Solubilization and separation of Ca2+-ATPase from the Ca2+-ryanodine receptor complex

Heavy sarcoplasmic reticulum (SR) preparations of rabbit skeletal muscle, which are enriched in Ca2+-release vesicles from the terminal cisternae (TC) and [3H]ryanodine receptor density, exhibit 60% of the Ca2+-ATPase activity, 58% of the EP level, and 30% of the steady state Ca2+ loading compared to membrane vesicles from the longitudinal SR. The Ca2+-ATPase of TC SR is solubilized and separated from the Ca2+-ryanodine receptor complex in the insoluble fraction on treatment with the detergent C12E9. However, a 50% decrease in receptor density is observed upon removal of the Ca2+-ATPase, suggesting a significant contribution of this protein to maintaining optimal receptor complex density.     

Kinetic properties of cyanase

Cyanase is an inducible enzyme in Escherichia coli that catalyzes the hydrolysis of cyanate. Bicarbonate is required for activity, perhaps as a substrate, and the initial product of the reaction is carbamate, which spontaneously breaks down to ammonia and bicarbonate [Anderson, P. M. (1980) Biochemistry 19, 2882]. The purpose of this study was to characterize the kinetic properties of cyanase. Initial velocity studies showed that both cyanate and bicarbonate act as competitive substrate inhibitors. A number of monovalent anions act as inhibitors. Azide and acetate appear to act as competitive inhibitors with respect to cyanate and bicarbonate, respectively. Chloride, bromide, nitrate, nitrite, and formate also inhibit, apparently as the result of binding at either substrate site. Malonate and several other dicarboxylic dianions at very low concentrations display "slow-binding", reversible inhibition which can be prevented by saturating concentrations of either substrate. The results are consistent with a rapid equilibrium random mechanism in which bicarbonate acts as a substrate, bicarbonate and cyanate bind at adjacent anion-binding sites, and both substrates can bind at the other substrate anion binding site to give a dead-end complex.     

Contributions of the Mac-1 glycoprotein family to adherence-dependent granulocyte functions: structure-function assessments employing subunit-specific monoclonal antibodies

MAb directed at the alpha-subunits of Mac-1 (alpha M), LFA-1 (alpha L), p150,95 (alpha X), or their common beta-subunit were used to characterize the contributions of the Mac-1 glycoprotein family to granulocyte adherence reactions. Inhibitory effects of these MAb in incubation experiments with normal granulocytes indicated distinct adhesive contributions of each subunit. Significantly greater adherence, and inhibition of adherence by anti alpha M, alpha X, and beta MAb, was observed under chemotactic conditions designed to "up-regulate" the surface expression of the alpha M beta and alpha X beta complexes. Adherence to protein-coated glass and binding of albumin-coated latex beads were significantly inhibited by anti-beta greater than anti-alpha M (OKM-10, M1/70, LM2/1.6 and OKM-1) greater than anti-alpha X greater than anti-alpha L MAb, but no effects of anti-HLA, AB, or anti-CR-1 MAb were evident. A similar rank order of inhibition was observed in granulocyte aggregation assays in response to C5a, PMA, or f-Met-Leu-Phe. Significant inhibition of directed migration by anti-beta or anti-alpha M (OKM-1 or OKM-10) MAb was observed in subagarose but not Boyden chemotaxis assays; inhibition was dependent on a continuous cell exposure to anti-Mac-1 alpha or beta during the assay, suggesting that a continuum of new Mac-1 expression is required for directed translocation. Phagocytosis of Oil-Red-O paraffin or zymosan selectively opsonized with C3-derived ligands was significantly inhibited by anti-alpha M MAb (OKM-10 greater than LM2/1.6 greater than M1/70 greater than OKM-1) or by combinations of anti-alpha M + anti-CR-1 MAb, but only minimal inhibitory effects of anti-beta MAb and no effects of anti-alpha L or anti-alpha X MAb were seen. Similarly, complement-dependent phagocytosis-associated lactoferrin release, ingestion, and intracellular killing of Staphylococcus aureus 502A, and binding of iC3b-opsonized SRBC, were significantly inhibited by anti-alpha M (OKM-10, M1/70) or combinations of anti-alpha M + anti-CR-1 MAb, but not by anti-beta, alpha L, or alpha X MAb. Notably, none of the anti-Mac-1 MAb demonstrated inhibitory effects in assays of adherence-independent functions including shape change, specific f-Met-Leu-3H-Phe binding, O-2 generation, chemiluminescence evolution, or lactoferrin release in response to PMA.(ABSTRACT TRUNCATED AT 400 WORDS)     

