Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: C30B5 .7
413,019 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have used oligonucleotide probes, based on a portion of the p60v-src autophosphorylation sequence, Glu-Asp-Asn-Glu-Tyr-Thr, to identify and characterize a cDNA from the human T-leukemia cell line, JURKAT. The JURKAT cDNA (designated ptk-JURKAT) was homologous to but distinct from the src, yes and fgr oncogenes, which encode protein-tyrosine kinases (ATP:protein phosphotransferase, EC 2.7.1.37). The ptk-JURKAT cDNA hybridized with a 2.2 kb RNA transcript from JURKAT cells and the human T-cell lymphoma line, MOLT-4, but failed to identify any transcript in two human B-cell lymphoma lines or a human erythroid-myeloid leukemia line, K562. Recently the nucleotide sequence has been established for the murine lymphocyte protein tyrosine kinase, p56LSTRA. The ptk-JURKAT cDNA appears to encode the human homolog of p56LSTRA.
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PMID:Human T lymphocytes express a protein-tyrosine kinase homologous to p56LSTRA. 348 86

Bacterial protein, staphylocoagulase, binds stoichiometrically to human prothrombin resulting in a coagulant complex, staphylothrombin. The enzymatic properties of staphylothrombin differ from those of alpha-thrombin in their substrate specificities toward natural and synthetic substrates, in addition to their interaction with protease inhibitors. In order to obtain information about the region of staphylocoagulase that interacts with human prothrombin, staphylocoagulase was cleaved by alpha-chymotrypsin. This limited alpha-chymotryptic cleavage of staphylocoagulase yielded three large fragments, fragments of 43, 30, and 20 kDa. The 43-kDa fragment exhibited a high affinity for human prothrombin (Kd = 1.7 nM), which is comparable to the affinity observed using intact staphylocoagulase (Kd = 0.46 nM). A complex of the 43-kDa fragment and prothrombin possessed both clotting and amidase activities essentially identical to those observed in a complex of intact staphylocoagulase and prothrombin. The 30-kDa fragment exhibited weaker affinity for prothrombin (Kd = 120 nM). While a complex of this fragment and prothrombin did not exhibit clotting activity, it nonetheless possessed a weak amidase activity. The 20-kDa fragment was found only to bind to prothrombin. The NH2-terminal sequence analyses of these fragments revealed that the 43-kDa fragment constitutes the NH2-terminal portion of staphylocoagulase, and contains the 30-kDa and 20-kDa fragments. It is therefore concluded that the functional region of staphylocoagulase for binding and activation of prothrombin is localized in the NH2-terminal region of the intact protein. The 43-kDa fragment contains 324 amino acids with a molecular weight of 38,098. The 43-kDa fragment has an unusual amino acid composition based on the sequence, in which the sum of Asp (28 residues), Asn (22), Glu (35), Gln (9), and Lys (52) residues accounts for more than 45% of the total residues. A comparison of the amino acid sequence of the 43-kDa fragment with that of streptokinase did not reveal any obvious sequence homology. There was also no sequence homology with those of trypsin, alpha-chymotrypsin, and elastase.
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PMID:Difference in enzymatic properties between "staphylothrombin" and free alpha-thrombin. 355 30

