SUMMARY Density gradient centrifugation and gel filtration have been used to study the formation ... more SUMMARY Density gradient centrifugation and gel filtration have been used to study the formation and some properties of stable complexes between seryl transfer ribonucleic acid synthetase and leucyl transfer ribonucleic acid synthetase and their cognate transfer RNA species (contained in unfractionated tRNA) from Escherichia coli. The molar ratio of tRNA to enzyme was measured as 0.8: 1 for tRNASer and seryl-tRNA synthetase. The complex formation is specific with the cognate tRNA. Five purified different isoaccepting E. coli tRNASe’ species have been shown to form complexes with the enzyme. The presence of high salt (0.5
The newly isolated strain Pseudomonas sp. ON-4a converts D,L-2-amino-delta2-thiazoline-4-carboxyl... more The newly isolated strain Pseudomonas sp. ON-4a converts D,L-2-amino-delta2-thiazoline-4-carboxylic acid to L-cysteine via N-carbamoyl-L-cysteine. A genomic DNA fragment from this strain containing the gene(s) encoding enzymes that convert D,L-2-amino-delta2-thiazoline-4-carboxylic acid into L-cysteine was cloned in Escherichia coli. Transformants expressing cysteine-forming activity were selected by growth of an E. coli mutant defective in the cysB gene. A positive clone, denoted CM1, carrying the plasmid pCM1 with an insert DNA of approximately 3.4 kb was obtained, and the nucleotide sequence of a complementing region was analyzed. Analysis of the sequence found two open reading frames, ORF1 and ORF2, which encoded proteins of 183 and 435 amino acid residues, respectively. E. coli DH5alpha harboring pTrCM1, which was constructed by inserting the subcloned sequence into an expression vector, expressed two proteins of 25 kDa and 45 kDa. From the analyses of crude extracts of E. coli DH5alpha carrying deletion derivatives of pTrCM1 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by enzymatic activity, it was found that the 25-kDa protein encoded by ORF1 was the enzyme L-2-amino-delta2-thiazoline-4-carboxylic acid hydrolase, which catalyzes the conversion of L-2-amino-delta2-thiazoline-4-carboxylic acid to N-carbamoyl-L-cysteine, and that the 45-kDa protein encoded by ORF2 was the enzyme N-carbamoyl-L-cysteine amidohydrolase, which catalyzes the conversion of N-carbamoyl-L-cysteine to L-cysteine.
... Frank F. Richards, WH Konigsberg, RW Rosenstein, Janos M. Varga ... Williamson rea-soned that... more ... Frank F. Richards, WH Konigsberg, RW Rosenstein, Janos M. Varga ... Williamson rea-soned that ifone counted the total number of clones transferred, and then identified from the IEF pattern, how many identical or "repeating" clones there were in that number, it would be ...
Abstract In separate experiments, involving chymotryptic and peptic digestion of the f2 coat prot... more Abstract In separate experiments, involving chymotryptic and peptic digestion of the f2 coat protein, a sufficient number of overlap peptides were isolated to permit us to propose an unambiguous arrangement of the tryptic peptides in the intact coat protein. The assignment of linkages between tryptic peptides was made by comparing the amino acid compositions of chymotryptic and peptic peptides containing lysine and arginine with the compositions of the tryptic peptides. In some cases these assignments were confirmed by digesting the overlap peptides with trypsin and by end group determinations. The order proposed for the tryptic peptides is T11-T8-T4-T3-T7-T9-T5-T2-T10-T6-T1. Two chymotryptic peptides were isolated which were derived from the NH2-terminal region of the protein. The order and amino acid sequence of these peptides were determined, making it possible to establish the sequence of the first 7 residues from the NH2-terminal end of the protein. This sequence is: Ala-Ser-Asn-Phe-Thr-Gln-Phe.
SummaryTissue factor (TF) is a membrane anchored glycoprotein that initiates blood coagulation by... more SummaryTissue factor (TF) is a membrane anchored glycoprotein that initiates blood coagulation by forming a complex with circulating factor VII or VIIa. TF has been identified in atherosclerotic plaques and may possibly trigger thrombosis after spontaneous plaque rupture as seen in acute myocardial infarction or angioplasty. We have previously developed an atherosclerotic rabbit model for study of the acute and chronic outcomes following angioplasty. As a first step in developing inhibitors of TF, we have isolated and characterized a rabbit cDNA coding for the mature TF. The sequence comparison of rabbit TF cDNA with those of human and mouse TFs show considerable similarity at both the nucleotide and amino acid levels. The TF cDNA when expressed in E. coli demonstrates a procoagulant activity comparable to that of native rabbit brain TF. The TF activity can be blocked by a polyclonal antibody against rabbit TF.
The bacteriophage T4 regA protein (M(r) = 14,6000) is a translational repressor of a group of T4 ... more The bacteriophage T4 regA protein (M(r) = 14,6000) is a translational repressor of a group of T4 early mRNAs. To identify a domain of regA protein that is involved in nucleic acid binding, ultraviolet light was used to photochemically cross-link regA protein to [32P]p(dT)16. The cross-linked complex was subsequently digested with trypsin, and peptides were purified using anion exchange high performance liquid chromatography. Two tryptic peptides cross-linked to [32P]p(dT)16 were isolated. Gas-phase sequencing of the major cross-linked peptide yielded the following sequence: VISXKQKHEWK, which corresponds to residues 103-113 of regA protein. Phenylalanine 106 was identified as the site of cross-linking, thus placing this residue at the interface of the regA protein-p(dT)16 complex. The minor cross-linked peptide corresponded to residues 31-41, and the site of cross-linking in the peptide was tentatively assigned to Cys-36. The nucleic acid binding domain of regA protein was further e...
