Kinetics and regulation of the ankyrin-band 3 interaction of the human red blood cell membrane

In an attempt to identify potential regulatory mechanisms for erythrocyte membrane-cytoskeletal interactions, the kinetics and pH dependence of the band 3-ankyrin interaction were investigated. Association of 125I-ankyrin with KI-stripped inside-out erythrocyte membrane vesicles was found to proceed in two kinetic phases. The initial, fast phase (t1/2 approximately 15-30 min) involved predominantly the binding of ankyrin to low affinity sites (KD approximately 130 nM) in a pH-dependent manner. The apparent pKa values describing this reversible pH dependence (7.2 +/- 0.1 and 9.2 +/- 0.1) defined states of band 3 with high, moderate, and no capacity to bind ankyrin (in order of increasing pH). Since the cytoplasmic domain of band 3 also exists in 3 distinct conformational states characterized by apparent pKa values of 7.2 and 9.2, it was hypothesized that the reversible structural equilibrium in band 3 could influence ankyrin binding. The second or slow phase of ankyrin binding to band 3 involved the conversion of low to high affinity sites (KD approximately 13 nM). This phase, which was largely temperature and pH independent, required roughly an order of magnitude longer to reach completion than the fast phase. Unfortunately, even though the slow phase could be cleanly separated from the fast phase at low pH, insufficient data were available to formulate a physical interpretation of its origin. Significantly, however, even after completion of the slow phase under the most quantitative binding conditions identified, a maximum of only 26% of the band 3 was found to bind ankyrin in situ. Although higher ankyrin-band 3 stoichiometries may be achievable with the isolated cytoplasmic fragment of band 3, we interpret the above 1:4 stoichiometry to suggest that the tetramer of band 3 constitutes the predominant ankyrin binding oligomer of band 3 on the membrane.     

Identification of DNA topoisomerase-II activity in terminally differentiated mammalian organs and in non-growing cultured cells

DNA topoisomerase-II activity was measured in a variety of rat organs and in two types of cultured mammalian cells at different stages of growth. The assay for enzyme activity is based on the ability of DNA topoisomerase II to catenate relaxed, circular double-stranded [3H]DNA into huge networks of interlocked circles which can be selectively trapped on a nitrocellulose filter. This catenation requires ATP and provides a sensitive, specific, and quantitative way to measure topoisomerase-II activity in crude extracts of nuclei. The level of type-II topoisomerase activity showed little variation at different stages of growth in either Chinese hamster ovary cells or human skin fibroblasts. In both cell types, growth-arrested cells contain levels of topoisomerase II very similar to those seen in actively growing cells. In addition, substantial levels of type-II topoisomerase are found not only in those rat organs expected to contain large populations of growing cells (testis, spleen), but also in organs composed primarily of cells in G0 (brain, liver, lung). These data indicate that total nuclear type-II topoisomerase activity does not vary dramatically with the state of cell growth or degree of cell differentiation.     

Control of cell volume in the J774 macrophage by microtubule disassembly and cyclic AMP

We have explored the possibilities that cell volume is regulated by the status of microtubule assembly and cyclic AMP metabolism and may be coordinated with shape change. Treatment of J774.2 mouse macrophages with colchicine caused rapid microtubule disassembly and was associated with a striking increase (from 15-20 to more than 90 percent) in the proportion of cells with a large protuberance at one pole. This provided a simple experimental system in which shape changes occurred in virtually an entire cell population in suspension. Parallel changes in cell volume could then be quantified by isotope dilution techniques. We found that the shape change caused by colchicine was accompanied by a decrease in cell volume of approximately 20 percent. Nocodozole, but not lumicolchicine, caused identical changes in both cell shape and cell volume. The volume loss was not due to cell lysis nor to inhibition of pinocytosis. The mechanism of volume loss was also examined. Colchicine induced a small but reproducible increase in activity of the ouabain-sensitive Na(+), K(+)-dependent ATPase. However, inhibition of this enzyme/transport system by ouabain did not change cell volume nor did it block the colchicines-induced decrease in volume. One the other hand, SITS (4’acetamido, 4-isothiocyano 2,2’ disulfonic acid stilbene), an inhibitor of anion transport, inhibited the effects of colchicines, thus suggesting a role for an anion transport system in cell volume regulation. Because colchicine is known to activate adenylate cyclase in several systems and because cell shape changes are often induced by hormones that elevate cyclic AMP, we also examined the effects of cyclic AMP on cell volume. Agents that act to increase syclic AMP (cholera toxin, which activates adenylate cyclase; IBMX, and inhibitor of phosphodiesterase; and dibutyryl cyclic AMP) all caused a volume decrease comparable to that of colchicine. To define the effective metabolic pathway, we studied two mutants of J774.2, one deficient in adenylate cyclase and the other exhibiting markedly reduced activity of cyclic AMP-dependent protein kinase. Cholera toxin did not produce a volume change in either mutant. Cyclic AMP produced a decrease in the cyclase-deficient line comparable to that in wild type, but did not cause a volume change in the kinase- deficient line. This analysis established separate roles for cyclic AMP and colchicine. The volume decrease induced by cyclic AMP requires the action of a cyclic AMP-dependent protein kinase. Colchicine, on the other hand, induced a comparable volume change in both mutants and wild type, and thus does not require the kinase.     

