Biomedical subjects
Publications and source records attributed to M S Urdea.
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This investigation demonstrates the use of substitution-inert metal ions as site-specific amino acid modifying reagents. The approach involves the production of a chelating agent at the site of interest with the subsequent in situ oxidation of substitution-labile cobalt(II) to exchange-inert cobalt(III) with H2O2. We have produced the chelate complex ethylenediamine-N,N'-diacetato(arsanilazotyrosinato-248 carboxypeptidase A)cobalt(III) [CoIII(EDDA)(AA-CPA-Zn)]. Model CoIII(EDDA)(azophenolate) complexes have helped to define the reaction conditions necessary to produce the enzyme derivative and have proved invaluable in the spectral analysis of the cobalt(III)-enzyme complex. The modified enzyme contains one active-site zinc and one externally bound cobalt per enzyme monometer. Circular dichroism and visible spectra of the derivative and apoenzyme substantiate the site-specific nature of the incorporation. Concimitant with CoIIIEDDA incorporation, the enzyme loses its peptidase activity yet maintains with FeIIEDTA returns the original properties of the arsanilazotyrosine-248 enzyme.
The residue Tyr 248 of carboxypeptidase A (CPA) is thought to play a role in catalysis by contributing a proton to the incipient amine anion generated during cleavage of peptide substrates. To test this hypothesis we have modified the rat CPA cDNA by site-directed mutagenesis so that the codon for Tyr 248 is replaced by that for Phe. Here, we report the expression of the cDNAs for proCPA and its Tyr-to-Phe variant in yeast via the alpha-factor system. Following zymogen activation by trypsin, wild-type CPA (CPA-WT) and variant CPA (CPA-Phe 248) were purified to homogeneity and characterized enzymatically. CPA-Phe 248 displays essentially undiminished values for the catalytic constant (kcat) towards various peptide and ester substrates. However, the Michaelis constants (Km values) of peptide substrates and the inhibition constant (Ki) of the potato carboxypeptidase inhibitor are increased 6-fold and 70-fold, respectively. These data suggest that the phenolic hydroxyl of Tyr 248 does not act as the requisite general acid catalyst but participates in ligand binding.
The amino-acid sequence of the precursor of the human tumour cell line-derived platelet-derived growth factor (PDGF) A-chain has been deduced from complementary DNA clones and the gene localized to chromosome 7. The protein shows extensive homology to the PDGF B-chain precursor. Expression of the PDGF A-chain gene is independent of that of the PDGF B-chain in a number of human tumour cell lines, and secretion of a PDGF-like growth factor of relative molecular mass 31,000 correlates with expression of A- but not B-chain messenger RNA.
Hepatitis C virus RNA (HCV-RNA) and serological markers of HCV infection were measured in 30 patients with chronic hepatitis C who had been treated with interferon (IFN). Patients were classified into four groups according to serum alanine aminotransferase (ALT) levels after treatment. These were: as complete responders (CR); partial responders (PR); transient responders (TR); and non-responders (NR). In all 11 patients in the CR group, HCV-RNA disappeared from serum for at least 24 months and anti-c100-3 decreased progressively during this time. In the PR group, four of five patients were positive for HCV-RNA in spite of the improvement of ALT levels and decline of anti-c100-3. In the TR and NR groups, HCV-RNA disappeared transiently or remained persistently positive. The results indicate that IFN-mediated improvement of ALT and decrease of anti-HCV (anti-c100-3) were not always related to the disappearance of HCV-RNA from serum. On the other hand, sustained disappearance of HCV-RNA from serum was demonstrated in the patients who did not have post-treatment ALT relapse. This indicates that IFN can eradicate HCV from serum in some patients and provide a clinical remission of chronic hepatitis C.