Search PubMed⌕ Search

Biomedical subjects

J Tang

Publications and source records attributed to J Tang.

At least 631 records · Page 35Linked to original sources
PubMed1976

Thermal fragmentation of Escherichia coli beta-galactosidase. Isolation and characterization of an alpha-complementing and two non-complementing polypeptide fractions.

Carboxymethylated Escherichia coli beta-galactosidase EC 3.2.1.23 could be broken to polypeptides of fairly uniform size (average molecular weight about 22,000 daltons) by heating for less than or equal to 8 h at 100 degrees C and pH 7.5 IN 8 M-urea. Using phosphocellulose chromatography in NaCl-urea gradients, the resulting polypeptide mixture could be resolved in three fractions essentially homogeneous by disc gel electrophoresis in urea at several pH values, and by isoelectric focusing. One of these fractions was active as alpha-donor in in vitro complementation of beta-galactosidase activity with Escherichia coli mutant M15; this activity was largely retained after CNBr cleavage. All three fractions carried arginine as carboxyl-terminal amino acid. No significant amount of any specific amino could be detected in NH2-terminal position.

Amino Acids↗
PubMed1975

Primary structure of porcine pepsin. I. Purification and placement of cyanogen bromide fragments and the amino acid sequence of fragment CB5.

Fragments resulting from the cyanogen bromide cleavage of reduced and aminoethylated porcine pepsin were purified. Only four of the five fragments theoretically present could be accounted for in major yield when the cyanogen bromide reaction was carried out at room temperature. The NH2-terminal fragment, CB2, contained an internal homoserine which was not cleaved to any significant extent. The amino acid sequence around this internal homoserine was determined by isolating and partially determining the sequence of an alpha-chymotryptic peptide. Cleavage at this methionine was increased by 50% when the cyanogen bromide reaction was carried out at 37 degrees. The NH2- and COOH-terminal sequences of five major fragments were determined. The placement of these fragments in the native pepsin molecule was demonstrated. The amino acid sequence of one of the fragments, CB5, was determined. This fragment contains 44 residues with an internal disulfide bridge. The COOH-terminal methionine of this fragment was connected to another 37-residue cyanogen bromide fragment of known sequence. Together these two fragments formed the COOH-terminal 81 residues of porcine pepsin.

Amino Acid Sequence↗
PubMed1975

Primary structure of porcine pepsin. II. Amino acid sequence of two cyanogen bromide fragments, CB3 and CB4.

The amino acid sequences of two cyanogen bromide fragments from porcine pepsin have been determined. Fragment CB3 which represents the NH2-terminal 80 residues of pepsin was assembled from the peptides purified from proteolytic digests of this fragment using alpha-chymotrypsin, thermolysin, and staphylococcal protease. Two chymotryptic peptides were isolated from the NH2-terminal region of this fragment. One of these contains 2 extra residues, Ala-Leu-, at the NH2 terminus. This peptide is apparently derived from a different cleavage site of pepsinogen in its conversion to pepsin. The second cyanogen bromide fragment, CB4, contains 47 residues. The sequence was established from the peptides resulting from proteolytic digests using alpha-chymotrypsin, alpha-lytic protease, and thermolysin. An isoleucyl residue at position 29 of fragment CB4 appears to be absent in some molecules. This represents a structural variant of pepsin.

Amino Acid Sequence↗
PubMed1975

Primary structure of porcine pepsin. III. Amino acid sequence of a cyanogen bromide fragment, CB2A, and the complete structure of porcine pepsin.

