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P Tempst

Publications and source records attributed to P Tempst.

155 records · Page 9Linked to original sources
PubMed1984

Detection of c-sis transcripts and synthesis of PDGF-like proteins by human osteosarcoma cells.

Platelet-derived growth factor (PDGF) has been previously shown to be homologous to the transforming gene of simian sarcoma virus (v-sis), and inappropriate expression of the cellular counterpart of the v-sis gene (c-sis) has been implicated in the generation of mesenchymal tumors. The U-2 OS human osteosarcoma line was shown to contain multiple c-sis transcripts. Immunoprecipitation experiments with antiserum to PDGF identified a variety of polypeptides ranging in size from 18,000 to 165,000 daltons that were immunoprecipitated specifically from U-2 OS cell extracts. The osteosarcoma also was shown to secrete a 29,000-dalton protein having the serological and structural characteristics of PDGF.

Cell Line↗
PubMed1984

Separation of peptides by reverse-phase high-performance liquid chromatography using propyl- and cyanopropylsilyl supports.

The separation of peptides and proteins by reverse-phase high-performance liquid chromatography with cyanopropylsilyl and large-pore propylsilyl supports, together with aqueous trifluoroacetic acid/acetonitrile gradients, was studied. Operating parameters (trifluoroacetic acid concentration, flow rate, and gradient slope) were evaluated using different enzymatic digests of horse cytochrome c and bovine serum albumin. Peptides ranging in size from five amino acids to 68 kDa could be separated on the propylsilyl column in a single chromatographic run. The cyanopropylsilyl column is suitable as a supplement to the use of the large-pore column for medium size (5-20 amino acids) peptides. The chromatographic supports and conditions presented here offer a simple, sensitive, and rapid separation system for a wide size range of peptides and proteins. They extend the versatility of separation methodology for these molecules.

Chromatography, High Pressure Liquid↗
PubMed1984

Purification of bovine bone morphogenetic protein by hydroxyapatite chromatography.

Bovine bone morphogenetic protein (bBMP) induces differentiation of mesenchymal-type cells into cartilage and bone. bBMP has an apparent Mr of 18,500 +/- 500 and represents less than 0.001% of the wet weight of bone tissue. A Mr 34,000 protein resembling osteonectin is separated by extraction with Triton X-100. A Mr 24,000 protein and about half of a Mr 22,000 protein are disassociated from bBMP by precipitation in 1.5 M guanidine hydrochloride. Aggregates of bBMP and a Mr 14,000 protein are insoluble in aqueous media; the bBMP becomes soluble when the Mr 14,000 protein is disassociated in 6 M urea and removed from the solution by ultrafiltration. Three separate molecular species with apparent Mrs 18,500, 17,500, and 17,000 are eluted at 0.10, 0.15, and 0.20 M phosphate ion concentrations, respectively, from a hydroxy-apatite column. The Mr 18,500 protein has the amino acid composition of acidic polylpeptide and includes four half-cystine residues; the pI is 4.9-5.1. The Mr 22,000 component is a chromoprotein resembling ferritin. The NH2-terminal amino acid sequence of the Mr 17,500 protein simulates histone H2B. The Mr 17,000 protein may possess calmodulin activity. Aggregates of the Mr 18,500 and other proteins induce formation of large deposits of bone; the Mr 18,500 protein alone is rapidly absorbed and induces formation of small deposits. None of the other proteins induces bone formation.

Amino Acid Sequence↗
PubMed1983

The amino acid sequence of cytochrome c-556 from Agrobacterium tumefaciens strain Apple 185.

The evidence for the amino acid sequence of cytochrome c-556 from Agrobacterium tumefaciens strain Apple 185 is reported. The sequence was determined by manual Edman degradation of tryptic and chymotryptic peptides using the DABITC/PITC double-coupling method; some peptides were further cleaved by partial acid hydrolysis and with Staphylococcus aureus protease. The sequence overlaps 13-15, 83-85 and 106-108 as well as the region 113-118 involving the haem-binding sequence Cys-Xaa-Xaa-Cys-His were deduced by homology with cytochrome c-556 from Agrobacterium tumefaciens strain B2a. The identity of histidine at position 6 has been inferred from fast-atom bombardment experiments on the N-terminal tryptic peptide, and Asp-63 was deduced from the electrophoretic mobility of the peptides in which it occurs. The cytochrome from A. tumefaciens Apple 185 contains 125 amino acids of which 71 are identical in the protein from strain B2a. Together with cytochrome c-556 from the photosynthetic prokaryote Rhodopseudomonas palustris strain 2.1.37, the presently studied protein is the third known example of a monohaem class II cytochrome of the low-spin type having the single haem group covalently linked near the C terminus of the polypeptide chain. The only methionine residue in the Apple protein, methionine-13, is the most likely candidate to be the sixth haem ligand and therefore to be responsible for the low-spin character of the haem iron.