alpha 2-Macroglobulin and the complement components C3 and C4 each contain a metastable binding site that is essential for covalent attachment. Two cyclic peptides are useful models of these unusual protein sites. Five-membered lactam 1 (CH3CO-Gly-Cys-Gly-Glu-Glp-Asn-NH2) contains an internal residue of pyroglutamic acid (Glp). Fifteen-membered thiolactone 2 (CH3CO-Gly-Cys-Gly-Glu-Glu-Asn-NH2 15-thiolactone) contains a thiol ester bond between Cys-2 and Glu-5. These isomeric hexapeptides are spontaneously interconverted in water. Competing with the two isomerization reactions are three reactions involving hydrolysis of 1 and 2. These five processes were found to occur simultaneously under physiologic conditions (phosphate-buffered saline, pH 7.3, 37 degrees C). Best estimates of the five rate constants for these apparent first-order reactions were obtained by comparing the observed molar percentages of peptides 1-4 with those calculated from a set of exponential equations. Both isomerization reactions (ring expansion of 1 to 2, k1 = 6.4 X 10(-5) s-1; ring contraction of 2 to 1, k-1 = 69 X 10(-5) s-1) proceeded faster than any of the hydrolysis reactions: alpha-cleavage of 1 with fragmentation to form dipeptide 3 (k2 = 3.3 X 10(-5) s-1), gamma-cleavage of 1 with ring opening to yield mercapto acid 4 (k3 = 0.35 X 10(-5) s-1), and hydrolysis of 2 with ring opening to give 4 (k4 = 1.9 X 10(-5) s-1). The isomerization rate ratio (k1/k-1 = 10.9) agreed with the isomer ratio at equilibrium (1:2 = 11 starting from 1 and 10 starting from 2). The alpha/gamma regioselectivity ratio (k2/k3 = 9.7) for hydrolysis of the internal Glp residue of 1 was consistent with results for model tripeptides. Part of the chemistry of the protein metastable binding sites can be explained by similar isomerization and hydrolysis reactions.
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PMID:Peptide models of protein metastable binding sites: competitive kinetics of isomerization and hydrolysis. 376 39

Fumarases in the mitochondrial and cytosolic fractions of rat liver were separately purified and crystallized. These two fumarases were not distinguishable in physicochemical, catalytic, or immunochemical properties. The sequences of seven amino acids in the C-terminal portions of the two fumarases were shown using carboxypeptidase P to be identical, i.e.-Val-Asp-Glu-Thr-Ala-Leu-Lys-. The amino acid sequence of the N-terminal portion of the mitochondrial fumarase was determined by the Edman method as Ala-Gln-Gln-Asn-Phe-Glu-Ile-Pro-Asp-, but that of the cytosolic fumarase could not be determined by the Edman method, since the N-terminal amino acid was blocked. The N-terminal amino acid of the cytosolic fumarase was identified as N-acetyl-alanine by analysis of the acidic amino acid produced by digestion of the enzyme protein with pronase E, carboxypeptidase A and B. Then the sequence of five amino acids in the N-terminal portion was determined by analyzing the acidic peptide obtained by limited proteolysis of the enzyme protein with carboxypeptidase A as Ac-Ala-Ser-Gln-Asn-Ser-. Peptide mapping of the tryptic peptides obtained from the mitochondrial and cytosolic fumarases showed no difference in the amino acid sequences of the two except in their N-terminal portions. The turnover rates of the mitochondrial and cytosolic fumarases were determined by injecting L-[U-14C]leucine into rat and following the decay of specific radioactivity incorporated into immunoprecipitates from the partially purified enzyme. The half-life of the cytosolic fumarase was estimated as 4.8 days from the decay curve of its specific radioactivity. The decay curve of the specific radioactivity of the mitochondrial fumarase, obtained after a single injection of L-[U-14]leucine, was quite unusual: its specific radioactivity remained constant for about 7 days after pulse labeling, and then decreased exponentially with a half-life of 9.7 days. Similar amounts of cytosolic and mitochondrial fumarase were found in the livers of the rat, mouse, rabbit, dog, chicken, snake, frog, and carp, respectively. Similar subcellular distributions of the enzyme were also found in the kidney, heart, and skeletal muscle of rats, and in hepatoma cells (AH-109A). However, in rat brain no fumarase activity was detected in the cytosolic fraction. Two putative precursor polypeptides of rat liver fumarase were synthesized when rat liver RNA was translated in vitro in a rabbit reticulocyte lysate system.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Mechanism of synthesis and localization of mitochondrial and cytosolic fumarases in rat liver. 381 85