Dual fluorochrome analysis of human B lymphocytes: phenotypic examination of resting, anti-immunoglobulin stimulated, and in vivo activated B cells

B cell-enriched preparations were prepared from human peripheral blood and lymphoid tissues by the depletion of T cells and monocytes. Only B cells by virtue of their staining with anti-B1 conjugated to fluorescein were additionally examined. Dual fluorescence staining and flow cytometric analysis demonstrated that the majority of "resting" human peripheral blood and splenic B cells co-express the B cell-restricted B1 and B2 antigens and lack B5, a B cell-restricted activation antigen, and interleukin 2 receptor (IL 2R). In contrast, nearly 2/3 and 1/3 of B1+ cells isolated from lymph node expressed IL 2R and B5 antigens, respectively. When B1+ B cells from peripheral blood and spleen were "activated" by anti-Ig, they lost the B2 antigen and acquired the B5 and/or IL 2R antigens. 2/3 of B1+ cells strongly expressed IL 2R, and up to 1/2 of B1+ cells co-expressed B5. Delineation of increased numbers of B1+ cells that co-express B5 and/or IL 2R within lymphoid tissues obtained from patients with diseases characterized by "activated" B cells provides in vivo confirmation that these phenotypic changes correlate with B cell activation. We believe that the identification and isolation of these and similar subsets of cells defined by differing cell surface phenotypes should further our understanding both of normal B cell activation and the pathophysiology of B cell disease states.     

Multiple sequence alignment

A method has been developed for aligning segments of several sequences at once. The number of search steps depends only polynomially on the number of sequences, instead of exponentially, because most alignments are rejected without being evaluated explicitly. A data structure herein called the "heap" facilitates this process. For a set of n sequence segments, the overall similarity is taken to be the sum of all the constituent segment pair similarities, which are in turn sums of corresponding residue similarity scores from a Table. The statistical models that test alignments for significance make it possible to group sequences objectively, even when most or all of the interrelationships are weak. These tests are very sensitive, while remaining quite conservative, and discourage the addition of "misfit" sequences to an existing set. The new techniques are applied to a set of five DNA-binding proteins, to a group of three enzymes that employ the coenzyme FAD, and to a control set. The alignment previously proposed for the DNA-binding proteins on the basis of structural comparisons and inspection of sequences is supported quite dramatically, and a highly significant alignment is found for the FAD-binding proteins.     

Study of the amniotic fluid from smokers and non-smokers in the Ames test

Amniotic fluid from smokers and non-smokers was tested by the Salmonella/mammalian microsome test. Concentrated amniotic fluid from heavy smokers at term showed an increase in the number of revertants with increasing exposure to tar. However, some of the non-smokers had a higher number of revertants than the smokers. No significant differences were found between second-trimester samples from smokers and non-smokers, but the limited volumes available at this stage of pregnancy may be a source of error.     

Production of native, correctly folded bovine pancreatic trypsin inhibitor by Escherichia coli

A gene for bovine pancreatic trypsin inhibitor (BPTI) was fused to the coding sequence for the Escherichia coli alkaline phosphatase signal peptide and expressed in E. coli under the control of the alkaline phosphatase promoter. When induced in phosphate-depleted medium such cells produced a trypsin inhibitor that was indistinguishable from native, properly folded BPTI. In particular, the BPTI produced by E. coli had three disulfide bonds that appeared to be identical to those found in native BPTI, as assayed by sensitivity to iodoacetate, dithiothreitol, and urea. This expression/secretion system will make possible the production of variant BPTI molecules, thus allowing the perturbing effects of amino acid substitutions on BPTI folding, structure, and function to be assessed.     

