Activation of ATP hydrolysis by an uncoupler in mutant mitochondria lacking an intrinsic ATPase inhibitor in yeast

An intrinsic ATPase inhibitor and 9-kDa protein are regulatory factors of mitochondrial ATP synthase in Saccharomyces cerevisiae. A gene encoding the ATPase inhibitor was isolated from a yeast genomic library with synthetic oligonucleotides as hybridization probes and was sequenced. The deduced amino acid sequence showed that the precursor protein contains an amino-terminal presequence of 22 amino acid residues. Mutant strains that did not contain the inhibitor and/or the 9-kDa protein were constructed by transformation of cells with their in vitro disrupted genes. The disruption of the chromosomal copy in recombinant cells was verified by Southern blot analysis, and the absence of the proteins in the mutant cells was confirmed by Western blot analysis. All the mutants could grow on a nonfermentable carbon source and the oxidative phosphorylation activities of their isolated mitochondria were the same as that of normal mitochondria. However, an uncoupler, carbonylcyanide-m-chlorophenylhydrazone, induced marked ATP hydrolysis in the inhibitor-deficient mitochondria, but not in normal mitochondria. These observations suggest that the ATPase inhibitor inhibits ATP hydrolysis by F1F0-ATPase only when the membrane potential is lost.     

Concanavalin A affects beta-tubulin mRNA expression during neuritic processes of mouse neuroblastoma N18TG2 cells in a different manner from colchicine

Addition of concanavalin A (Con A) to mouse neuroblastoma N18TG2 cells cultured with dibutyryl-cAMP which can stimulate neurite outgrowth, stopped the neuritic processes effectively. The extended neurites showed a gradual retraction for at least 8 hrs after addition of Con A, while addition of colchicine caused rapid retraction of the neurites. Immunocytochemistry showed that the addition of Con A did not disorganize the microtubules but the addition of colchicine did. The increase in beta-tubulin mRNA expression which was observed after cell culture and after stimulation by dB-cAMP was suppressed by the addition of Con A. Con A did not affect the beta-tubulin mRNA expression when the cells had already been cultured, while colchicine drastically decreased it. Thus, Con A appeared to affect the beta-tubulin mRNA expression in a different manner from colchicine, probably through inhibition of cell movement.     

Primary structure of the heparin-binding site of type V collagen

The abilities of collagens, type I, II, III, IV, and V, to bind heparin were examined by heparin-affinity chromatography and binding studies with [35S]heparin. At a physiological pH and ionic strength, only type V collagen bound to heparin. Collagens type I and II showed higher affinities than types III and IV for heparin, but did not bind to a heparin column at a physiological ionic strength. The heparin binding site of type V collagen was located in a 30 kDa CNBr fragment of the alpha 1(V) chain, and the amino acid sequence of this fragment was determined. The 30 kDa fragment contained a cluster of basic amino acid residues, and enzymatic cleavage within this basic domain greatly reduced the heparin-binding activities of the resulting peptides. Thus this basic region is probably the heparin-binding site of type V collagen.     

Production of tropane alkaloids in cultured cells of Hyoscyamus niger

Tests for calluses rich in tropane alkaloids were made with newly induced calluses of Atropa belladonna, Datura stramonium and Hyoscyamus niger. Only calluses of H. niger gave an alkaloid-positive test.A Hyoscyamus cell line had the highest total alkaloid content of all the calluses screened by the cell-squash alkaloid assay. Both hyoscyamine and scopolamine were identified in the cultured cells of this line by TLC, GLC and GC-MS.Abbreviations NAA 1-Naphthaleneacetic acid - BA Benzyladenine - BSA N,O-Bis(trimethylsilyl) acetamide     

Amino acid sequence at the ATP-binding site of cGMP-dependent protein kinase

The amino acid sequence at the ATP-binding site on the cGMP-dependent protein kinase has been determined. For this determination the enzyme was labeled covalently by 5'-p-fluorosulfonyl[14C]benzoyladenosine and fragmented using cyanogen bromide or digested by trypsin after succinylation. The 14C-labeled peptides were purified by gel filtration and high performance liquid chromatography. The amino acid sequence around the site was found to be: -Val-Glu-Leu-Val-Gln-Leu-Lys-Ser-Glu-Glu-Ser-Lys-Thr-Phe-Ala-Met-*Lys-Ile-Leu-Lys--Lys-Arg-His-Ile-Val-Asp-Thr-Arg-Gln-Gln-Glu-His-Ile-Arg-Ser-Glu-Lys-, in which *Lys is the lysine residue that was modified by the affinity reagent. When this sequence was compared with that of the ATP-binding site of the catalytic subunit of cAMP-dependent protein kinase, a high degree of structural homology was observed for this site in the two proteins.     

