An electrogenic proton pump in plasma membranes from the cellular slime mould Dictyostelium discoideum

Plants and fungi possess an outwardly directed plasma membrane proton pump that may regulate intracellular pH. We provide the first demonstration that amoebae of the slime mould Dictyostelium discoideum also possess a similar proton pump. It can be assayed either as an ATPase activity in highly purified plasma membranes or as a proton pump, after solubilization and reconstruction into liposomes. The pump is inhibited by vanadate, diethylstilbestrol (DES) and miconazole but not by azide or ouabain. The proton pump described here may represent the target for the action of DES and miconazole, both of which have previously been shown to induce stalk cell formation during the in vitro development of Dictyostelium.     

Conservation of interphase chromatin nonhistone antigens as components of metaphase chromosomes

The degree of conservation of HeLa interphase chromatin nonhistone antigens among the nonhistones of isolated metaphase chromosomes was determined with immunological procedures. Proteins were separated on SDS-polyacrylamide gels and electrophoretically transferred to diazophenylthioether (DPT)-paper, which was then overlaid with antiserum to chromatin from interphase nuclei. The bound antibodies were detected with 125I-labeled protein A. Alternatively, polyacrylamide gels were directly overlaid with antiserum and with 125I-protein A. Densitometry of autoradiograms and stained gels revealed the degree of conservation of nonhistone antigenic determinants from interphase to metaphase to be over 90% for chromatin.     

Purification and properties of a plasmid-encoded 2,4-dichlorophenol hydroxylase

2,4-Dichlorophenol hydroxylase, an enzyme involved in the bacterial degradation of the herbicide 2,4-dichlorophenoxyacetate (2,4-D) was purified from two bacterial strains that harbored the same 2,4-D plasmid, pJP4. The purified enzymes (Mr 224 000) from the two transconjugants were indistinguishable; they contained FAD and were composed of non-identical subunits, Mr 67 000 and 45 000, respectively. Various substituted phenols were hydroxylated, using either NADH or NADPH. The amino acid composition of the native enzyme was determined.     

Regulatory properties of rabbit red blood cell hexokinase at conditions close to physiological

The true level of hexokinase in rabbit erythrocytes was determined by three different methods, including the spectrophotometric glucose-6-phosphate dehydrogenase coupled assay and a new radioisotopic assay. The value found at 37°C (pH 7.2) was 10.23±1.90 μmol/h per ml red blood cells, which is lower than previously reported values. More than 40 cellular components of the rabbit erythrocytes were tested for their effects on the enzyme. Their intracellular concentrations were also determined. Several of these compounds were found to be competitive inhibitors of the enzyme with respect to Mg·ATP^2?. Furthermore, reduced glutathione at a concentration of 1 mM was able to maintain hexokinase in the reduced state with full catalytic activity. The ability of orthophosphate to remove the inhibition of some phosphorylated compounds was examined under conditions similar to cellular (pH 7.2 and 50 μM of orthophosphate) and found to be of no practical interest. In contrast, the binding of ATP^4? and 2,3-diphosphoglycerate to the rabbit hemoglobin significantly modifies their intracellular concentrations and the formation of the respective Mg complexes. The pH-dependence of the reaction velocity and of the kinetic properties of the enzyme in different buffer systems were also considered. This information was computerized, and the rate of glucose phosphorylation in the presence of the mentioned compounds was determined. The value obtained, 1.94±0.02 μmol/h per ml red blood cells, is practically identical to the measured rate of glucose utilization by intact rabbit erythrocytes (1.92±0.3 μmol/h per ml red blood cells). These results provide further evidence for the central role of hexokinase in the regulation of red blood cell glycolysis.     

ATP and the processivity of Xenopus laevis DNA polymerase α

We use specific restriction fragments as defined primers for DNA synthesis on single-stranded circular phage fd DNA. These structures are relatively poor templates for a highly purified DNA polymerase α from Xenopus laevis eggs. However, DNA synthesis is stimulated about 5-fold by addition of ATP to the reaction mixture. We show that the deoxynucleotide polymers, synthesized in the presence of ATP, are significantly longer than those produced in the absence of ATP. We also show that this effect is due to a more tenacious binding of DNA polymerase α to DNA and conclude that ATP increases the processivity of the enzyme.     

Resolution of multiple fluorescence lifetimes in heterogeneous systems by phase-modulation fluorometry

Procedures are described for the treatment of phase and modulation lifetime data in fluorescent systems having multiexponential decay. All computer procedures (called FIT programs) arise from the lifetime resolution theory for phase-modulation measurements (Weber, G (1981) J. Phys. Chem. 85, 949–953). The programs most successful in resolving heterogeneous lifetimes use a Monte Carlo approach in which phase and modulation lifetime data at three modulation frequencies are simultaneously utilized. These programs are shown to have more utility than the final closed form procedure presented by Weber (1981). The FIT routines are simple and require little computer time while yielding excellent results. To illustrate the applicability of these programs, defined binary (carbazole and pyrene) and ternary systems (carbazole, pyrene and POPOD) were examined. In most cases, the resolved lifetimes were within 5% of the independently measured value and the fractional fluorescence contributions were within 10% of that expected. These results demonstrate that phase-modulation measurements analyzed by appropriate computer programs are capable of solving for lifetimes in both binary and, in selected cases, ternary systems. An example is given from the recent literature (Dalbey, R., Weiel, J. and Yount, R.G. (1983) Biochemistry 22, 4696–4706) in which the above programs allowed the resolution of both binary and ternary lifetimes of a dansyl label on myosin, where Förster energy transfer was occuring. These lifetimes] were used to quantify changes in distances between two activity-related thiols on myosin upon the addition of Mg-ATP or its analogs.     

