Na+ and K+ transport at basolateral membranes of epithelial cells. II. K+ efflux and stoichiometry of the Na,K-ATPase

Changes of 42K efflux (J23K) caused by ouabain and/or furosemide were measured in isolated epithelia of frog skin. From the kinetics of 42K influx (J32K) studied first over 8-9 h, K+ appeared to be distributed into readily and poorly exchangeable cellular pools of K+. The readily exchangeable pool of K+ was increased by amiloride and decreased by ouabain and/or K+-free extracellular Ringer solution. 42K efflux studies were carried out with tissues shortcircuited in chambers. Ouabain caused an immediate (less than 1 min) increase of the 42K efflux to approximately 174% of control in tissues incubated either in SO4-Ringer solution or in Cl-Ringer solution containing furosemide. Whereas furosemide had no effect on J23K in control tissues bathed in Cl-rich or Cl-free solutions, ouabain induced a furosemide-inhibitable and time-dependent increase of a neutral Cl-dependent component of the J23K. Electroconductive K+ transport occurred via a single-filing K+ channel with an n' of 2.9 K+ efflux before ouabain, normalized to post-ouabain (+/- furosemide) values of short-circuit current, averaged 8-10 microA/cm2. In agreement with the conclusions of the preceding article, the macroscopic stoichiometry of ouabain-inhibitable Na+/K+ exchange by the pump was variable, ranging between 1.7 and 7.2. With increasing rates of transepithelial Na+ transport, pump-mediated K+ influx saturated, whereas Na+ efflux continued to increase with increases of pump current. In the usual range of transepithelial Na+ transport, regulation of Na+ transport occurs via changes of pump-mediated Na+ efflux, with no obligatory coupling to pump-mediated K+ influx.     

In vitro induction of suppressor cells by plasma of rats with Trypanosoma brucei rhodesiense infection

Mitogenic responses of B and T lymphocytes from spleens of rats infected with Trypanosoma brucei rhodesiense were suppressed. Plasma from infected rats suppressed the mitogenic responses of B and T lymphocytes from spleens of normal uninfected rats. Removal of immune complexes from plasma of infected rats significantly reduced the suppressive effect of the plasma on splenic lymphocytes of normal uninfected rats. Normal thymus cells treated with plasma from infected rats and added to cultures of normal spleen lymphocytes inhibited the mitogenic responses of B and T lymphocytes. We suggest that the interaction of immune complexes and Fc or C3b receptors of T lymphocytes resulted in the in vitro induction or activation of T suppressor lymphocytes.     

Cloning and expression of a tylosin resistance gene from a tylosin-producing strain of Streptomyces fradiae

Summary A gene conferring high-level resistance to tylosin in Streptomyces lividans and Streptomyces griseofuscus was cloned from a tylosin-producing strain of Streptomyces fradiae. The tylosin-resistance (Tyl^r) gene (tlrA) was isolated on five overlapping DNA fragments which contained a common 2.6 Kb KpnI fragment. The KpnI fragment contained all of the information required for the expression of the Tyl^r phenotype in S. lividans and S. griseofuscus. Southern hybridization indicated that the sequence conferring tylosin resistance was present on the same 5 kb SalI fragment in genomic DNA from S. fradiae and several tylosin-sensitive (Tyl^s) mutants. The cloned tlrA gene failed to restore tylosin resistance in two Tyl^s mutants derived by protoplast formation and regeneration, and it restored partial resistance in a Tyl^s mutant obtained by N-methyl-N-nitro-N-nitrosoguanidine (MNNG) mutagenesis. The tlrA gene conferred resistance to tylosin, carbomycin, niddamycin, vernamycin-B and, to some degree, lincomycin in S. griseofuscus, but it had no effect on sensitivity to streptomycin or spectinomycin, suggesting that the cloned gene is an MLS (macrolide, lincosamide, streptogramin-B)-resistance gene. Twenty-eight kb of S. fradiae DNA surrounding the tlrA gene was isolated from a genomic library in bacteriophage Charon 4. Introduction of these DNA sequence into S. fradiae mutants blocked at different steps in tylosin biosynthesis failed to restore tylosin production, suggesting that the cloned Tyl^r gene is not closely linked to tylosin biosynthetic genes.     

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.     

