Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: C30B5 .7
 413,019 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A scheme is proposed for generating the intact Val-448-Phe-545 polypeptide of human plasminogen which contains the fifth kringle domain of the plasmin heavy chain. The procedure is based on a pepsin fragmentation of miniplasminogen and involves the purification of the kringle 5-containing fragment by gel filtration and ion-exchange chromatography. The final product is characterized by amino acid analysis, N- and C-terminal analyses, and high-resolution 1H-NMR spectroscopy at both 300 MHz and 611 MHz. We detect a (40:60%) Asp/Asn heterogeneity at site 452 of the Glu-plasminogen molecule. In the conventional kringle numbering system, the kringle 5 domain extends from Cys-1 to Cys-80, which corresponds to Cys-461 to Cys-540 in plasminogen. A preliminary 1H-NMR characterization of kringle 5 focuses on the global conformational features of the polypeptide. Assignments are given for a number of resonances, including the Tyr-72, the His imidazoles' and the Trp indoles' spin systems. Comparison with human plasminogen kringles 1 and 4 shows that the kringle 5 conformation is highly structured and very similar to that of the homologous domains. This conservancy is particularly striking in the environment surrounding Leu-46 and in the overall features of the aromatic spectrum. There are some differences, particularly in the buried His-33 imidazole group, whose H2 resonance is shifted to 9.67 ppm. A preliminary study of benzamidine-binding shows that the ligand interacts weakly (Ka approximately equal to 1.7 mM -1) mainly through the amidino functional group. Trp-62 and Tyr-72 are significantly perturbed by benzamidine, suggesting that these residues are part of the ligand-binding site.
 ...
PMID:Isolation, purification and 1H-NMR characterization of a kringle 5 domain fragment from human plasminogen. 303 Apr 35

A human liver cDNA library enriched for full-length clones was screened for plasminogen cDNA using a synthetic 24-nucleotide probe derived from a reported partial cDNA sequence. 12 positive clones were identified and one of these was characterized in detail. The 2.7 kb insert contains the complete coding region. At 5 positions, it gives residues different from those reported in a previous amino acid sequence analysis of the protein. The present results show an extra Ile at position 65, Gln instead of Glu at positions 53 and 342, Asn at position 88 instead of Asp, and Asp at position 453 rather than Asn. In the 3'-non-coding region an extension of 29 bases is found which does not contain any structure compatible with a known polyadenylation signal. Instead, the consensus signal AATAAA is placed at a distance of 46 bases upstream of the poly(A)-tail.
 ...
PMID:Molecular cloning and characterization of a full-length cDNA clone for human plasminogen. 303 Aug 13

The nucleotide affinity label 5'-p-fluorosulfonylbenzoyl adenosine reacts at the active site of rabbit muscle pyruvate kinase, with irreversible inactivation occurring concomitant with incorporation of about 1 mol of reagent/mol of enzyme subunit (Annamalai, A. E., and Colman, R. F. (1981) J. Biol. Chem. 256, 10276-10283). Purified peptides have now been isolated from 70% inactivated enzyme containing 0.7 mol of reagent/mol of enzyme subunit. Rabbit muscle enzyme labeled with radioactive 5'-p-fluorosulfonylbenzoyl adenosine was digested with thermolysin. Nucleosidyl peptides were purified by chromatography on phenylboronate-agarose and reverse-phase high performance liquid chromatography. After amino acid and N-terminal analysis, the peptides were identified by comparison with the primary sequences of chicken and cat muscle enzyme. About 75% of the reagent incorporated was distributed equally among three O-(4-carboxybenzenesulfonyl)tyrosine-containing peptides: Leu-Asp-CBS-Tyr-Lys-Asn, Val-CBS-Tyr, and Leu-Asp-Asn-Ala-CBS-Tyr. These tyrosines are located in a 28-residue segment of the 530-amino acid sequence. The remainder of the incorporation was found in two N epsilon-(4-carboxybenzenesulfonyl)lysine-containing peptides. Leu-CBS-Lys and Ala-CBS-Lys-Gly-Asp-Tyr-Pro. Modification in the presence of MnATP or MnADP resulted in a marked decrease in labeling of these peptides in proportion to the decreased inactivation. It is suggested that these modified residues are located in the region of the catalytically functional nucleotide binding site of pyruvate kinase.
 ...
PMID:Identification of tyrosine and lysine peptides labeled by 5'-p-fluorosulfonylbenzoyl adenosine in the active site of pyruvate kinase. 308 67

