Exchange of recA protein between adjacent recA protein-single-stranded DNA complexes

We have examined the exchange of recA protein between stable complexes formed with single-stranded DNA (ssDNA) and (a) other complexes and (b) a pool of free recA protein. We have also examined the relationship of ATP hydrolysis to these exchange reactions. Exchange was observed between two different recA X ssDNA complexes in the presence of ATP. Complete equilibration between two sets of complexes occurred with a t1/2 of 3-7 min under a set of conditions previously found to be optimal for recA protein-promoted DNA strand exchange. Approximately 200 ATPs were hydrolyzed for every detected migration of a recA monomer from one complex to another. This exchange occurred primarily between adjacent complexes, however. Little or no exchange was observed between recA X ssDNA complexes and the free recA protein pool, even after several hundred molecules of ATP had been hydrolyzed for every recA monomer present. ATP hydrolysis is not coupled to complete dissociation or association of recA protein from or with recA X ssDNA complexes under these conditions.     

Isolation and characterization of a new Mr 18,000 protein with calcium vector properties in amphioxus muscle and identification of its endogenous target protein

A new Ca2+-binding protein, called CaVP, has been detected in muscle of the cephalochordate amphioxus and purified to electrophoretic homogeneity. The Mr 18,000 protein (pI = 4.9) binds 2 Ca2+ atoms in a noncooperative way with an intrinsic binding constant of 8.2 X 10(6) M-1. Ca2+, but not Mg2+, induces a 10% increase in alpha-helical content in the metal-free protein. CaVP does not interact with chlorpromazine, but forms a Ca2+-dependent complex with melittin. In situ, CaVP forms a high affinity Ca2+-dependent complex with an Mr 36,000 protein present in muscle extracts of amphioxus. This complex has been purified by gel filtration and ion exchange chromatography, and the target protein further purified after dissociation of the complex in the presence of Ca2+-chelating agents and 6 M urea. The nearly pure Mr 36,000 protein also forms a Ca2+-dependent complex with calmodulin which, however, is less stable during electrophoresis than the CaVP-Mr 36,000 protein complex. Amphioxus CaVP does not substitute for calmodulin in a specific enzyme assay nor for troponin C in restoring Ca2+ sensitivity to skinned muscle fibers. Its polyclonal antibody does not cross-react with the latter two activators. No immunological cross-reacting counterpart of CaVP was found in organs of fish and rat. Its relative abundance in amphioxus muscle indicates that CaVP must underlie an important new limb of Ca2+ regulation in this particular muscle.     

von Willebrand factor. A reduced and alkylated 52/48-kDa fragment beginning at amino acid residue 449 contains the domain interacting with platelet glycoprotein Ib

We have purified a reduced and alkylated tryptic fragment of von Willebrand factor (vWF) which migrated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a 52/48-kDa doublet, but behaved as a single 46-kDa species after partial deglycosylation. After extensive treatment with denaturants, the 52/48-kDa polypeptide retained its ability to inhibit ristocetin-induced platelet aggregation in the presence of native vWF, as well as aggregation induced by desialylated vWF alone. Therefore, the 52/48-kDa polypeptide interacts with the platelet glycoprotein Ib receptor even in the absence of ristocetin. Both the 52/48- and the 46-kDa species inhibited ristocetin-induced binding of the intact molecule to platelets, but did not affect thrombin-induced binding. Determination of the NH2-terminal sequence of both members of the doublet gave identical results: VTLNPSDPEHCQ. This provided additional evidence that differences between the doublet constituents were only of carbohydrate composition and established the position of this peptide within the vWF polypeptide chain of approximately 2050 amino acid residues as beginning with the residue tentatively designated 449. These studies suggest that native conformation is not necessary for binding of vWF to platelets at the glycoprotein Ib receptor and that a linear amino acid sequence following residue 449 defines a domain responsible for this interaction.     

Complementation between uncF alleles affecting assembly of the F1F0-ATPase complex of Escherichia coli.

A mutant affected in the b subunit (coded by the uncF gene) of the F1F0-ATPase in Escherichia coli was isolated by a localized mutagenesis procedure in which a plasmid carrying the unc genes was mutagenized in vivo. The biochemical properties of cells carrying the uncF515 allele were examined in a strain carrying the allele on a multicopy plasmid and a mutator-induced polar unc mutation on the chromosome. The strain carrying the mutant unc allele was uncoupled with respect to oxidative phosphorylation. Membrane-bound ATPase activity was very low or absent, and membranes were somewhat proton permeable. It was concluded that the F0 sector was assembled. Determination of the DNA sequence of the uncF515 allele showed it differed from wild type in that a G----A substitution occurred at position 392, resulting in glycine being replaced by aspartate at position 131. Genetic complementation tests indicated that the uncF515 allele complemented the uncF476 allele (Gly 9----Asp). Two-dimensional gel electrophoresis of membrane preparations indicated that the uncF515 and uncF476 alleles interrupted assembly of the F1F0-ATPase at different stages.     

