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Biomedical subjects

C House

Publications and source records attributed to C House.

47 records · Page 3Linked to original sources
PubMed1988

Characteristics of the porcine kidney cell line IB-RS-2 clone D10 (IB-RS-2 D10) which is free of hog cholera virus.

A Brazilian stock of clone C17 of the IB-RS-2 porcine kidney cell line which was contaminated with hog cholera virus (HCV) was cloned. One clone designated IB-RS-2 D10 was determined to be free of HCV, 20 other viruses, and Mycoplasma. IB-RS-2 D10 cells possessed the same viral susceptibility pattern as the contaminated parent cells to the viruses of foot-and-mouth disease, swine vesicular disease, vesicular exanthema of swine, transmissible gastroenteritis, and several other viruses. The IB-RS-2 D10 cells had a median chromosome count of 34, were morphologically epithelioid cells, and were resistant to HCV infection. Freedom from HCV affords advantages for vaccine production and avoids laboratory contamination.

Animals↗
PubMed1987

Protein kinase C contains a pseudosubstrate prototope in its regulatory domain.

The regulatory domain of protein kinase C contains an amino acid sequence between residues 19 and 36 that resembles a substrate phosphorylation site in its distribution of basic residue recognition determinants. The corresponding synthetic peptide (Arg19-Phe-Ala-Arg-Lys-Gly-Ala25-Leu-Arg-Gln-Lys-Asn-Val-His -Glu-Val-Lys-Asn36) acts as a potent substrate antagonist with an inhibitory constant of 147 +/- 9 nM. It is a specific inhibitor of protein kinase C and inhibits both autophosphorylation and protein substrate phosphorylation. Substitution of Ala25 with serine transforms the pseudosubstrate into a potent substrate. These results demonstrate that the conserved region of the regulatory domain (residues 19 to 36) of protein kinase C has the secondary structural features of a pseudosubstrate and may be responsible for maintaining the enzyme in the inactive form in the absence of allosteric activators such as phospholipids.

Amino Acid Sequence↗
PubMed1987

The influence of basic residues on the substrate specificity of protein kinase C.

The substrate specificity of protein kinase C has been examined using a series of synthetic peptide analogs of glycogen synthase, ribosomal protein S6, and the epidermal growth factor receptor. The glycogen synthase analog peptide Pro1-Leu-Ser-Arg-Thr-Leu-Ser-Val-Ala-Ala10 was phosphorylated at Ser7 with a Km of 40.3 microM. Peptide phosphorylation was strongly dependent on Arg4. When lysine was substituted for Arg4 the Km was increased approximately 20-fold. Addition of basic residues on either the NH2-terminal or COOH-terminal side of the phosphorylation site of the glycogen synthase peptide improved the kinetics of peptide phosphorylation. The analog Pro-Leu-Ser-Arg-Thr-Leu-Ser-Val-Ala-Ala-Lys-Lys was phosphorylated with a Km of 4.1 microM. Substitution of Ser7 with threonine increased the apparent Km to 151 microM. The truncated peptide Pro1-Leu-Ser-Arg-Thr-Leu-Ser-Val8 was phosphorylated with similar kinetic constants to the parent peptide, however, deletion of Val8 increased the apparent Km to 761 microM. The ribosomal peptide S6-(229-239) was phosphorylated with a Km of approximately 0.5 microM predominantly on Ser236 and is one of the most potent synthetic peptide substrates reported for a protein kinase. The apparent Km for S6 peptide phosphorylation was increased by either deletion of the NH2-terminal 3 residues Ala229-Arg-231 or by substitution of Arg238 on the COOH-terminal side of the phosphorylation site with alanine. This analog peptide, [Ala238]S6-(229-239) was phosphorylated with an approximate 6-fold reduction in Vmax and a switch in the preferred site of phosphorylation from Ser236 to Ser235. These results support the concept that basic residues on both sides of the phosphorylation site can have an important influence on the kinetics of phosphorylation and site specificity of protein kinase C.

Animals↗
PubMed1984

Synthetic peptide substrates for the membrane tyrosine protein kinase stimulated by epidermal growth factor.

The substrate specificity of the epidermal-growth-factor-stimulated tyrosine protein kinase of A431 cell membranes has been studied using a series of synthetic peptide analogs of the sequence around the phosphorylated tyrosine-419 of pp60src. The nine-residue peptide Leu-Ile-Glu-Asp-Ala-Glu-Tyr-Thr-Ala was phosphorylated on tyrosine with an apparent Km of 0.4 mM and a V of 5.7 nmol X min-1 X mg-1. Synthetic peptide tyrosine phosphorylation was stimulated by epidermal growth factor but not by insulin or relaxin. Extension of the nine-residue peptide to include the basic residues, arginine-412, arginine-422 and lysine-423 led to an increased apparent Km. Substitution of glutamic-418 by leucine also increased the apparent Km. In the model peptide Ile-Xaa-Xaa-Ala-Ala-Tyr-Thr-Ala a lower apparent Km was obtained when Xaa was glutamic rather than aspartic acid. Poly(aspartic acid) and poly(glutamic acid) had only weak effects on peptide tyrosine phosphorylation. The results support the concept that acidic residues and not basic residues are important specificity determinants for the epidermal-growth-factor-stimulated tyrosine protein kinase.

