Chicken skeletal muscle has three Ca2+-dependent proteinases

Chicken breast muscle has three Ca2+-dependent proteinases, two requiring millimolar Ca2+ (m-calpain and high m-calpain) and one requiring micromolar Ca2+ (mu-calpain). High m-calpain co-purifies with mu-calpain through successive DEAE-cellulose (steep gradient), phenyl-Sepharose, octylamine agarose, and Sephacryl S-300 columns, but elutes after mu-calpain when using a shallow KCl gradient to elute a DEAE-cellulose column. The mu- and m-calpains have 80 and 28 kDa polypeptides and are analogous to the mu- and m-calpains that have been purified from bovine, porcine and rabbit skeletal muscle. High m-calpain, which seems to be a new Ca2+-dependent proteinase, is still heterogeneous after the DEAE-cellulose column eluted with a shallow KCl gradient. Additional purification through two successive HPLC-DEAE columns and one HPLC-SW-4000 gel permeation column produces a fraction having six major polypeptides and 6-8 minor polypeptides on SDS-PAGE. A 74-76 kDa polypeptide in this fraction reacts in Western blots with monospecific, polyclonal anti-calpain antibodies that react with both the 80 kDa and the 28 kDa polypeptides of mu- or m-calpain. High m-calpain also is related to mu- and m-calpain in that it causes the same limited digestion of skeletal muscle myofibrils, has a similar pH optimum near pH 7.9-8.4, requires Ca2+ for activity, and reacts with the calpain inhibitor, calpastatin, and a variety of serine and cysteine proteinase inhibitors in a manner identical to mu- and m-calpain. High m-calpain differs from mu- and m-calpain in its elution off DEAE-cellulose columns and its requirement of 3800 microM Ca2+ for one-half maximal activity compared with 5.35 microM Ca2+ for mu-calpain and 420 microM Ca2+ for m-calpain. The physiological significance of high m-calpain in unclear. The presence of mu-calpain in chicken breast muscle suggests that all skeletal muscles contain both mu- and m-calpain, although the relative proportions of these two proteinases may vary in different species.     

Transformed glucocorticoid receptors consist of multiple subspecies with differing capacities to bind DNA-cellulose

The glucocorticoid receptor can be transformed into a DNA-binding protein by a process that is both hormone and temperature dependent. We have used a modification of the conventional method of anion-exchange chromatography to separate and analyze a variety of receptor subspecies that result from this transition. One receptor form (peak A) was found to have a capacity to bind DNA-cellulose which was significantly greater than that of the other species. Under conditions of mild heating (15 degrees C), the relative abundance of peak A in the receptor population and the rate of receptor transformation were both increased as a result of incubating samples with alkaline phosphatase. The mechanism appears to involve the conversion of the more "acidic" forms into that of peak A. The results indicate that receptor transformation is a multistep process which may be promoted by the removal of phosphate from either the receptor or a receptor-bound regulatory factor.     

Association of folic acid with rat liver microsomes

Summary Shin et al. (Biochim Biophys Acta 444: 794–801, 1976) described the subcellular location of [³H]folic acid after injection into rats. The microsomal fraction of the liver contained relatively large amounts of tracer initially but lower amounts at later times. Because of the heterogeneous nature of the microsomal fraction of the liver we re-examined the nature of the folate binding fraction. The location of injected [³H]folic acid resembled that of the microsomes derived from the plasma membrane, where ultracentrifugal analysis was conducted in the presence and absence of cesium ions. The location of the folate did not resemble that of microsomes derived from the endoplasmic reticulum (ER). One of the marker enzymes of the ER was the vitamin K-dependent carboxylase. A simple method for reducing vitamin K is described.     

Flavonoid aglycones from the leaf surfaces of someLabiatae species

The distribution of excreted flavonoid aglycones within the familyLabiatae was studied and differences were found, especially in the A-ring substitution patterns. Thus, 5,7-dihydroxy-6-methoxyflavones with substituted B-rings are characteristic of species ofSalvia (sect.Salvia),Rosmarinus andOcimum; 5,7-dihydroxy-6,8-dimethoxyflavones occur only inOcimum and 5,6-dihydroxy-7,8-dimethoxyflavones inThymus and related species. Members of the two subfamiliesLamioideae andNepetoideae produce exudate flavonoids, but some genera are devoid of these compounds. There is a correlation between the habitat where the plant grows and production of these compounds, the species from (semi-)arid habitats being those which generally accumulate external flavonoids.     

Codon usage patterns in Escherichia coli, Bacillus subtilis, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Drosophila melanogaster and Homo sapiens; a review of the considerable within-species diversity.

The genetic code is degenerate, but alternative synonymous codons are generally not used with equal frequency. Since the pioneering work of Grantham's group it has been apparent that genes from one species often share similarities in codon frequency; under the "genome hypothesis" there is a species-specific pattern to codon usage. However, it has become clear that in most species there are also considerable differences among genes. Multivariate analyses have revealed that in each species so far examined there is a single major trend in codon usage among genes, usually from highly biased to more nearly even usage of synonymous codons. Thus, to represent the codon usage pattern of an organism it is not sufficient to sum over all genes as this conceals the underlying heterogeneity. Rather, it is necessary to describe the trend among genes seen in that species. We illustrate these trends for six species where codon usage has been examined in detail, by presenting the pooled codon usage for the 10% of genes at either end of the major trend. Closely-related organisms have similar patterns of codon usage, and so the six species in Table 1 are representative of wider groups. For example, with respect to codon usage, Salmonella typhimurium closely resembles E. coli, while all mammalian species so far examined (principally mouse, rat and cow) largely resemble humans.     

Production of the penicillin precursor δ-(L-α-aminoadipyl)-L-cysteinyl-D-valine (ACV) by cell-free extracts from Streptomyces clavuligerus

Abstract Glycerol-stabilised cell extracts of Streptomyces clavuligerus contain an enzyme activity which synthesises ACV from the individual amino acids L -α-aminoadipic acid, L -cysteine and L -valine. Enzyme activity was optimum in reaction mixtures containing 1 mM ATP together with an ATP regenerating system. The ACV synthetase enzyme formed ACV analogs when provided with L - carboxymethylcysteine in place of L -α-aminoadipic acid or when provided with L - allo isoleucine or L -α-aminobutyrate in place of L -valine. Multistep conversion of individual amino acids to penicillin and cephalosporin antibiotics was restricted as a result of the inhibitory effects of L -α-aminoadipic acid and L -cysteine on isopenicillin N synthetase.     

Dissolved oxygen control of a maltose feed to batch fermentations ofStreptomyces clavuligerus: Effect on cephamycin C biosynthesis

Summary Compared to controls, a maltose-fed fermentation ofStreptomyces clavuligerus showed a 2-fold reduction in desacetoxycephalosporin C synthase activity and in the production of the antibiotic, cephamycin C. Accumulation of the pathway intermediate, penicillin N occurred in the control fermentations but not in the maltose-fed culture, indicating that the carbon source was also regulating steps earlier in the pathway.Since the dissolved oxygen concentration was effectively maintained at almost constant levels in both the controls and maltose-fed fermentations, the observed maltose interference with cephamycin C biosynthesis was not related to the aeration condition of the actively growingS. clavuligerus culture.