A procedure of large-scale isolation of homogeneous ribonuclease Th1 from cultural filtrates of Trichoderma harzianum with a yield over 50% has been developed. Three ion-exchange chromatographies on CM- and DEAE-cellulose gave 7500 fold purification of the protein with a specific activity of ca. 4500 U/mg. The RNase Th1 is shown to be a basic protein (pI 9.5) with Mr 10,747; it contains 106 amino acid residues (2 Asp, 6 Asn, 9 Thr, 12 Ser, 2 Glu, 1 Gln, 4 Pro, 16 Gly, 14 Ala, 4 Cys, 7 Val, 5 Ile, 2 Leu, 7 Tyr, 6 Phe, 2 His, 4 Lys, 3 Arg). The total amino acid sequence of RNase Th1 was determined and, on comparison with other guanyl-specific fungal RNases, showed a significant degree of homology, thus indicating probability of a common origin. By means of the equilibrium dialysis, crystals of RNase Th1 were obtained with the space group P3(2)21, a = b = 55.7, c = 80.1 A. A preliminary X-ray study of RNase Th1 was undertaken.
 ...
PMID:[Isolation, analysis of amino acid sequence and crystallization of the extracellular ribonuclease Th1 from Trichoderma harzianum-01]. 313 1

NaDodSO4/PAGE analysis of in vitro translation products of rat submaxillary gland (SMG) mRNAs has revealed an important sexual dimorphism. Moreover, most of the rat male-specific major translation products differ in size from those translated from male mouse SMG mRNAs. To characterize proteins accumulated in the rat SMG under androgen control, a cDNA library was constructed. Here we report the nucleotide sequence of a 0.7-kilobase mRNA that is 1000-3000 times more abundant in male rats than in female rats. The predicted corresponding protein, SMR1, has a molecular weight of 16,000 and contains a signal peptide for secretion and potential signals for glycosylation. An interesting feature of SMR1 is the presence, in a hydrophilic region, of the tetrapeptide Gln-His-Asn-Pro surrounded by two pairs of basic residues that represent potential cleavage sites for maturation enzymes. In rats, the tissue distribution of the SMR1 mRNA is restricted to the SMG and the prostate. Only very low amounts of SMR1 mRNA can be detected in the SMG of male or female mice. Southern blot analysis indicates the presence of three genes in rats but only one in mice. Hypotheses on the physiological role of SMR1-derived peptides in male rats are discussed.
 ...
PMID:High level of accumulation of a mRNA coding for a precursor-like protein in the submaxillary gland of male rats. 318 44

The soluble venom of the scorpion of Colima, Mexico, was fractionated by Sephadex G-50 gel filtration followed by separation of the toxic fraction (number II) with carboxymethyl-cellulose columns in 20 mM ammonium acetate buffer, pH 4.7. Of 24 fractions five were toxic to mice and were further separated in 50 mM phosphate buffer, pH 6.0 using the same ion exchange resin. Final separation included chromatography of the toxic subfractions in the same resin, but in 75 mM ammonium acetate buffer, pH 5.0. By this procedure at least five distinct toxins were obtained in pure form according to gel electrophoresis analysis. Amino acid composition of toxin 1 (component II.20.3.4) and toxic component II.22.5 is included. The N-terminal amino acid sequence of component II.22.5 was shown to be: Lys-Glu-Gly-Tyr-Ile-Val-Asn-Tyr-His-Thr-Gly-Cys-Lys-Tyr-Thr-Cys-Ala-Lys- Leu-Gly - Asp-Asn-Asp-Tyr-Cys-Leu-Arg-Glu-Cys-Lys-.
 ...
PMID:Isolation of several toxins from the venom of the scorpion Centruroides limpidus tecomanus Hoffmann. 320 84

A yolk protein, egg-specific protein, synthesized and accumulated in the developing ovaries of Bombyx mori serves not only as the nutritive source for embryogenesis but also for the reorganization of the yolk system through limited degradation. Using the purified egg-specific protein as a substrate, a protease responsible for its limited hydrolysis was identified in embryonating eggs and purified to homogeneity. The protease had an apparent molecular mass of 30,500 with one subunit of 29,000 daltons. It hydrolyzes synthetic substrates at carbonyl bonds of Arg or Lys residues, and the hydrolysis is strongly inhibited by diisopropylfluorophosphate, phenylmethanesulfonyl fluoride, and leupeptin, suggesting that it is a trypsin-like protease. The protease shows an extremely high degree (over 2,000-fold) of specificity for egg-specific protein compared to other yolk proteins. Intact egg-specific protein is cleaved into three fragments in two steps; the first releases a 8.7-kDa peptide as an end product and a 55-kDa peptide intermediate, and in the second the intermediate is cleaved into 36- and 17.2-kDa peptides. By relating the NH2-terminal amino acid sequences of these peptides to the sequence of the intact egg-specific protein, the protease was shown to cleave first at a Lys-Asn site and secondly at Arg-Asp. Proteolytic activity abruptly appears mid-way in embryogenesis and increases steeply during completion of larval differentiation.
 ...
PMID:A unique protease responsible for selective degradation of a yolk protein in Bombyx mori. Purification, characterization, and cleavage profile. 327 55