Smooth muscle myosin light chain kinase contains a 64 residue sequence that binds calmodulin in a Ca2+-dependent manner (Guerriero, V., Jr., Russo, M. A., and Means, A. R. (1987) Biochemistry, in press). Within this region is a sequence with homology to the corresponding sequence reported for the calmodulin binding region of skeletal muscle myosin light chain kinase (Blumenthal, D. K., Takio, K., Edelman, A. M., Charbonneau, H., Titani, L., Walsh, K. A., and Krebs, E. G. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 3187-3191). Inspection of these sequences reveals that they both share a similar number and spatial arrangement of basic residues with those present in the myosin light chain substrate. We have synthesized a 22-residue peptide corresponding to residues 480-501 (determined from the cDNA) of the smooth muscle myosin light chain kinase. This peptide, Ala-Lys-Lys-Leu-Ser-Lys-Asp-Arg-Met-Lys-Lys-Tyr-Met-Ala-Arg-Arg-Lys-Trp- Gln-Lys-Thr-Gly, inhibited calmodulin-dependent activation of the smooth muscle myosin light chain kinase with an IC50 of 46 nM. At saturating concentrations of calmodulin, the 22-residue peptide inhibited myosin light chain and synthetic peptide substrate phosphorylation competitively with IC50 values of 2.7 and 0.9 microM, respectively. An 11-residue synthetic peptide analog, corresponding to part of the calmodulin-binding sequence in skeletal muscle myosin light chain kinase, Lys-Arg-Arg-Trp-Lys-Lys-Asn-Phe-Ile-Ala-Val, also competitively inhibited synthetic peptide substrate phosphorylation with a Ki of 1 microM. The competitive inhibitory activity of the calmodulin binding regions is similar to the apparent Km of 2.7 microM for phosphorylation of the 23-residue peptide analog of the smooth muscle myosin light chain and raises the possibility that the calmodulin binding region of the myosin light chain kinase may act as a pseudosubstrate inhibitor of the enzyme.
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PMID:The calmodulin binding domain of chicken smooth muscle myosin light chain kinase contains a pseudosubstrate sequence. 381 8

A modified procedure for the purification of the colony-stimulating factors (CSFs) in mouse L-cell-conditioned medium is used to isolate two forms of CSF, which are separable by reversed-phase high performance liquid chromatography with 300-A pore size supports. The specific biological activity of these CSFs (2 X 10(9) colonies/mg) was considerably higher than has been achieved by other methods. Even at high concentration (200 pM) both molecules stimulated predominantly more macrophage than granulocyte colonies; however, the less hydrophobic form appeared to stimulate the formation of more pure granulocytic colonies. Almost twice as much of the less hydrophobic CSF was recovered from L-cell-conditioned medium. Analysis using sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that both forms of L-cell CSF had apparent molecular masses of approximately 70,000 daltons. However, on reduction with 2-mercaptoethanol, while both forms generated a 39,000-dalton subunit, the less hydrophobic form also yielded a 32,000-dalton subunit. Storage of either form of L-cell CSF at pH 2.1, in the presence of acetonitrile or isopropanol, destroyed the biological activity. Electrophoretic analysis of the L-cell CSFs stored under these conditions indicated that this was associated with a spontaneous dissociation of the CSF dimer into the inactive subunits. There was some charge heterogeneity (pI 3.5-4.7) indicating different degrees of glycosylation. The unique N-terminal amino acid sequences of both forms of CSF were the same: (Lys-Glu-Val-Ser-Glu-His-X-Ser-His-Met-Ile-Gly-Asn). Thus, the polypeptide chains appear to be identical for the subunits of both forms of L-cell CSF.
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PMID:Purification of two forms of colony-stimulating factor from mouse L-cell-conditioned medium. 387 28