Dehydrodieugenol and its monomethyl ether from Virola carinata

The constituents of Virola carinata were established as dehydrodieugenol, its monomethyl ether and sitosterol.     

Contribution from the chirality of the growing chain to the enantiomer selectivity in activated-NCA polymerization of α-amino acid N-carboxyanhydride

As a model compound for the growing chain in the activated-NCA type of polymerization of α-amino acid N-carboxyanhydride (NCA), 3-[ω-acetylglycyl-poly(α-amino acid) acyl]-α-amino acid NCA (called the prepolymer) having various degrees of polymerization (DPs) was synthesized by the polymerization of Phe, Val, Glu(OEt), and Asp(OBzl) NCA in the presence of AcGly NCA by the tertiary amine. Activated (S)-Phe, Val, Glu(OEt), and Asp(OBzl) NCA were added to the terminal cyclic group of the corresponding (S)- or (R)- prepolymer, and the enantiomer selectivity in the reaction was investigated. With prepolymers having DPs ranging from 1 to 15, the addition reaction always took place preferentially between species having the same configuration, and the degree of the enantiomer selection increased with increasing DP of the prepolymer. With prepolymers having DP = 1 and 2, we found contributions from the chiral terminal unit and the chiral penultimate unit to the enantiomer selection, respectively. Prepolymer having DP = 5 was shown to take a β-type conformation, which led to higher enantiomer selection; and prepolymers having DP = 10 and 15 were shown to take an α-helix conformation, which led to much higher enantiomer selection than did the β-type conformation. In the present investigation the mechanisms of terminal-unit control, penultimate-unit control and conformational control of the enantiomer selection in the activated-NCA type of polymerization were clearly observed.     

Induction of a novel long-chain acyl-CoA hydrolase in rat liver by administration of peroxisome proliferators

The activity of long-chain acyl-CoA hydrolase in rat liver was increased by the administration of peroxisome proliferators, such as ethyl p-chlorophenoxyisobutyrate, di(2-ethylhexyl)phthalate or acetylsalicylic acid. The induced activity was mainly confined in the soluble fluid after the subcellular fractionation. The enzyme was purified nearly to homogeneity from livers of rats treated with di(2-ethylhexyl)phthalate. The specific activity of the final preparation was 247 mumol palmitoyl-CoA hydrolyzed min-1 mg protein-1. The molecular weight of the native enzyme was estimated to be 150 000 by gel filtration and that of the subunits was 41 000 by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The activity of the enzyme was not increased but inhibited by bovine serum albumin or Triton X-100. The molecular and catalytic properties of the enzyme suggest that the induced enzyme was different from mitochondrial and microsomal long-chain acyl-CoA hydrolyses in liver.     

Substrate utilization kinetic model for biological treatment process

The applicability of Contois' kinetic equation to aerobic and anaerobic treatments of organic wastes is investigated. A refractory coefficient to account for the nonbiodegradable portion of the organic substrates in the digester is incorporated into the kinetic equation. The kinetic equation is applied to the data for aerobic digestions of organic substrates and for anaerobic treatment of dairy wastes. They all show a very good fit of the kinetic equation to the data. Furthermore, the kinetic parameters and the refractory coefficients are shown to be independent of influent organic substrate concentration. This study confirms previous reports that the effluent quality of biological treatment systems for organic wastes depends on influent organic waste concentration. The effect of temperature on the kinetic parameters and the refractory coefficient for anaerobic treatment of sewage sludge are studied. It shows that the kinetic parameters vary with temperature, while the refractory coefficient remains fairly constant. Equations to predict biodegradable treatment efficiency and volumetric substrate utilization rate are also briefly discussed.