Amino Acid Sequence↗
PubMed1983

The complete amino-acid sequence of the low-spin class II cytochrome c-556 from Agrobacterium tumefaciens strain B2a.

The amino acid sequence of the soluble monohaem cytochrome c-556 from Agrobacterium tumefaciens, strain B2a, has been determined. The sequence was derived from peptides obtained by digestion of the apoprotein with trypsin and chymotrypsin, and by subdigestion of some of the peptides with Staphylococcus aureus protease and thermolysin. Sequencing of the various peptides was achieved by a combination of manual dansyl-Edman degradation and automatic liquid-phase sequence analysis. The main characteristic of this cytochrome is that the haem-binding sequence Cys-Xaa-Yaa-Cys-His occurs in the C-terminal region of the polypeptide chain, the first cysteine being located 11 residues ahead of the C-terminal lysine-122. As such, the protein belongs to cytochrome c sequence class II (sensu Ambler). The cytochrome c-556 is the first example known of a class II cytochrome of the low-spin type isolated from an obligate aerobic organism.

Amino Acid Sequence↗
PubMed1982

Metal coordination centres of class II cytochromes c.

The class II cytochromes Rhodospirillum molischianum cytochrome c', Rhodopseudomonas palustris cytochrome C556 and Agrobacterium tumefaciens (B2a) cytochrome c556 have been investigated with a variety of spectroscopic techniques. The cytochrome c' was found to be high-spin and the two cytochromes c556 were found to be mainly low-spin and sx-coordinate with the fifth and sixth ligands being histidine and methionine. The implications of the different types of iron coordination are discussed.

Cytochrome c Group↗
PubMed1980

Cytochromes c-556 from three genetic races of Agrobacterium. Purification and comparison of their properties.

1. The soluble cytochromes c-556 from three strains of Agrobacterium tumefaciens, B6, II Chrys and Apple 185 have been purified to homogeneity. The strains are representative members of the three main genetic races of Agrobacterium. The purity of the final preparations was established by electrophoresis with an without sodium dodecyl sulphate, by analytical isoelectric focusing and ultracentrifugation, and by N-terminal analysis. 2. Properties of these cytochromes were compared wih those of cytochrome c-556 from A. tumefaciens, strain B2a, a member of the same genetic race as strain B6. The four cytochromes are monohaem proteins with molecular weights of about 12300 (determined by four different methods). The isoelectric points of those from strains B6 and B2a are identical at pH 5.5, but they differ from the cytochromes of the other genetic races: cytochrome c-556 from strain Apple 185 is more acidic (ph 5.2) and that from strain II Chrys more basic (pH 6.2). The cytochromes from strains b6 and B2a have very similar but not identical amino acid compositions; both of them differ more from Apple 185 than from II Chrys c-556. 3. Comparison of the tryptic, chymotryptic and thermolytic fingerprints of cytochrome c-556 from strains B2a and II Chrys reveals strong homology between the primary structures of these cytochromes. Therefore and because of the sequence identity of the first eight residues, the cytochromes c-556 from strains II Chrys, B6 and B2a are most likely C-terminal haem-bound, of the same type as the cytochrome c' from photosynthetic bacteria.

Amino Acid Sequence↗
PubMedDate not supplied

A microchemical facility for the analysis and synthesis of genes and proteins.

A series of automated instruments that use state-of-the-art chemical methods has been developed for high-sensitivity protein sequencing, DNA synthesis and peptide synthesis. These instruments have been integrated into a centralized microchemical facility in order to promote their use for the study of a variety of biologically interesting problems. This facility has as one of its major functions the development of new chemistries and instrumentation for the structural analysis and synthesis of genes and proteins.

Amino Acid Sequence↗
PubMedDate not supplied

Facile and sensitive assay for monitoring proteolytic activities with defined specificities: studies on amyloid beta-protein processing in Alzheimer's disease.

A novel approach was taken to screen for the presence of highly specific proteases in tissue extracts and to monitor their purification. We successfully used this method to identify proteolytic activity in brain which may be responsible for the generation of the N-terminus of the beta-protein, the major protein in the brain amyloid deposits of Alzheimer's disease. A radiolabeled synthetic decapeptide, spanning this putative cleavage site, was used as an artificial substrate. Digestion was monitored by TLC separation of the reaction products followed by autoradiography. Mobility shifts indicate cleavage; the identity of the fragments can be inferred from synthetic markers or from comparison to control digests with known proteases. When using highly purified or semi-purified proteases, larger amounts of unlabeled peptide substrates can be used and the resulting fragments identified by sequencing. The assay is fast, sensitive (femtomole level), compatible with crude extracts, allows simultaneous analysis of multiple samples and is highly selective with respect to the specificity of the enzyme. The method is generally applicable to the search for proteases with known, putative or even desired specificity.

Alzheimer Disease↗