X-ray analyses have shown that the glucopyranose rings of GlcNAc-Asn [4-N-(2-acetamido-2-deoxy-beta-d-glucopyranosyl)-l-asparagine] and Glc-Asn [4-N-(beta-d-glucopyranosyl)-l-asparagine] both have the C-1 chair conformation and also that the glucose-asparagine linkage of each molecule is present in the beta-anomeric configuration. The dimensions (the estimated standard deviations of the last digit are in parentheses) of the glycosidic bond in GlcNAc-Asn and Glc-Asn are, respectively, C((1))-N((1)) 0.1441(6)nm, 0.146(2)nm; angle O((5))-C((1))-N((1)) 106.8(3) degrees , 105.7(8) degrees ; angle C((2))-C((1))-N((1)) 111.1(4) degrees , 110.4(9) degrees ; angle C((1))-N((1))-C((9)) 121.4(4) degrees , 120.5(9) degrees . The glycosidic torsion angle C((9))-N((1))-C((1))-C((2)) is 141.0 degrees and 157.6 degrees in GlcNAc-Asn and Glc-Asn respectively. Hydrogen-bonding is extensive in these two crystal structures and does affect one torsion angle in particular. Two very different values of chi(1)(N-C(alpha)-C(beta)-C(gamma)) occur for the asparagine residue of the two different molecules; the values of chi(1), -69.0 degrees in GlcNAc-Asn and 61.9 degrees in Glc-Asn, correspond to two different staggered conformations about the C(alpha)-C(beta) bond as the NH(3) (+) group is adjusted to different hydrogen-bonding patterns. The two trans-peptide groups in GlcNAc-Asn show small distortions in planarity whereas that in Glc-Asn is more non-planar. The mean plane through the atoms of the amide group at C((2)) in GlcNAc-Asn is approximately perpendicular (69 degrees ) to the mean plane through the C((2)), C((3)), C((5)) and O((5)) atoms of the glucose ring and that at C((1)) is less perpendicular (65 degrees ). The mean plane through the atoms of the amide group in Glc-Asn makes an angle of only 55 degrees with the mean plane through these same four atoms of the glucose ring. The N((1))-H bond of the amide at C((1)) is trans to the C((1))-H bond in these two compounds; the N((2))-H bond of the amide at C((2)) is trans to the C((2))-H bond in GlcNAc-Asn. The values of the observed and final calculated structure amplitudes have been deposited as Supplementary Publication SUP 50035 (26 pages) at the British Library (Lending Division), (formerly the National Lending Library for Science and Technology), Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies may be obtained on the terms given in Biochem. J. (1973) 131, 5.
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PMID:The molecular and crystal structures of 4-N-(2-acetamido-2-deoxy-beta-D-glucopyranosyl)-L-asparagine trihydrate and 4-N-(beta-D-glucopyranosyl)-L-asparagine monohydrate. The x-ray analysis of a carbohydrate-peptide linkage. 446 50

A growth hormone releasing factor of a human pancreatic islet tumor (hpGRF) of an acromegalic patient was purified and subjected to Edman degradation in a spinning cup sequencer. Approximately 0.7-1.2 nmol of peptide was applied to the cup without any pretreatment, after coupling to 3-sulfophenyl isothiocyanate or after cleavage with cyanogen bromide, staphylococcal protease, or trypsin. On the basis of the analytical data, the N-terminal sequence of 39 residues is established to be H-Tyr-Ala-Asp-Ala-Ile-Phe-Thr-Asn- Ser-Tyr-Arg-Lys-Val-Leu-Gly-Gln-Leu-Ser-Ala-Arg-Lys- Leu-Leu-Gln-Asp-Ile-Met-Ser-Arg-Gln-Gln-Gly-Glu-Ser- Asn-Gln-Glu-Arg-Gly-. It is proposed that alanine is residue 40 and represents (as free acid) the C terminus of hpGRF. Synthetic hpGRF(1-40)-OH is highly potent in stimulating GH secretion from the rat anterior pituitary in vitro and in vivo. The C-terminal sequence of hpGRF does not appear to contribute significantly to the biologic intrinsic activity and potency of hpGRF, as demonstrated by the fact that the natural product and the synthetic peptides hpGRF(1-40)-OH, hpGRF(1-40)-NH2, and hpGRF(1-29)-NH2 show equivalent in vitro activities. On the basis of sequence homologies, hpGRF is closely related to members of the glucagon secretin family, especially to the porcine gut peptide PHI.
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PMID:Sequence analysis of a growth hormone releasing factor from a human pancreatic islet tumor. 629 53