Different interactions used by Cro repressor in specific and nonspecific DNA binding

The mode of interaction of Cro repressor with specific and nonspecific sites on DNA was explored by chemical modification and protection of lysine and tyrosine residues. Cro has 8 lysines. In the presence of DNA, lysines 32 and 56 are fully protected and lysines 21, 62, and 63 are partially protected from alkylation. However, the terminal amino group and lysines 8, 18, and 39 are not protected. Location of the protected and unprotected lysines on the three-dimensional Cro structure defines a DNA-binding region. The results provide direct experimental support for a mode of interaction between Cro and DNA, in which Cro buries its 2-fold related alpha-helices in consecutive DNA major grooves (Anderson, W. F., Ohlendorf, D. H., Takeda, Y., and Matthews, B. W. (1981) Nature 290, 754-758; Ohlendorf, D. H., Anderson, W. F., Fisher, R. G., Takeda, Y., and Matthews, B. W. (1982) Nature 298, 718-723). In the model, the carboxyl-terminal part of Cro was tentatively presumed to interact with the DNA minor groove. Protection of lysines 62 and 63 confirms the involvement of the carboxyl terminus in DNA binding. Although nonspecific and specific DNA protect the same lysine residues, there are differences in the nature of the interaction of Cro with nonspecific and specific DNA. Cro-nonspecific DNA interaction is salt-sensitive, suggesting that the interaction is predominantly electrostatic. On the other hand, Cro-specific DNA interaction is salt-resistant, suggesting that the interaction may include nonelectrostatic components (hydrogen bonds and hydrophobic interactions) as well. Protection experiments of tyrosine residues (against iodination) suggest that the conformation of Cro repressor changes in two stages: first, when Cro binds at nonspecific sites, and, second, when Cro binds to specific sites on DNA.     

Pertussis toxin prevents homologous desensitization of adenylate cyclase in cultured renal epithelial cells

The biochemical mechanisms of adenylate cyclase desensitization in arginine vasopressin-responsive epithelial cells remain unclear. Preincubation of cultured rabbit renal cortical collecting tubular cells with arginine vasopressin leads to a 30-100% decline in arginine vasopressin-stimulated adenylate cyclase activity. This loss of adenylate cyclase activity is time- and arginine vasopressin concentration-dependent. Preincubation with arginine vasopressin does not result in significant changes in basal, NaF-, forskolin-, isoproterenol- or cholera toxin-stimulated adenylate cyclase activity. Preincubation of cells with chlorophenylthio-cAMP, forskolin, and cholera toxin does not result in loss of arginine vasopressin-stimulated adenylate cyclase activity. Since products of cyclo-oxygenase inhibit arginine vasopressin action, cells were preincubated with indomethacin. Arginine vasopressin-induced adenylate cyclase desensitization is not reversed by indomethacin. By contrast, incubation with pertussis toxin prevents arginine vasopressin-induced adenylate cycle desensitization. These data demonstrate that arginine vasopressin induces homologous desensitization in membranes from cultured rabbit cortical collecting tubular cells and suggest that this desensitization is mediated, at least in part, by pertussis toxin substrate. These observations provide a unifying mechanism for desensitization of adenylate cyclase-coupled hormone receptors.     

Mutagenicity of drinking water detected by the Tradescantia micronucleus test

Spring Lake reservoir of Macomb, Illinois, is a typical model of the drinking water supply of some midwestern towns of the United States. Water samples collected periodically in 1980 and 1981 from this lake were tested for mutagenicity using the Tradescantia micronucleus (Trad-MCN) test, a highly sensitive mutagen-detecting bioassay. Water samples from 1981 were also analyzed chemically. The micronucleus (MCN) frequency peaked (12-14 MCN/100 tetrads) in mid-July in both years, as compared with the average frequency (5 MCN/100 tetrads) of the base-line control that was maintained in nutrient solution (prepared with distilled water and pure chemicals). Drinking water from the tap was tested in parallel with lake water, and its mutagenicity tended to fluctuate with the mutagenicity of the lake water.