A rapid colorimetric assay for heparinase activity.

A rapid, sensitive, assay for enzymes that degrade heparin is described. The procedure is based on the interference of heparin with color development during the interaction of protein with the dye Coomassie brilliant blue. The loss of this property when the glycosaminoglycan is degraded by heparinase can be used to quantify activity of the enzyme in pure form, or in complex biological samples such as tissue homogenates or serum. The assay is also suitable for studying dependence of heparinase activity under conditions such as varying pH and temperature.     

Influence of latitude, water depth, day v. night and wet v. dry periods on the species composition of reef fish communities in tropical Western Australia

Trap sampling over reefs in deep (mean = 20 m) and shallow (mean = 10 m) waters along c. 1500 km of coastline in tropical north‐western Australia during both day and night and in wet and dry periods yielded 23 377 fishes, representing 32 families, 58 genera and 119 species. Individuals of the Serranidae, Lutjanidae, Lethrinidae and Carangidae contributed 88·9% to the total catch. The ichthyofaunal compositions of the Kimberley, Canning and Pilbara bioregions were relatively discrete. Species composition was influenced far more by location (latitude) than by water depth, period and time of day, and underwent a gradational change southwards. The latter change reflected differences in the trends exhibited by the relative abundances of certain species with increasing latitude and the confinement of other species largely to particular regions. The three most abundant species, i.e. Lethrinus sp. 3, Lutjanus carponotatus and Lethrinus laticaudis contributed 34·8, 20·8 and 11·6% to the total catch, respectively. The first species was rarely recorded in the two most northern locations and was abundant in the four most southern locations, whereas the last two species were relatively more abundant in northern than in southern locations. Lutjanus bitaeniatus and Lutjanus johnii were found exclusively at the two locations in the Kimberley region, whereas Abalistes stellatus, Pentapodus emeryii and Lethrinus nebulosus were not caught in this region but were found in both locations of the Canning and Pilbara regions. The species composition in deep and shallow waters at each location almost invariably differed significantly between day and night and between dry and wet periods, with species such as L. bitaeniatus, L. johnii, Lutjanus sebae and A. stellatus being more abundant over deep reefs, whereas L. carponotatus, L. laticaudis, Siganus fuscescens and Lethrinus lentjan were more numerous over shallow reefs. Species such as L. johnii and Lethrinus atkinsoni were relatively more important in night‐time than daytime catches, whereas the reverse applied to Lethrinus lentjan, L. laticaudis and Choerodon cyanodus. Lethrinus sp. 3 and L. laticaudis were relatively more important in catches during the dry than wet period.     

Nuclear events during early chondrogenesis: phosphorylation of the precartilage 35.5-kDa domain-specific chromatin protein and its regulation by cyclic AMP

During chondrogenesis in vivo and in vitro, a family of nonhistone proteins (Mr 35,500), designated PCP 35.5, is lost from the nuclei of precartilage mesenchyme cells. A basic subcomponent of this family, designated PCP 35.5b, is phosphorylated during the first few hours of chondrogenesis in vitro by a phosphorylating system whose activity is enhanced 12- to 15-fold by exposure of differentiating precartilage cells to dibutyryl cyclic AMP. This phosphorylating system is present in isolated precartilage cell nuclei, where it retains its dependence on cyclic AMP and its specificity for PCP 35.5b. Assays for nuclear cyclic AMP inhibitable protein phosphatase activity capable of dephosphorylating PCP 35.5b were negative, indicating that the system responsible for phosphorylating this protein is a cyclic AMP-dependent protein kinase. Chromatin fractionation studies indicate that PCP 35.5b is localized at sites previously shown to be closely associated with DNase I-sensitive domains of precartilage cell chromatin. These studies define PCP 35.5b as a strategically located component of precartilage cell chromatin which is the major or sole chromatin target of cyclic AMP-dependent phosphorylation during chondrogenesis. This chromatin modification occurs prior to overt cartilage differentiation and may therefore play a regulatory role in the acquisition of the cartilage cell phenotype.     

Regulatory and essential light-chain-binding sites in myosin heavy chain subfragment-1 mapped by site-directed mutagenesis

Site-directed mutagenesis of the cloned subfragment-1 (S-1) region of the unc-54 gene, encoding the myosin heavy chain B (MHC B) from Caenorhabditis elegans, has been used to locate binding sites for the regulatory and essential light chains. MHC B S-1 synthesized in Escherichia coli co-migrated with rabbit skeletal muscle myosin S-1 (Mr 90,000), was recognized by anti-nematode myosin antiserum on immunoblots, and specifically bound to 125I-labelled regulatory and essential light chains in a gel overlay assay. Deletion of 102 residues from the C terminus (mutant 655) reduced regulatory and essential light-chain binding to about 30% and 20% of wild-type levels, respectively. Similar reductions in relative binding of the two light chains were seen with mutant 534, in which 38 residues were deleted from the C terminus. Potential binding sites within 75 residues of the C terminus of S-1 were mapped by construction of five other mutant S-1 clones (398, 399, 400, 409 and 411) containing internal deletions of ten to 12 amino acid residues. These showed up to 30% reductions in their ability to bind essential light chains, but did not differ significantly from wild-type in their ability to bind regulatory light chains. Another mutant, 415, containing a deletion of a conserved acidic hexapeptide, E-D-I-R-D-E, showed enhancement of binding of regulatory and essential light chains to 150% and 165% of wild-type levels. Hence, the major binding sites for both light chains are within 38 amino acid residues of the C terminus.