Study of a series of analogs of salmon calcitonin in which alanine replaces leucine

Leucine residues at positions 12, 16 and 19 of salmon calcitonin were systematically replaced by alanine either one, two or three at a time. Substitution of Ala at positions 12 or 16 had the greatest effect on the structure of the peptide and on the ability of the peptide to attain structures of higher helical content in the presence of lipid. Despite the similar effects on the conformational properties, the effects on activity are markedly different for analogs with Ala substitutions at positions 12 and 16. The Ala12 derivatives are much less active while the Ala16 derivatives are slightly more active than the parent hormone. In contrast to the effects of substitutions at these positions, substitution of Ala at position 19 has relatively little effect on activity or on conformation. These results demonstrate that different regions of the calcitonin molecule have different conformational requirements for maximal activity.     

A sensitive procedure for determination of cathepsin D: activity in alveolar and peritoneal macrophages

Summary Several new synthetic substrates fulfilling the specificity requirements of cathepsin D were synthesized. One of these D-Phe-Ser(O-CH₂-C₆H₅)-Phe-Phe-Ala-Ala-pAB (pAB = p-aminobenzoate) proved to be highly sensitive and convenient for measuring activity. Enzyme determination was carried out in a two-step reaction. In the first step the enzyme hydrolyzes the Phe-Phe bond of the substrate at pH 3.4. In the second step aminopeptidase M (EC 3.4.11.2) degrades one of the products Phe-Ala-Ala-pAB at pH 7 to 8 with the release of free pAB, which is then determined by a diazotization procedure. Activity can be measured in as little as 1 to 5 µg of macrophage protein. The activity of cathepsin D in rat alveolar macrophages was almost ten times higher than in resident peritoneal macrophages, and more than 25 times higher than in blood monocytes. The data indicate that transformation of blood monocytes into macrophages is associated with a much greater increase of cathepsin D activity in alveolar than peritoneal macrophages.Abbreviations BOC tert-butoxycarbonyl - H₄-furan tetrahydrofuran - DMF dimethylformamide - HPLC high pressure liquid chromatography - pNA p-nitroanilide - pAB p-aminobenzoate - -CH₂C₆H₅ benzyl - Bz benzoyl - Et₃N triethylamine - CF₃CO₂II trifluoroacetic acid     

A robust method to screen detergents for membrane protein stabilization,revisited

This report is a follow up of our previous paper (Lund, Orlowski, de Foresta, Champeil, le Maire and Møller (1989), J Biol Chem 264:4907–4915) showing that solubilization in detergent of a membrane protein may interfere with its long-term stability, and proposing a protocol to reveal the kinetics of such irreversible inactivation. We here clarify the fact that when various detergents are tested for their effects, special attention has of course to be paid to their critical micelle concentration. We also investigate the effects of a few more detergents, some of which have been recently advertised in the literature, and emphasize the role of lipids together with detergents. Among these detergents, lauryl maltose neopentyl glycol (LMNG) exerts a remarkable ability, even higher than that of β-dodecylmaltoside (DDM), to protect our test enzyme, the paradigmatic P-type ATPase SERCA1a from sarcoplasmic reticulum. Performing such experiments for one's favourite protein probably remains useful in pre-screening assays testing various detergents.     

Regulation of translation rate during morphogenesis in the fungus Mucor

The present study was undertaken in order to elucidate the molecular mechanisms responsible for regulating changes in the specific rate of protein synthesis during the yeast-to-hyphae morphogenesis in the fungus, Mucor racemosus. The distribution of ribosomes between active polysomes and monosomes and inactive subunits was determined by means of pulse-labeling and density gradient fractionation techniques. The percentage of ribosomes active in protein synthesis was observed to decrease throughout the morphological transition. The rate of amino acid addition to nascent polypeptide chains was calculated and the transit time of messenger RNA translation was measured. The results showed a significant increase in the velocity of ribosome movement along the message which was continuously adjusted throughout hyphal development.