Enrico Polastro

Cytochrome C methylation

In this review, protein methylation is outlined in general terms, highlighting the major amino acids that are methylated and some of the proteins in which they are found. The majority of the review examines the methylation of cytochrome c at Lys-77 of lower eukaryotes as a possible model for methylation studies. Early work involving the purification and characterization of the methyltransferase responsible for this methylation indicated cytochrome c was methylated posttranslationally, yet prior to import into the mitochondria. Methylation in vitro occurred only at the in vivo methylation site and only on cytochrome c. Later studies using in vitro translated apocytochrome c revealed that methylated, as compared with unmethylated, apocytochrome c was imported preferentially into yeast, but not rat liver, mitochondria. Efforts to discover the reasons for this preference have shown that methylation of apocytochrome c dramatically lowers its isoelectric point (against a predicted increase) and decrease its Stokes radius. A possible mechanism for these differences involving the disruption of hydrogen bonds is presented here with space-filling models. Finally, the in vivo significance of this modification is also discussed.

Purification and some molecular properties of protein methylase II from equine erythrocytes

Abstract Protein methylase II (S-adenosyl-L-methionine: protein carboxyl-O-methyltransferase; E.C. 2.1.1.24) has been purified 28 000 fold from equine erythrocytes. The purified enzyme is homogeneous on polyacrylamide gel electrophoresis performed either in presence or in absence of SDS, and on analytical ultracentrifugation. It appears constituted of a single polypeptidic chain of a molecular weight very close to 25 000 Daltons. Other enzymatic properties of the protein are quite similar to those previously reported for similar enzymes. The amino acid analysis of the enzyme is presented. The single cysteine residue, the enzyme contains, is essential for the enzymatic activity. Other amino acids apparentely involved in catalysis are tentatively identified.

Study of the biological significance of cytochrome methylation I. Thermal, acid and guanidinium hydrochloride denaturations of baker's yeast ferricytochromes c

The iso-cytochromes c from bakers yeast: iso-1 methylated and unmethylated forms and iso-2 have been purified and their stabilities towards denaturants compared to that of horse heart cytochrome c. Thermal, acid and guanidinium hydrochloride denaturations were followed using fluorescence emission of their tryptophan 59 and/or the absorbance in the Soret region as the physical parameters. Very few differences could be evidenced among the ferricytochromes investigated in this study insofar as the acid denaturations are concerned. This is to be contrasted with the conclusions of the thermal and guanidinium hydrochloride denaturations studies which clearly showed the ferricytochrome from horse heart to be much more stable than those from bakers yeast. No appreciable differences could be measured among the methylated and unmethylated forms of iso-1 cytochrome c nor among iso-1 and iso-2 cytochromes from bakers yeast. Our results suggest that a stabilizing effect of methylation on the tridimensional structure of ferricytochrome c must probably be discarded. Other possible physiological roles of methylation are suggested taking into account the relative instability of ascomycetess cytochromes as compared to mammalian ones.

Evidence that trimethylation of iso-I-cytochrome c from Saccharomyces cerevisiae affects interaction with the mitochondrion

Cytochrome c is a protein occurring in all eucaryotic organisms. Its function and gross conformation has remained the same through evolution as indicated by the constancy of properties like its absorption spectrum, biological activity and redox potential [l]. Of the 103 amino acid ‘core sequence’ common to all types of cytochromes c so far sequenced 35 residues are found to be constant [2]. The longest invariant sequence is the undecapeptide comprising residues 70-80. This segment is thought to be very important for the function of the haemoprotein: indeed the sulfur atom of methionine 80 is most probably the sixth ligand to the haeme iron, tyrosine 74 participates to the mechanism of reduction of the haemoprotein by succinate cytochrome c oxidoreductase [3] and lysines 72 and 79 appear to be involved in binding with cytochrome c oxidase [4]. Yet, the undecapeptide 70-80 is not strictly invariant in all cytochromes c, since cytochrome c from Ascomycetes and higher plants has the lysine 72 e-N-trimethylated [5 1. The biological role of this posttranslational modification is still unclear and has been proposed [6] that methylation of lysine 72 is more coincidental than specific. In investigations comparing the methylated and

Biological significance of methylation of cytochromes from ascomycetes and plants

Abstract The K m and V max values characterizing the reaction of bakers yeast iso -I-cytochrome c , whether tri-methylated or not at lysine residue 72, with crude preparations of cytochrome c peroxidase, cytochrome c oxidase and succinate cytochrome c oxidoreductase from Saccharomyces cerevisiae are similar. These results, as well as the redox potential values, the auto-oxidability parameters and the circular dichroism spectra, strongly suggest that the biological methylation of yeast cytochrome c does not alter its functional properties. The functional characteristics of bakers yeast iso -I-cytochrome c are similar to those of horse heart cytochrome c and yeast iso -2-cytochrome c.

Carboxypeptidase Y from Saccharomyces cerevisiae: Circular dichroism and fluorescence studies

Carboxypeptidase Y was isolated fromSaccharomyces cerevisiae and its molecular structure investigated. The enzyme in the native state possesses 40% of its amino acid residues in a β-conformation. Its tryptophan residues seem to be largely buried in an apolar and unsymmetrical environment.