Amino Acid Sequence↗
PubMed1983

Role of basic residues in the phosphorylation of synthetic peptides by myosin light chain kinase.

The substrate specificity of the chicken gizzard myosin light chain kinase has been studied by using a series of synthetic peptide analogs of the NH2-terminal sequence of the chicken gizzard myosin light chain (Mr = 20,000). An 18-residue synthetic peptide, (sequence in text) corresponding to the sequence reported by Maita et al. [Maita, T., Chen, J. I. & Matsuda, G. (1981) Eur. J. Biochem. 117, 417-424], was phosphorylated with a 22-fold higher Km and a Vmax that was decreased to 1% of the native protein substrate. This peptide was also an inferior substrate when compared with an 18-residue synthetic peptide with an alternative sequence, (sequence: see text) which was phosphorylated with an apparent Km of 6.9 microM, comparable to the native protein substrate of 8.6 microM, and a Vmax of 3.9 mumol X min-1 X mg-1, 11% of that for the protein substrate. The kinetic of phosphorylation of shortened peptides corresponding to both sequences, together with peptides with appropriate substitutions, indicated that basic residues were the primary determinants of specificity for the smooth muscle myosin light chain kinase. In the latter peptide sequence, lysine residues 11 and 12 and the arginine at position 13 had a major influence on the kinetics of peptide phosphorylation.

Amino Acid Sequence↗
PubMed1982

Phosphorylation of a synthetic heptadecapeptide by smooth muscle myosin light chain kinase.

A synthetic heptadecapeptide corresponding to part of the NH2-terminal 17 residues of chicken gizzard myosin light chain (Mr = 20,000), Ser-Ser-Lys-Thr-Thr-Lys-Arg-Pro-Gln-Arg-Ala-Thr-Ser-(P)-Asn-Val-Phe-Ser-NH2, was readily phosphorylated by the myosin light chain kinase isolated from the same tissue. The synthetic peptide was phosphorylated stoichiometrically at serine 13, the same residue phosphorylated in the parent protein. The apparent Km and Vmax for peptide phosphorylation was 90 microM and 1.3 mumol min-1 mg-1 compared to 10 microM and 22 mumol min-1 mg-1, respectively, for the myosin light chain. The synthetic heptadecapeptide acted as a competitive inhibitor for myosin light chain phosphorylation with Ki approximately 600 microM. Acetylation of the heptadecapeptide alpha-amino group of serine 1 had little effect on Vmax (0.8 mumol min-1 mg-1) and increased the apparent Km 2-fold. The smooth muscle myosin light chain kinase did not phosphorylate the synthetic heptadecapeptide analog of the corresponding skeletal muscle myosin light chain (Mr = 18,500), nor did it phosphorylate synthetic peptide substrates specific for the cAMP-dependent protein kinase or phosphorylase b kinase. These findings support the idea that the myosin light chain kinase has particular protein substrate specificity requirements and that some of these are derived from the region of primary structure around the phosphorylation site in its native substrate.

Amino Acid Sequence↗
PubMedDate not supplied

Rapid and sensitive detection of foot-and-mouth disease virus in tissues by enzymatic RNA amplification of the polymerase gene.

Direct detection of foot-and-mouth disease (FMD) virus from infected bovine and porcine tissue was investigated using a modified polymerase chain reaction (PCR) technique. A high degree of conservation was found in the genomic region coding for the viral RNA polymerase among the seven FMD viral (FMDV) serotypes. An oligomeric primer pair and probe were constructed from consensus sequence data within this area. First strand cDNA was synthesized using random hexamers and Moloney MuLV reverse transcriptase. The oligomeric primers used for PCR of the random primed cDNA yielded a 454-base-pair target amplification product. The PCR product was sized by agarose gel electrophoresis and hybridized strongly with the consensus sequence oligomeric probe. The PCR product was further examined by digestion with NcoI, confirming the predicted internal restriction enzyme site. All seven serotypes of FMDV RNA were amplified in a few hours and the PCR product tested positive. The sensitivity of the enzymatic amplification for detection of FMDV was 10 TCID50 by gel electrophoresis and less than 1 TCID50 when combined with hybridization to a labeled probe. The technique was specific, as determined by examination of at least 12 other viruses, including enteroviruses and other agents of vesicular disease. In vitro enzymatic amplification of cDNA from FMDV RNA using the modified PCR technique is highly specific, rapid and at least as sensitive as presently used procedures for FMDV laboratory diagnosis.

Animals↗