The native F0F1-inhibitor protein complex from beef heart mitochondria and its reconstitution in liposomes

A functional F₀F₁ ATP synthase that contains the endogenous inhibitor protein (F₀F₁I) was isolated by the use of two combined techniques [Adolfsen, R., McClung, J.A., and Moudrianakis, E. N. (1975).Biochemistry 14, 1727–1735; Dreyfus, G., Celis, H., and Ramirez, J. (1984).Anal. Biochem. 142, 215–220]. The preparation is composed of 18 subunits as judged by SDS-PAGE. A steady-state kinetic analysis of the latent ATP synthase complex at various concentrations of ATP showed aV _max of 1.28mol min^–1 mg^–1, whereas theV _max of the complex without the inhibitor was 8.3mol min^–1 mg^–1. In contrast, theK _m for Mg-ATP of F₀F₁ I was 148M, comparable to theK _m value of 142M of the F₀F₁ complex devoid of IF₁. The hydrolytic activity of the F₀F₁I increased severalfold by incubation at 60C at pH 6.8, reaching a maximal ATPase activity of 9.5mol min^–1 mg^–1; at pH 9.0 a rapid increase in the specific activity of hydrolysis was followed by a sharp drop in activity. The latent ATP synthase was reconstituted into liposomes by means of a column filtration method. The proteoliposomes showed ATP-Pi exchange activity which responded to phosphate concentration and was sensitive to energy transfer inhibitors like oligomycin and the uncouplerp-trifluoromethoxyphenylhydrazone.     

Composition and properties of Albizia lebbeck gum exudate

An analytical study has been made of six gum specimens from Albizia lebbeck, Leguminosae. Galactose, mannose, arabinose, glucuronic acid and its 4-O--metyl analogue are present in all the specimens studied. Rhamnose was not detected according to sugar analysis and spectral data. The absence of this sugar, the high acidity and the relatively low limit viscosity number contrast with the values reported previously for one African sample of A. lebbeck. The lead content is much higher than that recorded for other Albizia gums. ¹³C-NMR spectrum of this gum, in deuterium oxide, shows good resolution.     

Structural variability in the genome of phage ?H of Halobacterium halobium

Summary The partially circularly permuted, terminally redundant structure of the DNA of phage H has been confirmed by a cleavage map for the restriction enzymes PstI, ClaI, BglII, HindIII, and, partially, BamHI.Six variants of phage H have been isolated from 71 single plaques. Their genomes differ by several insertions, a deletion, and an inversion of a DNA segment with a minimal length of 11 kb. The inversion occurs with high frequency in variants carrying at the flanks of the invertible DNA in verted repeats of a 1.8 kb DNA element which shares sequence homology with the DNA of H. halobium and may be involved in the extreme variability of its genome.     

Biosynthesis in Escherichia coli of sn-glycerol 3-phosphate, a precursor of phospholipid. Palmitoyl-CoA inhibition of the biosynthetic sn-glycerol-3-phosphate dehydrogenase.

Homogeneous biosynthetic sn-glycerol-3-phosphate dehydrogenase (EC 1.1.1.8) of Escherichia coli was potently inhibited by palmitoyl-CoA and other long chain acyl-CoA thioesters. The concentration dependence of this inhibition was not cooperative. Enzyme activity was inhibited 50% at 1 microM palmitoyl-CoA; thus, this inhibition occurred at concentrations below the critical micellar concentration of palmitoyl-CoA. Palmitoyl-CoA was a reversible, noncompetitive inhibitor with respect to both NADPH and dihydroxyacetone phosphate. Palmitoyl-CoA did not affect the quaternary structure of the enzyme. This inhibition could be prevented or reversed by the addition of phospholipid vesicles prepared from E. coli phospholipids. Palmitoyl-CoA did not alter the kinetics of inhibition by sn-glycerol 3-phosphate, which is a proven physiological regulator of this enzyme. Decanoyl-CoA, dodecanoyl-CoA, myristoyl-CoA, palmitoyl-(1,N6-etheno)CoA, stearoyl-CoA, and oleoyl-CoA inhibited sn-glycerol-3-phosphate dehydrogenase at concentrations below their critical micellar concentrations. Palmitate inhibited sn-glycerol-3-phosphate dehydrogenase activity 50% at 200 microM. Palmitoyl-carnitine, deoxycholate, taurocholate, and dodecyl sulfate were more potent inhibitors than Triton X-100, Tween-20, or Tween-80. Palmitoyl-acyl carrier protein at concentrations up to 50 microM had no effect on sn-glycerol-3-phosphate dehydrogenase activity. The possible physiological role of long chain fatty acyl-CoA thioesters in the regulation of sn-glycerol 3-phosphate and phospholipid biosynthesis in E. coli is discussed.     

Glucocorticoid induction of tyrosine hydroxylase in a continous cell line of rat pheochromocytoma.