The importance of carboxyl groups near the active site zinc for the catalytic function of alcohol dehydrogenase I from Saccharomyces cerevisiae was examined by directed mutagenesis and steady state kinetics. Asp-49 was changed to asparagine and Glu-68 to glutamine (residue numbering as for horse liver enzyme). The catalytic efficiencies (V/Km) for ethanol oxidation and acetaldehyde reduction were decreased by factors of 1000 with the Asn-49 mutant and 100 with the Gln-68 enzyme. For the Asn-49 mutant, dissociation constants for coenzymes increased 7-fold, and Michaelis and inhibition constants for substrates and substrate analogs increased by factors of 20-50. The turnover numbers were reduced 50-fold for ethanol oxidation and 15-fold for acetaldehyde reduction. Product and dead-end inhibition studies and kinetic isotope effects showed that the mechanism with NAD+ and ethanol was rapid equilibrium random, in contrast to the ordered mechanism of wild-type enzyme. Alcohol dehydrogenase I and the Asn-49 mutant had similar CD spectra and 2 zinc atoms/subunit, but slightly different UV absorption and fluorescence spectra. The Gln-68 mutant resembled the wild-type enzyme in most kinetic constants, but the turnover number for ethanol oxidation decreased 35-fold, and Kd for NAD+ and Km for acetaldehyde increased by factors of 4 and 50, respectively. The pK values for V1 and V1/Km for ethanol oxidation were shifted from 7.7 (wild-type) to 6.8 in the Gln-68 and 6.2 in the Asn-49 mutant. The altered electrostatic environment near the active site zinc apparently decreases activities by hindering isomerizations of enzyme-substrate complexes.
 ...
PMID:Carboxyl groups near the active site zinc contribute to catalysis in yeast alcohol dehydrogenase. 328 40

Large amounts of a highly purified, extracellular elastolytic protease of Vibrio vulnificus were obtained by sequential ammonium sulphate precipitation and hydrophobic interaction chromatography with phenyl-Sepharose CL-4B. The protease had an Mr of about 50,500 (estimated by SDS-PAGE), a pI of 5.7, and a temperature optimum range of 55 to 60 degrees C. The pH optimum and the results of inactivation studies suggested that the enzyme was a neutral metalloprotease. The protease had about 429 amino acid residues, and the first 20 amino-terminal amino acid residues were Ala-Gln-Ala-Asn-Gly-Thr-Gly-Pro-Gly-Gly-Asn-Ser-Lys-Thr-Gly-Arg-Tyr-Glu- Phe-Gly . The purified protease was toxic for mice (about 1.5 mg kg-1 and 4.5 mg kg-1, intraperitoneal and intravenous LD50 values, respectively), and subcutaneous injection of the enzyme elicited rapid and extensive dermonecrosis.
 ...
PMID:Purification and characterization of an elastolytic protease of Vibrio vulnificus. 331 81

Nucleotide sequence analysis of a cDNA clone shown to direct the synthesis in Escherichia coli of a pI 6.5 form of dihydrofolate reductase (DHFR) with an apparent molecular weight of 21,000 has clarified the allelic nature of the DHFR genes present in the Chinese hamster lung cell line DC-3F. By comparison with other cDNAs encoding different forms of DHFR produced by these cells or by antifolate-resistant sublines derived from them (Melera, P.W., Davide, J.P., Hession, C.A., and Scotto, K.W (1984) Mol. Cell. Biol. 4, 38-48) and with the use of transcription vectors to generate homogeneous populations of specific DHFR mRNAs for subsequent translation in vitro, we demonstrate that, with respect to the proteins they encode, these alleles differ only at amino acid position 95; a conversion of Asp----Asn at this position is solely responsible for the electrophoretic mobility and pI differences between the Mr 21,000 pI 6.5 and Mr 20,000 pI 6.7 forms of the enzyme. We also show that the conversion of Leu to Phe at position 22 of the Mr 21,000 pI 6.5 enzyme results in a mutant form whose catalytic activity is equal to or greater than normal, but whose IC50 for methotrexate is 85 microM. Additionally, the in vitro translation experiments show that the minor pI forms of DHFR known to exist in Chinese hamster lung cells are generated by a translational or post-translational modification step. Preliminary evidence suggests that this modification may result from an acetylation of the N terminus of the protein.
 ...
PMID:Antifolate-resistant Chinese hamster cells. Molecular basis for the biochemical and structural heterogeneity among dihydrofolate reductases produced by drug-sensitive and drug-resistant cell lines. 333 1


<< Previous 1 2 3 4 5 6 7 8 9 10 Next >>