The complete amino acid sequence of porcine pepsin (EC 3.4.4.1) was constructed from the sequence of five cyanogen bromide fragments. The sequence of one of these fragments, CB2A, is reported here. The sequences of 4 other fragments are known from previous work. Porcine pepsin contains 327 residues with three structural variants. The active center aspartyl residue, which reacts with 1,2-epoxy-3-(p-nitrophenoxy)propane (Chen, K. C. S., and Tang, J. (1972) J. Biol. Chem. 247, 2566-2574), is located at residue 32. Another active site aspartyl residue, which reacts with diazo inactivators (Bayliss, R. S., Knowles, J. B., and Wybrandt, G. B. (1969) Biochem. J. 113, 377-386, IS LOCATED AT RESIDUE 215. The sequences around these 2 aspartyl residues are apparently homologous to each other. The sequences around the tryptophanyl residues at positions 39, 141, 181, and 300 are also homologous to one another. These homologous sequences could be genetic in origin. Fragment CB2A which contains 119 residues was constructed from the peptide sequences resulting from six proteolytic digestions and chemical cleavage at tryptophanyl bonds.

Amino Acid Sequence↗
PubMed1975

Conversion of pepsinogen to pepsin. Further evidence for intramolecular and pepsin-catalyzed activation.

Exposure of pepsinogen to acid for less than 2 min yields a product with proteolytic activity. This activity is due to intramolecular and intermolecular formation of pepsin from pepsinogen. We find no evidence for intermolecular proteolytic activity in the zymogen. These conclusions are based upon two sets of experiments. First, chemical cleavage of pepsinogen during short activation is demonstrated by quantitative analysis of the NH2-terminal 2 residues of the pepsin and pepsinogen in an activation mixture. In addition, quantitative NH2-terminal analyses after activation under different conditions confirm our previous inference that the product of unimolecular pepsinogen activation is homogeneous whereas bimolecular activation produces a pepsin product with a variety of NH2 termini. Second, spectral changes which occur upon acidification of a pepsinogen solution and are reversed by neutralization are shown to be consistent with the chemical cleavage of pepsinogen during acidification. The first order rate constant for pepsinogen activation, calculated from these spectral experiments, agrees well with the value we had determined previously.

Amino Acid Sequence↗
PubMed1975

Fragments of beta-galactosidase from Escherichia coli. Fragmentation, purification, characterization and in vitro complementation.

Thermal fragmentation of the beta-galactosidase was studied in different buffer solutions and at different temperatures. Fragmentation of the subunits in small size polypeptides could be observed directly. The fragmentation proceeded in buffer solution, pH 7.0, at either 75 degrees C or 100 degrees C in the presence of sodium dodecyl sulfate. The elevated temperature appeared to accelerate this process. At 100 degrees C, pH 7.2, the fragmentation proceeded in the absence of sodium dodecyl sulfate, but in the presence of 8 M urea. Molecular weights, determined by sodium dodecyl sulfate disc gel electrophoresis were from 130,000 to about 20,000. Multiple bands were observed. After dissociation was complete at 37 degrees C, the fragments were purified by ion-exchange column chromatography. Of the five fragments thus obtained, four were homogeneous by disc-gel electrophoresis. Molecular weight of the homogeneous fragments were found to be near 25,000. The fifth comprised a mixture of four fragments having molecular weights from 29,000 to 72,000. Two of the fragments were active as the alpha-donor in in vitro complementation with mutant M15, which contains a deletion in the alpha-region of the z gene.

Chromatography, DEAE-Cellulose↗
PubMed1973

Amino-acid sequence of porcine pepsin.

As the culmination of several years of experiments, we propose a complete amino-acid sequence for porcine pepsin, an enzyme containing 327 amino-acid residues in a single polypeptide chain. In the sequence determination, the enzyme was treated with cyanogen bromide. Five resulting fragments were purified. The amino-acid sequence of four of the fragments accounted for 290 residues. Because the structure of a 37-residue carboxyl-terminal fragment was already known, it was not studied. The alignment of these fragments was determined from the sequence of methionyl-peptides we had previously reported. We also discovered the locations of activesite aspartyl residues, as well as the pairing of the three disulfide bridges. A minor component of commercial crystalline pepsin was found to contain two extra amino-acid residues, Ala-Leu-, at the amino-terminus of the molecule. This minor component was apparently derived from a different site of cleavage during the activation of porcine pepsinogen.

Amino Acid Sequence↗