Regulation of human leukocyte microsomal hydroxymethylglutaryl-CoA reductase activity by a phosphorylation and dephosphorylation mechanism

The activity of microsomal 3-hydroxy-3-methylglutaryl coenzyme A reductase (EC 1.1.1.34), obtained from cultured human IM-9 lymphoid cells or freshly isolated human peripheral blood leukocytes, is modulated by a phosphorylation/dephosphorylation mechanism. Addition of MgATP + ADP to IM-9 cell microsomal reductase leads to a time-dependent loss of enzyme activity. Inactivated reductase is reactivated by rat liver reductase phosphatase. Kinase-dependent IM-9 cell microsomal reductase, prepared by heating IM-9 microsomes for 15 min at 50°C, is inactivated in the presence of MgATP and ADP only after addition of cytosolic reductase kinase from either IM-9 cells, freshly isolated leukocytes or rat liver. Inactivation is time-dependent and dependent on the cytosolic protein concentration. Inactivated reductase is reactivated by rat liver reductase phosphatase. For cultured IM-9 cells and freshly isolated leukocytes incubated with culture medium for 2 h, the ratios of active (unphosphorylated) to total (phosphorylated + unphosphorylated) reductase activity are 0.22 and 0.43, respectively. Thus, in addition to its regulation by changes in the amount of total enzyme protein, human leukocyte reductase activity is also modulated by a phosphorylation/dephosphorylation mechanism.     

Diacytosis of 125I-asialoorosomucoid by rat hepatocytesa. A non-lysosomal pathway insensitive to inhibition by inhibitors of ligand degradation

Diacytosis of ¹²⁵I-asialoorosomucoid by rat hepatocytes was studied by preincubating the cells with the labelled ligand at 37°C for 30 min or 18°C for 2 h, washing free of cell surface receptor-bound tracer at 4°C and then reincubating at 37°C. The cells preloaded at 37°C released a maximum of 18% of the total intracellular ligand as undegraded molecules after 1 h of incubation with an apparent first-order rate constant of 0.018 min^?1 ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 39}\text{min}$). When the preloaded cells were incubated in the presence of 100 μg/ml unlabelled asialoorosomucoid or 5 mM ethylene glycol bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid, the amount of the released ligand increased to 32 and 37%, respectively, without apparent change in kinetics, indicating that these agents prevented rebinding of the released ligand. In the presence of 5 μM colchicine, 20 μM cytochalasin B, 20 μM chloroquine, 10 mM NH₄Cl, 10 μM monensin or 20 μM leupeptin, degradation of the preloaded ligand was inhibited, whereas the release of the ligand was either slightly increased or unchanged. Similar effects of leupeptin, colchicine and asialoocrosomucoid were observed with cells preloaded at 18°C. These results indicate that diacytosis of ¹²⁵I-asialoorosomucoid occurs from a prelysosomal compartment via a route insensitive to inhibition by the inhibitors of ligand degradation.     

Callose deposition during gravitropism ofZea mays andPisum sativum and its inhibition by 2-deoxy-D-glucose

In etiolated corn (Zea mays L.) and etiolated pea (Pisum sativum L.) seedlings, a gravitropic stimulation induces the deposition of callose. In the corn coleoptiles this occurs within 5 min of gravity stimulation, and prior to the beginning of curvature. Both gravitropic curvature and callose deposition reach their maxima by 12 h. Within the first 2 h more callose is deposited on the upper (concave) side, but after 2–3 h, this deposition pattern is reversed. An inhibitor of protein glycosylation, 2-deoxy-d-glucose (DDG), inhibits callose production and considerably retards gravitropic bending in both species of plants. Mannose can relieve the inhibition of gravitropic bending by DDG. The pea mutant Ageotropum, which does not respond to gravity when etiolated, also fails to produce callose in response to a gravitic stimulus. These correlations indicate that callose deposition may be a biochemical component of gravitropism in plant shoots.Abbreviation DDG 2-deoxy-d-glucose     

Sulphides and furanones from steam volatile oils of Allium fistulosum and Allium chinense

The constituents of the steam volatile oils from two kinds of Allium fistulosum, A. fistulosum var. caespitosum and A. chinense, have been investigated by GC and spectral techniques (IR, UV, GC/MS, ¹H NMR and ¹³C NMR). The compounds identified from the neutral fraction of each volatile oil included sulphides, thiolanes, alcohols, aldehydes, ketones, furanones and others. Among the sulphur compounds, dipropyl disulphide comprised ca 28% of A. fistulosum oil, ca 23% of A. fistulosum var. caespitosum oil and ca 30% of A. chinense oil. A. fistulosum oil was characterized by a large quantity of tridecan-2-one (ca 52%) and 2,3-dihydro-2-octyl-5-methylfuran-3-one (ca 16%). Also, a large amount of 2,3-dihydro-2-hexyl-5-methylfuran-3-one (ca 20%) was isolated from A. chinense oil.