Use of micelles in studying drug-binding sites: simulation of the tubulin-bound fluorescence of colchicine

Enhancement of the fluorescence intensity of colchicine occurs in media of low polarity and appreciable viscosity; this is suggested to be the basis of the intensification of its fluorescence when it is bound to and immobilized in tubulin. We show here that the tubulin-bound fluorescence features of colchicine are largely reconstructed upon solubilizing it in chosen micellar aggregates that offer optimal polarities and microviscosities. Triton X-100 and bile salt micelles intensify the colchicine emission but the maximal effects are obtained with tetrameric aggregates of the peptide melittin. Estimates of the polarity, microviscosity and binding-site dimensions of colchicine are obtained using this mimetic approach. Our results suggest that well chosen micellar systems act as good models to reconstruct and analyze the spectral properties of molecules immobilized in their binding sites.     

Origin of life: A hypothesis for the origin of adaptor-mediated ordered synthesis of proteins and an explanation for the choice of terminating codons in the genetic code

Life can be defined as a system of self-sustained chemical processes springing from the ordered synthesis of proteins directed by nucleic acids. To the notoriously difficult problem of the origin of this basic process of nucleic acid-directed protein synthesis, we give a solution of molecular interactions between pentanucleotides and amino acids. A particular conformation of a pentanucleotide forms a double sided template, with its ‘inside’ capable of nestling an amino acid while the ‘outside’ acts as an adaptor to a ‘codon’ triplet on long-chain nucleic acids. This serves as a primitive decoding system. An important aspect of our postulate is that a dynamic interaction is triggered, by this decoding system, through which amino acids are brought to juxtaposition facilitating peptide bond formation. Almost all the important and unique features of contemporary protein-synthesizing machinery are seen to be a direct and natural consequence of our postulate. The emergence of the termination codons also fits in, as a natural consequence of this molecular mechanism.     

Mammalian sulfoconjugate metabolism

Sulfoconjugates occur ubiquitously as sulfopolysaccharides, sulfolipids and sulfoproteins. A variety of sulfotransferases catalyze the sulfation process with 3’-phosphoadenosine 5’-phosphosulfate as the sulfate donor. Sulfatases that catalyze the desulfation of different sulfoconjugates are known to be deficient in a number of genetic storage disorders.     

Thiol protease and cathepsin D activities in selected tissues and cultured cells from normal and dystrophic mice

Thiol protease and cathepsin D activities were studied in extracts from hindlimb muscle of 60-day-old normal and dystrophic mice, strain 129 ReJ, and from cultured normal and dystrophic cells. Total thiol protease activity in dystrophic muscle extracts was 3.5 times higher than in normal muscle extracts, while cathepsin D, activity was 2.2 times greater in dystrophic muscle compared with normal muscle. Activation (pH 4.5, 30 degrees C) of latent thiol protease activity in extracts of muscle occurred concomitant with the inactivation or dissociation of endogenous protease inhibitors. Thiol protease assays revealed a higher ratio of active to inactive protease activity in extracts from dystrophic muscle than from normal muscle. Cultured myoblasts (L69/1) were found to contain 30-fold more thiol protease(s) and 6-fold more cathepsin D activity than whole muscle. Cells established from dystrophic muscle and grown in culture for periods up to 6 months were more responsive to thiol protease activation conditions than similar cultures derived from normal muscle. From data on the rate and extent of thiol protease activation in extracts from dystrophic cells and hindlimb muscle compared with normal tissue, it appears that cells and tissues from dystrophic mice contain a lower level of protease inhibitors than cells and tissues from normal mice.     

Effects of prolactin and androgens on seminal vesicular lipids of castrated mature bonnet monkeys Macaca radiata

Effects of prolactin (PRL), bromocriptine (Br), testosterone propionate (TP), dihydrotestosterone (DHT) and the combination of these androgens with PRL/Br on the total lipid, total cholesterol, total glyceride glycerols, total phospholipid and their fractions in seminal vesicles of castrated mature monkeys were studied. Glyceride glycerols formed the major portion (50%) of total lipids in normal monkeys. Cholesterol and phospholipids were of equal share (25%). Esterified cholesterol formed major share (75%) of total cholesterol. Diacyl glycerol was the major (60%) glyceride glycerol and phosphatidyl choline and ethanolamine were the major phospholipid classes. Except triacyl glycerol castration markedly decreased all the lipid classes. PRL restored normal free and esterified cholesterol and phosphatidyl inositol but Br invariably decreased all the lipid classes. TP/DHT treatment stimulated the free and esterified cholesterol more than the control; it restored the normal glyceride glycerols. Phosphatidyl inositol, choline and ethanolamine were stimulated by androgens and other phospholipid classes were brought to normal. Addition of PRL + TP/DHT markedly increased esterified cholesterol, phosphatidyl inositol, choline, ethanolamine and phosphatidic acid. In all these aspects, Br counteracted the effects of androgens and PRL.     

Role of exogenous hemin in the synthesis of hemoproteins and nonheme proteins during glucose repression in Saccharomyces cerevisiae

Exogenous addition of hemin to glucose-repressed cells of Saccharomyces cerevisiae stimulates the incorporation of amino acid into cytoplasmic proteins twofold. There was no significant change in the synthesis of total cytoplasmic RNA whereas a 40% increase in the synthesis of poly(A)-containing RNA was observed upon hemin treatment. Cell-free translation of cytoplasmic mRNAs and immunoprecipitation analysis of the translated products with antibodies against subunit V of cytochrome oxidase and the alpha and beta subunits of F1-ATPase reveals that there is an eightfold enrichment of the mRNA for subunit V of cytochrome oxidase upon hemin treatment. The effect of hemin on the alpha and beta subunits of F1-ATPase is only marginal, suggesting a differential role for heme in the synthesis of hemoproteins and nonheme proteins during glucose repression.