Subunit structure of the dihydrolipoyl transacylase component of branched-chain alpha-keto acid dehydrogenase complex from bovine liver. Mapping of the lipoyl-bearing domain by limited proteolysis

To characterize the lipoyl-bearing domain of the dihydrolipoyl transacylase (E2) component, purified branched-chain alpha-keto acid dehydrogenase complex from bovine liver was reductively acylated with [U-14C] alpha-ketoisovalerate in the presence of thiamin pyrophosphate and N-ethylmaleimide. Digestion of the modified complex with increasing concentrations of trypsin sequentially cleaved the E2 polypeptide chain (Mr = 52,000) into five radiolabeled lipoyl-containing fragments in the order of L1 (Mr = 28,000), L2 (Mr = 24,500), L3 (Mr = 21,000), L4 (Mr = 15,000) to L5 (Mr = 14,000) as determined by the autoradiography of sodium dodecyl sulfate-polyacrylamide gel. In addition, a lipoate-free inner E2 core consisting of fragment A (Mr = 26,000) and fragment B (Mr = 22,000) was produced. Fragment A contains the active site for transacylation reaction and fragment B is the subunit-binding domain. Fragment L5 and fragment B were stable and resistant to further tryptic digestion. Mouse antiserum against E2 reacted only with fragments L1, L2, and L3, and did not bind fragments L4, L5, A, and B as judged by immunoblotting analysis. The anti-E2 serum strongly inhibited the overall reaction catalyzed by the complex, but was without effect on the transacylation activity of E2. Measurement of incorporation of [1-14C]isobutyryl groups into the E2 subunit indicated the presence of 1 lipoyl residue/E2 chain. Based on the above data, a model is proposed in which the lipoyl-bearing domain is connected to the inner E2 core via a trypsin-sensitive hinge. The lipoyl-bearing domain contains five consecutive tryptic sites (L1 to L5), with the L1 site in the hinge region, and the L5 site next to the terminal lipoyl-binding sequence. An exposed and antigenic region is located between L1 and L4 tryptic sites of the lipoyl-bearing domain. The region accounts for about 24% of the E2 chain length. Binding of antibodies to this region probably impairs the mobility of the lipoyl-containing polypeptide, resulting in an interruption of the active-site interactions that are necessary for the overall reaction. The lack of antigenicity and resistance to tryptic digestion indicate a highly folded conformation for fragment L5, the limit polypeptide carrying the single lipoyl residue.     

Directionality in FLP protein-promoted site-specific recombination is mediated by DNA-DNA pairing

The 2 mu plasmid of the yeast Saccharomyces cerevisiae encodes a site-specific recombination system consisting of plasmid-encoded FLP protein and two recombination sites on the plasmid. The recombination site possesses a specific orientation, which is determined by an asymmetric 8-base pair spacer sequence separating two 13-base pair inverted repeats. The outcome or directionality of site-specific recombination is defined by the alignment of two sites in the same orientation during the reaction. Sites containing point mutations or 1-base pair insertions or deletions within the spacer generally undergo recombination with unaltered sites at reduced levels. In contrast, recombination between the two identical mutant sites (where homology is restored) proceeds efficiently in all cases. Sites containing spacer sequences of 10 base pairs or more are nonfunctional under all conditions. A recombination site in which 5 base pairs are changed to yield an entirely symmetrical spacer sequence again recombines efficiently, but only with an identical site. This reaction, in addition, produces a variety of new products which can only result from random alignment of the two sites undergoing recombination, i.e. the reaction no longer exhibits directionality. These and other results demonstrate that both the efficiency and directionality of site-specific recombination is dependent upon homology between spacer sequences of the two recombining sites. This further implies that critical DNA-DNA interactions between the spacer region of the two sites involved in the reaction occur at some stage during site-specific recombination in this system. The specific spacer sequence itself appears to be unimportant as long as homology is maintained; thus, these sequences are probably not involved in recognition by FLP protein.     

Hydroxylation of biphenyl by Aspergillus parasiticus: Approaches to yield improvment in fermenter cultures

We carried out experiments designed to increase the rate of production of 4,4'-dihydroxybiphenyl (biphenol) from biphenyl by Aspergillus parasiticus. We show that 0.5 mg/ml biphenyl, the substrate for the reaction, significantly inhibits growth of the organism and that at 0.04 mg/ml, 2-hydroxybiphenyl or 4-hydroxybiphenyl (an intermediate of the reaction) strongly inhibit oxygen uptake, probably by inhibition of mitochondrial electron transport. Both factors may contribute to the low hydroxylation rates observed previously [J. H. Golbeck and J. C. Cox, Biotechnol. Bioeng., 26, 434 (1984)]. We therefore adapted the organism to the presence of 0.08 mg/ml 2- and 4-hydroxybiphenyl in the growth medium and found that cultures of adapted strains hydroxylated biphenyl at rates ca. three-fold faster than control cultures. Once the fungal mycelia were grown, they could be recycled at least twice into fresh fermentation broth. Recycled organisms were capable of hydroxylating biphenyl more rapidly than cells in the primary fermentation culture and there was no lag period between introduction of biphenyl and the onset of hydroxylation. Cell recycle thus results in a considerable saving in carbon costs and fermentation time.