kinases, cAMP and Ca2+-calmodulin exerted little influence on the activation reaction. Furthermore, microsomal PE methyltransferase activity was unaffected by purified preparations of cAMP-dependent protein kinase, calmodulin-dependent protein kinase, and casein kinase II, nor was methyltransferase activity influenced by the purified catalytic subunits of protein phosphatases 1 and 2A. Cytosol also contained inhibitors of PE methyltransferase which could overcome the Mg X ATP X NaF-mediated activation of the enzyme, but were not affected by the thermostable phosphatase inhibitors 1 and 2. Part of this inhibitory activity (apparent molecular mass of 15 X 10(3) daltons) was insensitive to trypsin and chymotrypsin, stimulated by Mn2+, and partly inhibited by NaF. Therefore, regulation of methyltransferase by reversible phosphorylation, while still a tenable hypothesis, is apparently more complex than previously proposed.
 ...
PMID:Regulation of rat liver phosphatidylethanolamine N-methyltransferase by cytosolic factors. Examination of a role for reversible protein phosphorylation. 301 87

Glycogen synthase I was purified from rat skeletal muscle. On sodium dodecyl sulfate polyacrylamide gel electrophoresis, the enzyme migrated as a major band with a subunit Mr of 85,000. The specific activity (24 units/mg protein), activity ratio (the activity in the absence of glucose-6-P divided by the activity in the presence of glucose-6-P X 100) (92 +/- 2) and phosphate content (0.6 mol/mol subunit) were similar to the enzyme from rabbit skeletal muscle. Phosphorylation and inactivation of rat muscle glycogen synthase by casein kinase I, casein kinase II (glycogen synthase kinase 5), glycogen synthase kinase 3 (kinase FA), glycogen synthase kinase 4, phosphorylase b kinase, and the catalytic subunit of cAMP-dependent protein kinase were similar to those reported for rabbit muscle synthase. The greatest decrease in rat muscle glycogen synthase activity was seen after phosphorylation of the synthase by casein kinase I. Phosphopeptide maps of glycogen synthase were obtained by digesting the different 32P-labeled forms of glycogen synthase by CNBr, trypsin, or chymotrypsin. The CNBr peptides were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and the tryptic and chymotryptic peptides were separated by reversed-phase HPLC. Although the rat and rabbit forms of synthase gave similar peptide maps, there were significant differences between the phosphopeptides derived from the N-terminal region of rabbit glycogen synthase and the corresponding peptides presumably derived from the N-terminal region of rat glycogen synthase. For CNBr peptides, the apparent Mr was 12,500 for rat and 12,000 for the rabbit. The tryptic peptides obtained from the two species had different retention times. A single chymotryptic peptide was produced from rat skeletal muscle glycogen synthase after phosphorylation by phosphorylase kinase whereas two peptides were obtained with the rabbit enzyme. These results indicate that the N-terminus of rabbit glycogen synthase, which contains four phosphorylatable residues (Kuret et al. (1985) Eur. J. Biochem. 151, 39-48), is different from the N-terminus of rat glycogen synthase.
 ...
PMID:Differences between glycogen synthases from rat and rabbit skeletal muscle as indicated by phosphopeptide maps. 310 44


1 2 3 Next >>