Volume Control by Muscle Fibers of the Blue Crab : Volume readjustment in hypotonic salines

Single isolated muscle fibers from the walking legs of the blue crab, Callinectes sapidus act as Boyle-van't Hoff osmometers with an osmotically inactive volume of 33 %. Fibers in hypotonic salines undergo a spontaneous volume readjustment toward the initial volumes of the cells found in isotonic salines. The volume readjustment is initiated by the increase in cell volume in hypotonic salines and appears to be dependent on the duration of exposure of the fiber to external sodium, the sodium concentration, and the pH of the external medium. The volume-readjusted cells continue to behave as osmometers, but with an increased relative osmotically inactive volume and a decreased internal resistivity. The decreases in cell volumes appear to be, in large part, due to losses of osmotically active nonelectrolytes from the cells.     

Calcium/Calmodulin-Dependent Protein Kinase II in Squid Synaptosomes

The Ca2+/calmodulin (CaM)-dependent protein kinase II system in squid nervous tissue was investigated. The Ca2+/CaM-dependent protein kinase II was found to be very active in the synaptosome preparation from optic lobe, where it was associated with the high-speed particulate fraction. Incubation of the synaptosomal homogenate with calcium, calmodulin, magnesium, and ATP resulted in partial and reversible conversion of the Ca2+/CaM-dependent protein kinase II from its calcium-dependent form to a calcium-independent species. The magnitude of this conversion reaction could be increased by inclusion of the protein phosphatase inhibitor NaF or by substitution of adenosine 5'-O-(3-thiotriphosphate) for ATP. When [gamma-32P]ATP was used, proteins of 54 and 58 kilodaltons (kDa) as well as proteins greater than 100 kDa were rapidly 32P-labeled in a calcium-dependent manner. Major 125I-CaM binding proteins in the synaptosome membrane fraction were 38 and 54 kDa. The Ca2+/CaM-dependent protein kinase II was purified from the squid synaptosome and was shown to consist of 54- and 58-60-kDa subunits. The purified kinase, like Ca2+/CaM-dependent protein kinase II from rat brain, catalyzed autophosphorylation associated with formation of the calcium-independent form. These studies, characterizing the Ca2+/CaM-dependent protein kinase II in squid neural tissue, are supportive of the putative role of this kinase in regulating calcium-dependent synaptic functions.     

Characterization of K Opioid Receptors in Neurosecretosomes from Bovine Posterior Pituitary

The binding properties of opioid receptors on isolated nerve terminals (neurosecretosomes) from bovine posterior pituitaries were characterized. Both [3H]etorphine and [3H]ethylketocyclazocine ([3H]EKC) showed high-affinity binding with complex binding isotherms, consistent with the presence of multiple classes of binding sites. [D-Ala2,D-Leu5]enkephalin showed no specific binding and failed to displace [3H]etorphine at high concentrations, indicating the absence of mu, delta, or benzomorphan (kappa 2) sites. Mathematical modelling of the data suggested the presence of three classes of binding sites. The first was of high affinity with Kd values of 0.9 and 2.0 nM for etorphine and EKC, respectively. The second class of sites appeared to bind etorphine with a KD of 150 nM, and EKC with extremely low affinity (unmeasurable binding). The third class of sites was characterized by KD values of 7 and 2 microM for etorphine and EKC, respectively. These results indicate that the nerve terminals of bovine posterior pituitary contain opioid binding sites of the kappa type. Furthermore, these binding sites appear heterogeneous, consisting of at least two and possibly more subtypes or states.     

Peptide hydrophobicity and partitioning in poly(ethylene glycol)/magnesium sulfate aqueous two-phase systems.

Several amino acids and peptides were partitioned in poly(ethylene glycol) (PEG)/magnesium sulfate (MgSO4) aqueous two-phase systems. The partition coefficients measured for amino acids and peptides were proportional to the difference in PEG concentration between the phases. The partitioning data were used to calculate the relative hydrophobicities of individual amino acids, which were then used to estimate the hydrophobicities of peptides. The partition coefficients of several dipeptides were predicted from these estimated hydrophobicities. A series of peptide fragments that compose the pentapeptide leucine enkephalin was also partitioned in the PEG/MgSO4 system. Again, the partitioning depended upon the hydrophobicities of the individual exposed amino acids.     

Steroids and oxygen solubility

Determinations of oxygen solubility in serum were made utilizing various concentrations of corticosteroids in the serum. Within a certain concentration range dexamethasone and betamethasone caused an increase in oxygen solubility. None of the other steroids tested demonstrated this property.     

Characterization of the CYP4A11 gene,a second CYP4A gene in humans

Comparison between the cDNA sequence of CYP4A11 and that deduced from a published genomic clone suggested the presence of an additional CYP4A gene in humans, CYP4A22. PCR amplification of genomic DNA yielded overlapping clones covering 13kb of genomic DNA and extending from 1003bp upstream from CYP4A11 translation initiation to 135bp upstream of the mRNA polyadenylation signal. Sequence and Southern blot analysis showed the presence in humans of two highly homologous CYP4A genes, CYP4A11 and CYP4A22. These two genes share 96% sequence identity and have similar intron/exon sizes and distribution. Short nucleotide insertions (< or =10bp) in introns 1, 3, 9, and 11, and deletions (< or =18bp) in introns 4, 6, and 11 differentiate the two genes. RT-PCR amplification of human kidney RNA followed by restriction fragment analysis showed that CYP4A11 is the predominant isoform expressed in kidney.     

Asymmetric synthesis with immobilized yeast in organic solvents: equilibrium conversion and effect of reactant partitioning on whole cell biocatalysis

A newly isolated strain of the yeast Saccharomyces cerevisiae is investigated for the biocatalytic reduction of ketones and the oxidation of alcohols in organic solvents. The yeast cells are immobilized by entrapment within calcium alginate beads and are found to possess the ability to stereoselectively reduce prochiral ketones and oxidize chiral alcohols to equilibrium conversions. The effect of reactant partitioning on the initial rate of the reactions is also investigated. The observed initial rates are found to vary inversely with reactant partitioning between the organic solvent and the biocatalyst beads. A kinetic model is developed to describe the initial reaction rate of hexanone reduction as a function of substrate concentration within the alginate beads.