Effects of combined administration ofl-tryptophan and tricyclic antidepressants on α2- and β-adrenoceptors and monoamine levels in rat brain

In order to test whether co-administration of a serotonin precursor with antidepressant drugs could potentiate the effects of the antidepressants on monoamines or adrenoceptors in rat brain,l-tryptophan (20 mg/kg) was administered to rats daily for 7 or 15 days, either alone or in combination with desipramine (10 mg/ kg) or amitriptyline (10 mg/kg). After treatment withl-tryptophan for 7 days, increases were observed in rat hypothalamic and frontal cortex 5-hydroxy-3-indoleacetic acid levels as well as in hypothalamic dopamine and nucleus accumbens 3,4-dihydroxyphenylacetic acid levels. After 15 days, hippocampal -adrenoceptor density was found to be decreased. There was no evidence of potentiation of desipramine or amitriptyline action whenl-tryptophan was administered in combination with the antidepressants. On the contrary, the antidepressants appeared to interact withl-tryptophan to reduce its effects.     

Effects of phorbol myristate acetate,phorbol dibutyrate,ethanol, dimethylsulfoxide,phenol, and seven metabolities of phenol on metabolic cooperation between chinese hamster v79 lung fibroblasts

The effect of phorbol myristate acetate, phorbol dibutyrate, ethanol, dimethylsulfoxide, phenol, and seven metabolites of phenol on metabolic cooperation were assessed as a function of mutant cell recovery from populations of cocultivated hypoxanthine-guanine phosphoribosyl transferase-deficient mutant (HGPRT–) and wild-type (HGPRT+) Chinese hamster V79 lung fibroblasts. Phorbol myristate acetate and phorbol diputyrate, two established tumor promoters, were potent inhibitors of metabolic cooperation. Ethanol and dimethylsulfoxide, solvents commonly used to prepare chemicals for testing, weakly inhibited metabolic cooperation. Phenol and phenylglucuronide had no effect on metabolic cooperation. Four oxidative metabolites (1,4-benzoquinone, catechol, hydroxyquinol and quinol) inhibited metabolic cooperation. Phenylsulfate weakly inhibited metabolic cooperation. Conversely, 2-methoxyphenol, a methylated derivative of catechol, appeared to enhance metabolic cooperation. These results generallyAbbreviations CAS Chemical Abstracts Service - DMSO dimethylsulfoxide - ETOH ethanol - HGPRT hypoxanthine-guanine phosphoribosyl transferase - HGPRT+ HGPRT-competent - HGPRT– HGPRT-te]deficient - MC metabolic cooperation - MC+ metabolic cooperation-competent - MC– metabolic cooperation-deficient - MEM minimum essential medium - PDBu phorbol dibutyrate - PMA phorbol myristate acetate - 6TG 6-thioguanine - 6TG^r 6-thioguanine-resistant - 6TG^s 6-thioguanine-sensitive - V79/MC assay Chinese hamster V79 lung fibroblast assay for metabolic cooperation     

Light-scattering investigation of the subunit structure and dissociation of Helix pomatia hemocyanin. Effects of salts and ureas

The effects of various salts of the Hofmeister and aliphatic acid salt series and hydrophobic reagents of the urea series on the subunit structure and the dissociation of Helix pomatia alpha-hemocyanin were investigated by employing light-scattering molecular weight methods. In moderate ranges of salt concentrations [0-1.0 M NaClO4, NaSCN, NaI, and guanidinium chloride (GdmCl) and 0-2.0 M NaBr], the dissociation reaction is essentially a two-step process characterized by the dissociation of whole hemocyanin molecules dissociating to half-molecules of decamers followed by the dissociation of the half-molecules to five dimeric fragments. The effectiveness of the salts and relative ineffectiveness of the ureas and GdmCl as dissociating agents in the first step of the dissociation reaction suggest that the stabilization of the contact areas between half-molecules in solution is largely a nonhydrophobic energy process involving polar and ionic interactions. Hydrophobic forces appear to be important, however, for stabilization of the half-molecules through side to side contacts of the five dimeric units that make up each half-molecule. The analysis of our dissociation data by use of equations derived in our previous studies [Herskovits, T. T., & Harrington, J.P. (1975) Biochemistry 14, 4964-4971] gave apparent estimates of amino acid groups of about 60-150 for each of the contact areas between the cylindrically shaped half-molecules and 30-60 for each of the dimers in the half-molecules themselves.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence for specific binding of uncapacitated boar spermatozoa to porcine zonae pellucidae in vitro

Washed ejaculated boar sperm and sperm from the cauda epididymis bind to the zona pellucida of fixed porcine eggs in large numbers. Sperm incubated in the presence of dextran sulfate (8 K daltons or 500 K daltons) or fucoidan and then washed no longer bind to eggs. Other acid carbohydrates (heparin, chondroitin sulfates, inositol hexasulfate, carboxymethylcellulose) fail to block sperm-egg binding even when added directly to sperm-egg suspensions. Seminal plasma and the seminal vesicle secretion contain basic proteins which bind tightly to sperm and bind reversibly to eggs preventing sperm from binding to eggs. When dextran sulfate or fucoidan are mixed with the vesicular secretion, from which seminal plasma basic proteins originate (Hunt et al., '83), the secretion loses the capacity to prevent sperm from binding to eggs; this suggests that seminal vesicle proteins can bind to the same site on zonae as do sperm and thus seminal plasma may modify sperm-egg interactions. Corpus and cauda epididymal sperm also bind in large numbers to the zona pellucida of isolated eggs but high concentrations of caput sperm, which exhibit high motility in the presence of caffeine, bind only in few numbers. Thus a component that enhances sperm-zona binding is apparently formed on the plasma membranes of uncapacitated sperm during passage through the epididymis. This finding, and an earlier observation that antibodies raised against uncapacitated sperm plasma membranes block sperm-egg binding in vivo (Peterson et al., '83) suggest that this component may be involved in sperm zona interaction in vivo.