Dimeric T. flavoviridis phospholipase A2 has been studied in terms of the interaction with essential Ca2+ by equilibrium gel filtration, ultraviolet difference spectroscopy, fluorescence measurements, and chemical modifications with p-bromophenacyl bromide. The subunit bound to Ca2+ with a 1:1 molar ratio and no cooperative binding was observed. The hypochromic effect produced upon the binding of Ca2+ is due to perturbation of (a) specific tryptophan residue(s) located in the vicinity of the active site and appears to be characteristic of this enzyme. On the basis of the pH dependence of the dissociation constants, it has been found that the alpha-amino group (pKa 8.7) controls the binding of Ca2+. Deprotonation of the alpha-amino group is possibly accompanied by conformational transition to the active form which is able to bind Ca2+. This is in contrast to the case of bovine pancreatic phospholipase A2 in which Asp-49 (pKa 5.2) is responsible for the metal ion binding (Fleer et al. (1981) Eur. J. Biochem. 113, 283-288). Des-octapeptide(1-8)-phospholipase A2 (L-fragment) was found to be capable of binding Ca2+ under the control of a group with a pKa of 7.6. This pKa value was similar to an apparent pKa of 7.5 determined for the histidine residue in the active site of the native enzyme by way of p-bromophenacyl bromide modification. It appears that the N-terminal (octapeptide) sequence affects the binding mode of Ca2+, possibly because of conformational transition arising from its removal. The reinvestigation showed that the N-terminal octapeptide sequence is Gly-Leu-Trp-Gln-Phe-Glu-Asn-Met.
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PMID:Interaction of Trimeresurus flavoviridis phospholipase A2 and its fragment with calcium ion. 652 Jan 20

The enzyme transferring the oligosaccharide from DolPP-(GlcNAc)2(Glc)3 to asparagine residues of glycoproteins has been solubilized from yeast membranes by extraction with detergents. Enzyme activity was tested by measuring transfer of the glycosyl moiety from DolPP-[14C]saccharides to the hexapeptide Tyr-Asn-Leu-Thr-Ser-Val. The rate of transfer was linear for 20 min, with about 40% of dolichyl-diphosphate-bound radioactivity transferred to the peptide. The solubilized enzyme has been characterized as follows: 1. The enzyme is most efficiently solubilized (60% of the membrane-associated activity) by 0.5% Nonidet P40 at a protein/detergent ratio of 2. Octylglucoside solubilizes one third of the activity, but strongly inhibits the reaction if present in the test at a concentration of 1%. 2. Divalent cations are absolutely required. 1 mM Mn2+ is optimal; Mg2+ at a concentration of 10mM yields only one third the activity observed with Mn2+. 3. The enzyme transfers besides dolichyl-diphosphate-bound (GlcNAc)2(Man)9(Glc)3 also (GlcNAc)2(Man)1 and (GlcNAc)2; the rate decreases in this order. No transfer is observed from DolPP-(GlcNAc)2(Man)9 and from DolPP-GlcNAc. 4. The Km value for DolPP-(GlcNAc)2(Man)9(Glc)3 of 0.5 microM does not differ significantly from that for DolPP-(GlcNAc)2 of 1.2 microM. A broad pH-optimum for the reaction with both substrates was found between 6.5 and 7.7. 5. However, a clear difference in Km values for the hexapeptide was observed with difference dolichol-linked sugar derivatives. With DolPP-(GlcNAc)2 a peptide concentration of 0.6 mM resulted in half-maximal transfer rate, whereas 0.05 mM peptide were sufficient with DolPP-(GlcNAc)2(Man)9(Glc)3 as donor.
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PMID:N-Glycosylation of yeast proteins. Characterization of the solubilized oligosaccharyl transferase. 701 1


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