SUMMARY Density gradient centrifugation and gel filtration have been used to study the formation ... more SUMMARY Density gradient centrifugation and gel filtration have been used to study the formation and some properties of stable complexes between seryl transfer ribonucleic acid synthetase and leucyl transfer ribonucleic acid synthetase and their cognate transfer RNA species (contained in unfractionated tRNA) from Escherichia coli. The molar ratio of tRNA to enzyme was measured as 0.8: 1 for tRNASer and seryl-tRNA synthetase. The complex formation is specific with the cognate tRNA. Five purified different isoaccepting E. coli tRNASe’ species have been shown to form complexes with the enzyme. The presence of high salt (0.5
The newly isolated strain Pseudomonas sp. ON-4a converts D,L-2-amino-delta2-thiazoline-4-carboxyl... more The newly isolated strain Pseudomonas sp. ON-4a converts D,L-2-amino-delta2-thiazoline-4-carboxylic acid to L-cysteine via N-carbamoyl-L-cysteine. A genomic DNA fragment from this strain containing the gene(s) encoding enzymes that convert D,L-2-amino-delta2-thiazoline-4-carboxylic acid into L-cysteine was cloned in Escherichia coli. Transformants expressing cysteine-forming activity were selected by growth of an E. coli mutant defective in the cysB gene. A positive clone, denoted CM1, carrying the plasmid pCM1 with an insert DNA of approximately 3.4 kb was obtained, and the nucleotide sequence of a complementing region was analyzed. Analysis of the sequence found two open reading frames, ORF1 and ORF2, which encoded proteins of 183 and 435 amino acid residues, respectively. E. coli DH5alpha harboring pTrCM1, which was constructed by inserting the subcloned sequence into an expression vector, expressed two proteins of 25 kDa and 45 kDa. From the analyses of crude extracts of E. coli DH5alpha carrying deletion derivatives of pTrCM1 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by enzymatic activity, it was found that the 25-kDa protein encoded by ORF1 was the enzyme L-2-amino-delta2-thiazoline-4-carboxylic acid hydrolase, which catalyzes the conversion of L-2-amino-delta2-thiazoline-4-carboxylic acid to N-carbamoyl-L-cysteine, and that the 45-kDa protein encoded by ORF2 was the enzyme N-carbamoyl-L-cysteine amidohydrolase, which catalyzes the conversion of N-carbamoyl-L-cysteine to L-cysteine.
... Frank F. Richards, WH Konigsberg, RW Rosenstein, Janos M. Varga ... Williamson rea-soned that... more ... Frank F. Richards, WH Konigsberg, RW Rosenstein, Janos M. Varga ... Williamson rea-soned that ifone counted the total number of clones transferred, and then identified from the IEF pattern, how many identical or "repeating" clones there were in that number, it would be ...
Abstract In separate experiments, involving chymotryptic and peptic digestion of the f2 coat prot... more Abstract In separate experiments, involving chymotryptic and peptic digestion of the f2 coat protein, a sufficient number of overlap peptides were isolated to permit us to propose an unambiguous arrangement of the tryptic peptides in the intact coat protein. The assignment of linkages between tryptic peptides was made by comparing the amino acid compositions of chymotryptic and peptic peptides containing lysine and arginine with the compositions of the tryptic peptides. In some cases these assignments were confirmed by digesting the overlap peptides with trypsin and by end group determinations. The order proposed for the tryptic peptides is T11-T8-T4-T3-T7-T9-T5-T2-T10-T6-T1. Two chymotryptic peptides were isolated which were derived from the NH2-terminal region of the protein. The order and amino acid sequence of these peptides were determined, making it possible to establish the sequence of the first 7 residues from the NH2-terminal end of the protein. This sequence is: Ala-Ser-Asn-Phe-Thr-Gln-Phe.
SummaryTissue factor (TF) is a membrane anchored glycoprotein that initiates blood coagulation by... more SummaryTissue factor (TF) is a membrane anchored glycoprotein that initiates blood coagulation by forming a complex with circulating factor VII or VIIa. TF has been identified in atherosclerotic plaques and may possibly trigger thrombosis after spontaneous plaque rupture as seen in acute myocardial infarction or angioplasty. We have previously developed an atherosclerotic rabbit model for study of the acute and chronic outcomes following angioplasty. As a first step in developing inhibitors of TF, we have isolated and characterized a rabbit cDNA coding for the mature TF. The sequence comparison of rabbit TF cDNA with those of human and mouse TFs show considerable similarity at both the nucleotide and amino acid levels. The TF cDNA when expressed in E. coli demonstrates a procoagulant activity comparable to that of native rabbit brain TF. The TF activity can be blocked by a polyclonal antibody against rabbit TF.
The bacteriophage T4 regA protein (M(r) = 14,6000) is a translational repressor of a group of T4 ... more The bacteriophage T4 regA protein (M(r) = 14,6000) is a translational repressor of a group of T4 early mRNAs. To identify a domain of regA protein that is involved in nucleic acid binding, ultraviolet light was used to photochemically cross-link regA protein to [32P]p(dT)16. The cross-linked complex was subsequently digested with trypsin, and peptides were purified using anion exchange high performance liquid chromatography. Two tryptic peptides cross-linked to [32P]p(dT)16 were isolated. Gas-phase sequencing of the major cross-linked peptide yielded the following sequence: VISXKQKHEWK, which corresponds to residues 103-113 of regA protein. Phenylalanine 106 was identified as the site of cross-linking, thus placing this residue at the interface of the regA protein-p(dT)16 complex. The minor cross-linked peptide corresponded to residues 31-41, and the site of cross-linking in the peptide was tentatively assigned to Cys-36. The nucleic acid binding domain of regA protein was further e...
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