Interaction of calmodulin with the particulate fraction of cardiac muscle

Tritiated calmodulin (T-CM) was bound to the EGTA-treated particulate fraction of cardiac muscle in a calcium-dependent manner with half-maximal binding occurring between 0.8 to 1.2 microM calcium. Binding exhibited high specificity at an optimum pH of 7.4-7.6. An excess of parvalbumin and other globular proteins did not displace T-CM. The Kd for the interaction was 2.5 +/- 0.83 microM. Binding was trypsin-sensitive, inhibited by high ionic strength and was heat inactivated at a midpoint of 48 - 50 degrees C. Competitive displacement of T-CM occurred with unlabeled troponin C and calmodulin over the same concentration range. The first-order rate constant of T-CM dissociation was 3.27 min-1. Calcium-dependent binding of T-CM was inhibited equally by both mepacrine and trifluoperazine with 50 percent inhibition occurring at 70 microM.     

Comparison of most probable number and pour plate procedures for isolation and enumeration of sulphite-reducing Clostridium spores and group D faecal streptococci from oysters

A comparative study of methods to enumerate sulphite-reducing Clostridium spores and Group D faecal streptococci in oysters demonstrated that pour plate solid agar techniques gave higher counts than liquid broth most probable number procedures. Reinforced clostridial broth with supplements to detect sulphite reduction was compared with pour plates of egg yolk-free tryptose sulphite cycloserine agar incubated at 37 degrees C for 24 h. Azide dextrose broth was compared with pour plates using Slanetz and Bartley (SB) agar or KF-streptococcus agar at 37 degrees C. Most probable number procedures used for both groups of organisms gave excessive numbers of improbable tube combinations. For enumeration of Group D faecal streptococci, a pour plate technique using SB agar incubated at 37 degrees C for 48 h is recommended.     

Aerobactin utilization by Neisseria gonorrhoeae and cloning of a genomic DNA fragment that complements Escherichia coli fhuB mutations.

Aerobactin, a dihydroxamate siderophore produced by many strains of enteric bacteria, stimulated the growth of Neisseria gonorrhoeae FA19 and F62 in iron-limiting medium. However, gonococci did not produce detectable amounts of aerobactin in the Escherichia coli LG1522 aerobactin bioassay. We probed gonococcal genomic DNA with the cloned E. coli aerobactin biosynthesis (iucABCD), aerobactin receptor (iutA), and hydroxamate utilization (fhuCDB) genes. Hybridization was detected with fhuB sequences but not with the other genes under conditions which will detect 70% or greater homology. Similar results were obtained with 21 additional strains of gonococci by colony filter hybridization. A library of DNA from N. gonorrhoeae FA19 was constructed in the phasmid vector lambda SE4, and a clone was isolated that complemented the fhuB mutation in derivatives of E. coli BU736 and BN3307. These results suggest that fhuB is a conserved gene and may play a fundamental role in iron acquisition by N. gonorrhoeae.     

The human glucocerebrosidase gene has two functional ATG initiator codons.

Gaucher disease is due to a deficiency in the activity of the enzyme glucocerebrosidase. Glucocerebrosidase is a lysosomal enzyme that presumably requires a signal peptide for transport across the membrane of the rough endoplasmic reticulum and glycosylation for transport into lysosomes. Human glucocerebrosidase cDNA contains two potential ATG start codons in its long open reading frame. The signal peptides that are initiated from each ATG are quite different in their hydrophobicity. We demonstrate that either ATG can function independently to produce active glucocerebrosidase enzyme in cultured fibroblasts. The glucocerebrosidase activity produced from translation products initiated at either ATG is found predominantly in the lysosomes.     

Identification and partial characterization of two major proteins of Mr 47,000 synthesized by bovine retinal endothelial cells in culture.

Biosynthetic experiments with cultured bovine retinal endothelial cells have identified a glycoprotein of Mr 47,000 (Gp47) as a major component secreted into the medium. Gp47 is a non-collagenous glycoprotein with a pI of 4.6-5.5, which does not bind to either gelatin-Sepharose or heparin-Sepharose but is retained by concanavalin A-Sepharose. The Mr of this species decreases to approx. 42,000 in the presence of tunicamycin, indicating that it contains asparagine-linked oligosaccharides. A second protein of Mr 47,000 (P47) is present in the cell layer/matrix of these cultured cells. The electrophoretic mobility of P47 remains unaltered when synthesized in the presence of tunicamycin. Peptide-mapping experiments using N-chlorosuccinimide and Staphylococcus aureus V8 proteinase demonstrate that Gp47 and P47 are distinct proteins, and are not related to colligin, a membrane-bound collagen-receptor protein of similar size, or to SPARC, a major secreted product of parietal endodermal cells and sparse cultures of aortic endothelial cells.