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M Merola

Publications and source records attributed to M Merola.

24 records · Page 2Linked to original sources
PubMed1989

Stereospecificity of reactions catalyzed by bacterial D-amino acid transaminase.

The spectral shift from 420 to 338 nm when pure bacterial D-amino acid transaminase binds D-amino acid substrates is also exhibited in part by high concentrations of L-amino acids (L-alanine and L-glutamate) but not by simple dicarboxylic acids or monoamines. Slow processing of L-alanine to D-alanine was observed both by coupled enzymatic assays using D-amino acid oxidase and by high pressure liquid chromatography analysis employing an optically active chromophore (Marfey's reagent). When the acceptor for L-alanine was alpha-ketoglutarate, D-glutamate was also formed. This minor activity of the transaminase involved both homologous (L-alanine and D-alanine) and heterologous (L-alanine and D-glutamate) substrate pairs and was a function of the nature of the keto acid acceptor. In the presence of alpha-ketoisovalerate, DL-alanine was almost completely processed to D-valine; within the limits of the assay no L-valine was detected. With alpha-ketoisocaproate, 90% of the DL-alanine was converted to D-leucine. In the mechanism of this transaminase reaction, there may be more stereoselective constraints for the protonation of the quinonoid intermediate during the second half-reaction of the transamination reaction, i.e. the donation of the amino group from the pyridoxamine 5'-phosphate coenzyme to a second keto acid acceptor, than during removal of the alpha proton in the initial steps of the reaction pathway. Thus, with this D-amino acid transaminase, the discrete steps of transamination ensure fidelity of the stereospecificity of reaction pathway.

Bacillus↗
PubMed1989

Site-directed mutagenesis of the cysteinyl residues and the active-site serine residue of bacterial D-amino acid transaminase.

Each of the three cysteinyl residues per subunit in D-amino acid transaminase from a thermophilic species of Bacillus has been changed to a glycine residue (C142G, C164G, and C212G) by site-directed mutagenesis. The mutant enzymes were detected by Western blots and a stain for activity. After purification to homogeneity, each mutant protein had the same activity as the wild-type enzyme. Thus, none of the Cys residues are essential for catalysis. Each protein when denatured showed the expected titer of two SH groups per subunit. In the native state, each of the three mutant proteins exhibited nearly the same slow rate of titration of SH groups as the wild-type protein with about one SH group titratable over a period of 4 h. Conversion of Ser-146, adjacent to Lys-145 to which the coenzyme pyridoxal phosphate is bound, to an alanine residue (S146A) does not alter the catalytic activity but has a significant effect on the SH titration behavior. Thus, three to four of the six SH groups of S146A are titratable by DTNB. The rapid SH titration of S146A is prevented by the presence of D-alanine. This finding suggests that the change of Ser-146 to Ala at the active site promotes the exposure and rapid titration of a Cys residue in that region. The rapid SH titration of S146A by DTNB is accompanied by a loss of enzyme activity. Two of the mutant enzymes, C142G and S146A, lose activity at 4 degrees C and also upon freezing and thawing. The mutant enzymes C164G and C212G show the same degree of thermostability as the wild-type enzyme.

Bacillus↗
PubMed1989

Activity and spectroscopic properties of bacterial D-amino acid transaminase after multiple site-directed mutagenesis of a single tryptophan residue.

One of the three tryptophan residues per subunit of thermostable D-amino acid transaminase, Trp-139, is close to the active-site Lys-145 in the sequence of the protein. This tryptophan has been changed to several other types of residues by site-directed mutagenesis. The only mutant protein that was sufficiently active and stable for study had Phe substituted for Trp (W139F). The spectroscopic properties of this mutant enzyme differed from those of the wild-type transaminase. For example, denatured W139F showed the expected decrease in fluorescence emission intensity at 350 nm due to the deletion of one Trp residue, but the fluorescence emission of the wild-type and W139F enzymes in the native state did not differ in intensity. This result suggests that the fluorescence of Trp-139 in the native, wild-type enzyme is not manifested perhaps due to its proximity to the coenzyme, pyridoxal phosphate. Results of energy-transfer studies at several wavelengths could also be interpreted as due to the proximity of Trp-139 and the coenzyme. Circular dichroism studies indicated that the negative Cotton effect at 420 nm due to the coenzyme was still present in W139F. However, the 280-nm optically active band present in the wild-type enzyme was greatly diminished in W139F. The mutant protein with Asp at position 139 (W139D) could not be isolated presumably because it was degraded. The other mutant enzymes, W139P, W139A, and W139H, were isolated with partial activities (15-35%) that were slowly lost upon storage at 4 degrees C. Overall, these results indicate the importance of Trp-139 in the thermostable D-amino acid transaminase.

Bacillus↗
PubMedDate not supplied

Poly(ADP-ribose)synthetase from HeLa cell nuclei: purification and properties.

Poly(ADP-ribose)synthetase has been purified over 600-fold from HeLa cell nuclei. Upon electrophoresis on 9% polyacrylamide slab-gel in the presence of SDS, the purified enzyme resolved in two bands that showed similar apparent Mr values, 110,000 and 118,000. The aminoacid composition of the two components differed from compositions reported for the same enzyme from other sources. In accordance with other reports, the enzyme purified from HeLa cell nuclei exhibited an absolute dependence on DNA and its activity was modulated by changes in the histone concentration. Incubation of intact nuclei from HeLa cells with [14C]NAD resulted in the isolation of a poly(ADP-ribose)synthetase to which a definite radioactivity is bound. These results are taken as a further demonstration that the enzyme can be auto-ADP-ribosylated.

Adenosine Diphosphate Ribose↗
PubMedDate not supplied

Cancer of the uterine cervix: clinical value of squamous cell carcinoma antigen (SCC) measurements.

The clinical value of SCC levels has been evaluated in four groups of women affected by cervical carcinoma. Among the 116 newly diagnosed patients, the SCC pretreatment level was elevated in 57% of cases and was strictly correlated with clinical stage (Ib to IV: p = < 0.01) and histotype (squamous cell carcinoma versus others: p = 0.0005). No significant difference was found in relation to nodal status. For the 28 patients submitted to neoadjuvant chemotherapy clinical response was correlated with the change in serum SCC level: stable or rising serum level indicated that the disease was unchanged or progressive, respectively. In the group of 48 patients affected by recurrent carcinoma, a raised SCC level was found in 71% of cases, with a lead time ranging from 0 to 12 months. No identification of the site of recurrence could be extrapolated from the value of SCC. As to the 108 regularly monitored patients, no significant difference in the risk to develop recurrence was shown for patients with a raised SCC level at the time of primary diagnosis (NED) versus relapsed: p = > 0.05).

Adult↗