We have established a continous cell line (G1) in which the tyrosine hydroxylase specific activity is increased as much as 50-100-fold in response to dexamethasone. This response is specific for the glucocorticoid class of steroid hormones; it is elicited by dexamethasone, corticosterone, and triamcinolone, but not by estradiol, testosterone, progesterone, or deoxycorticosterone acetate. The increase in tyrosine hydroxylase specific activity is likely to be due to the increased synthesis of new enzyme protein rather than an activation of existing protein molecules, inasmuch as this increase is completely blocked by cycloheximide.     

Regulation of retinol-binding protein metabolism in cultured rat liver cell lines

Retinol-binding protein (RBP), the plasma transport protein for vitamin A, is synthesized and secreted by the liver. In vitamin A deficiency, RBP secretion is blocked, leading to low serum and high liver levels of RBP. Administration of retinol to the intact rat stimulates a rapid secretion of RBP from liver into serum. We explored the use of a liver cell culture system to study the regulation of the synthesis and secretion of RBP. We found two lines of differentiated rat hepatoma cells, MH₁C₁ and H₄ II EC₃ (H₄), that synthesized RBP during culture in vitro. The net synthesis of RBP was a function of the number of cells per dish and the duration of incubation. Both cell lines synthesized RBP when incubated in Neuman and Tytell's Serumless Medium (NTS medium), while the MH₁C₁ cells also synthesized RBP in Ham's F-12 medium with added serum. A relatively large proportion (14–56%) of the RBP was retained within the cells when they were incubated in the vitamin A-free NTS medium alone. Addition of serum to NTS medium stimulated the release of RBP from the cells into the medium and also increased the net synthesis of RBP. These effects were not due to the increased adhesion of the cells to the petri dish. Addition of retinol (at levels of 0.35 or 3.5 nmole/ml) to the NTS medium resulted in the stimulation of RBP secretion from the cells into the medium and an increase in the net synthesis of RBP. By contrast, retinol had no effect on either the net synthesis or the cell-to-medium distribution of rat serum albumin. The data from these cell lines in culture suggest that retinol has a specific regulatory effect on RBP metabolism. These cells thus resemble the normal rat liver cell in vivo in regard to the known regulation of RBP metabolism.     

Different structural requirements of endotoxic glycolipid for tumor regression and endotoxic activity

Endotoxic glycolipid extracted from the heptose-less mutant of $\text{Salmonella typhimurium}$ was treated with alkali and acid reagents. The glycolipid freed of all O-ester linked fatty acids by hydroxylamine had lost tumor regression activity and toxicity, whereas a partial removal of O-ester linked fatty acids by mild alkali did not impair with these activities. The glycolipid retained both activities after removal of 2-keto-3-deoxyotonate by sodium acetate (pH 4.5) but was rendered nontoxic while retaining antitumor activity when hydrolyzed by 0.1N HCl whereby 2-keto-3-deoxyoctonate and glycosidic phosphate was split off the glycolipid molecule. Nontoxic and tumor regressive fractions were separated by means of preparative thin layer chromatography of glycolipid hydrolyzed by mild acid. Thus, it was concluded that glycosidic bound phosphate and at least a portion of fatty acids of the lipid A moiety were essential for toxicity, but that this phosphate is not essential for tumor regression activity.     

Activity feedback to the mammalian circadian pacemaker: influence on observed measures of rhythm period length.

In the mouse, activity is precisely timed by the circadian clock and is normally most intense in the early subjective night. Since vigorous activity (e.g., wheel running) is thought to induce phase shifts in rodents, the temporal placement of daily exercise/activity could be a determinant of observed circadian rhythm period. The relationship between spontaneous running-wheel activity and the circadian period of free-running rhythms was studied to assess this possibility. With ad libitum access to a running wheel, mice exhibited a free-running period (tau) of 23.43 +/- 0.08 hr (mean +/- SEM). When running wheels were locked, tau increased (23.88 +/- 0.04 hr, p less than 0.03), and restoration of ad libitum wheel running again produced a shorter period (tau = 23.56 +/- 0.06 hr, p less than 0.05). A survey of free-running activity patterns in a population of 100 mice revealed a significant correlation between the observed circadian period and the time of day in which spontaneous wheel running occurred (r = 0.7314, p less than 0.0001). Significantly shorter periods were observed when running was concentrated at the beginning of the subjective night (tau = 23.23 +/- 0.04), and longer periods were observed if mice ran late in the subjective night (tau = 23.89 +/- 0.04), F (1, 99) = 34.96, p less than 0.0001. It was previously believed that the period of the circadian clock was primarily responsive to externally imposed tonic or phasic events. Systematic influences of spontaneous exercise on tau demonstrate that physiological and/or behavioral determinants of circadian timekeeping exist as well.