Specialized transduction with lambda plac5: dependence on recA and on configuration of lac and att lambda.

The construction of lambda plac5 transducing phages carrying various lacZ alleles is described. Genetically disabled (N- N- P-) lambda plac transducing the phages were used to study the dependence of specialized transduction on host RecA function and on the location of the lacZ gene in the recipient strain. In the absence of site-specific recombination at att lambda, transduction was completely dependent on host RecA function. Regardless of the configuration of att lambda, lambda plac transducing phages recombined at a 20- to 50-fold higher frequency with F42 lac than with a lac gene located in the cellular chromosome. Deletion mutants of lacZ in the recipient strain were used to show that the probability of lac recombination resulting from lambda plac infection is apparently proportional to the amount of homology between the parental lacZ genes.     

The effect of anesthetic charge on anesthetic-phospholipid interactions

Cationic and uncharged forms of a tertiary amine local anesthetic are reported to have different properties and potencies as nerve blocking agents. However, the relative capacities of each form of the local anesthetic to perturb the properties of different model membrane systems is unknown. For this reason we have studied the effects of uncharged lidocaine (high pH) and its quaternary amine analogue (W49091) on the phase transition properties of DMPS, DPPE and DPPC liposomes using high-sensitivity differential scanning calorimetry. We report that neutral lidocaine interacts similarly with all three phospholipids. This interaction results in a decrease in the temperature of the gel å liquid crystalline phase transition ($\text{T}{}_{\mathrm{<mtext>m</mtext>}}$), an increase in the enthalpy of the transition ($\text{ΔH}$), and a slight decrease in the cooperativity of melting. Quaternary lidocaine (W49091), on the other hand, interacts significantly with only DMPS; the result being again a decrease in the temperature of DMPS melting, an increase in $\text{ΔH}$, and a slight decrease in the cooperativity of the phase transition. These results are interpreted to indicate that uncharged lidocaine enters the membrane during the DPPE and DPPC phase transitions. In the case of DMPS, an influx of both charged forms of lidocaine must occur at $\text{T}{}_{\mathrm{<mtext>m</mtext>}}$. These anesthetic fluxes at the lipid's phase transition are suggested to be responsible for the observed elevated enthalpies of the respective transitions. The observation that the cationic form of lidocaine does not significantly modify the behavior of DPPC and DPPE liposomes suggests that these lipids are not important components of the anesthetic's site in nerve membranes. However, the dramatic perturbation of the properties of DMPS by W49091 suggests that phosphatidylserine may comprise part of this inhibitory site.     

The toxin-agglutinin fold. A new group of small protein structures organized around a four-disulfide core

The three-dimensional structures of the snake venom postsynaptic neurotoxins and of the domains in wheat germ agglutinin show a remarkably similar overall folding pattern, consisting of equivalently placed, but variably sized loops which are held together by four similarly positioned disulfide bonds. Furthermore, occurrence of this wheat germ agglutinin-neurotoxin domain fold is predicted not only in the snake venom cardiotoxins and cytotoxins with neurotoxin-matched half-cystine sequence positions, but also for two small plant proteins, hevein and ragweed pollen allergen Ra5, on the basis of a nearly exact match of their half-cystine, sequence positions with those of the wheat germ agglutinin domain.     

Regulation of the biosynthesis of aminoacyl-transfer ribonucleic acid synthetases and of transfer ribonucleic acid in Escherichia coli. V. Mutants with increased levels of valyl-transfer ribonucleic acid synthetase.

Spontaneous revertants of a temperature-sensitive Escherichia coli strain harboring a thermolabile valyl-transfer ribonucleic acid (tRNA) synthetase were selected for growth at 40 degrees C. Of these, a large number still contain the thermolabile valyl-tRNA synthetase. Three of these revertants contained an increased level of the thermolabile enzyme. The genetic locus, valX, responsible for the enzyme overproduction, is adjacent to the structural gene, valS, of valyl-tRNA synthetase. Determination (by radioimmunoassay) of the turnover rates of valyl-tRNA synthetase showed that the increased level of valyl-tRNA synthetase is due to new enzyme synthesis rather than decreased rates of protein degradation.     

Specific release of plasma membrane enzymes by a phosphatidylinositol-specific phospholipase C.

The release of plasma membrane ecto-enzymes by a phosphatidylinositol-specific phospholipase C from Staphylococcus aureus was investigated. There was no effect on L-leucyl-beta-naphthylamidase, alkaline phosphodeisterase I and Ca2+- or MG2+-ATPase, but substantial proportions of the alkaline phosphatase and 5-nucleotidase were released. There was no simultaneous release of phospholipid and the solubilized enzymes were not exluded from Sepharose 6-B. It was therefore concluded that release was not a secondary consequence of membrane vesiculation but occurred as a result of the disruption of specific interactions involving the phosphatidylinositol molecule.     

Morphological and histochemical evidence of mitoribosomes in Manduca sexta.

The mitochondria found in the neurons of the frontal ganglion of Manduca sexta contained numerous mitoribosomes. The mitochondria of the glial and perineural cells did not contain mitoribosomes. The mitoribosomes were digested in RNase whereas phospholipase C digested the cellular membranes but had no effect on the mitoribosomes.