Characterization of a region of the primary sequence of troponin C involved in calcium ion-dependent interaction with troponin I.

1. The CNBr digest of troponin C from rabbit fast skeletal muscle was shown to possess many of the functional properties of the whole troponin C molecule. 2. A peptide corresponding to residues 83-134 was isolated, which forms a Ca(2+-dependent complex with troponin I and neutralizes the inhibition by troponin I of the Mg(2+-stimulated adenosine triphosphatase of desensitized actomyosin. 3. The peptide inhibits the phosphorylation of fast-skeletal-muscle, but not cardiac-muscle, troponin I, by 3' :5'-cyclic AMP-dependent protein kinase. In this property it was as effective as whole skeletal-muscle troponin C when compared on a molar basis. 4. Biological activity was also present in other fractions obtained from the CNBr digest. 5. By gel filtration and affinity chromatography of the whole CNBr digest of troponin C, two peptides, one of which was identified as representing residues 83-134, were shown to form Ca(2+-dependent complexes with troponin I. 6. The significance of these findings for the mechanism of interaction of troponin C and troponin I is discussed.     

Alteration of the Intracellular Energetic and Ionic Conditions by Mengovirus Infection of Ehrlich Ascites Tumor Cells and Its Influence on Protein Synthesis in the Midphase of Infection

Mengovirus infection of Ehrlich ascites tumor cells caused a change of the intracellular ATP concentration. It increased by 35% within the first 3 h postinfection and then declined to zero within the next 5 h. The decrease in the ATP concentration was due, at least in part, to leakage of ATP into the medium, where it could be demonstrated by the luciferin-luciferase assay. Gross leakage of ATP was observed at 4.5 h postinfection, concomitant with the production of the first intracellular, infectious virus particles. A similar concentration decrease was detected for Mg(2+), the polyamines, and K(+), whereas an increase in the Na(+) concentration was observed. The intracellular Mg(2+) concentration varied synchronously with the ATP level, rising by 16% during the first 3 h postinfection and then progressively falling to lower values in the late period of the infectious cycle. After an initial slight enhancement, the putrescine, spermidine, and spermine concentrations declined at about 1.5 h postinfection. Wherease the intracellular K(+) concentration increased by 17% during the first hour postinfection, the Na(+) concentration diminished by the same value within the same time period, leaving the internal ionic strength unchanged early in infection. Three hours after the beginning of virus infection, there was a rapid decline of K(+) and enhancement of Na(+) within the cell. These alterations of the intracellular energetic and ionic conditions seem to be, at least in part, responsible for the cessation of virus-specific protein synthesis in mengovirus-infected Ehrlich ascites tumor cells commencing 3 to 3.5 h postinfection.     

The catabolism of intravenously injected heparan N-[35S]sulphate in the rat

The metabolic fate of heparan N-[(35)S]sulphate was studied in rats. Heparan sulphate was obtained from either bovine aorta or lung and labelled with (35)S by desulphation and subsequent resulphation in vitro. Experiments in which heparan N-[(35)S]sulphate was administered intravenously to either free-range or wholly anaesthetized rats with ureter cannulae established that substantial desulphation occurs in vivo, with elimination of inorganic [(35)S]sulphate in urine. Oligosaccharides labelled with (35)S, possible intermediates in heparan sulphate degradation, could not be detected in urine or blood. The general distribution of radioactivity after administration of heparan N-[(35)S]sulphate, as demonstrated by whole-body radioautography, suggested that desulphation was not restricted to one organ in particular. Support for this view was obtained in experiments in which heparan N-[(35)S]sulphate was administered to animals after the removal of kidneys, liver, spleen, pancreas or gastrointestinal tract. In all cases inorganic [(35)S]sulphate was still produced. The ability of rats of desulphate heparan N-[(35)S]sulphate was progressively impaired by increasing concentrations of heparin administered simultaneously. It was concluded that heparan sulphate is metabolized at a number of sites in the body by a sequence of degradative events leading to the formation of inorganic sulphate. It is also concluded that at least some of these events are common to heparan sulphate and heparin.     

Lipids of the marine bacterium Flexibacter polymorphus

This paper reports on the total fatty acid composition of a marine bacterium representative of the genus Flexibacter. Flexibacter polymorphus is unusual in containing a high proportion of the polunsaturated acid C20:53 whilst the level of branched fatty acids is low. These facts suggest that the membrane flexibility necessary for its gliding motility is a function of the polyunsaturated fatty acid composition. Biosynthetic precursors to the C20:5 acid are present which are characteristic of an oxygen-dependent pathway. The fatty acid composition of the structural lipids is influenced by changes in the culture medium. Na₂S inhibits production of the C20:5 acid at levels much lower than that at which it is known to inhibit growth. The intracellular granules observable in F. polymorphus do not contain elemental sulphur, in contrast to Beggiatoa sps., but instead probably contain lipids.     

Utility of d4-PGE2 as an internal standard to quantify endogenous levels of PGE1, PGE2, 190H PGE1 and 190H PGE2 in human seminal fluid by GC-MS-SIM

Four prostaglandins-PGE₁, PGE₂, 190H PGE₁ and 190H PGE₂-were quantified in human seminal fluid by GC-MS-SIM using only the internal standard, d₄-PGE₂. Methods and calculations were developed to minize errors inherent in using only one internal standard for quantifying four closely related prostaglandins. Preliminary data concerning the statistical significance of the differences found between PGE and 190H PGE levels in fertile, azospermic and oligospermic men are reported.     

Enhanced production of Rauscher leukemia virus after infection of high-passaged JLS-V9 cells.

JLS-V9, a mouse bone marrow cell line infected with Rauscher leukemia virus at high passage level, produced larger amounts of virus than the standard JLS-V10 cells. The enhanced virus production was attributed to the increased saturation density of JLS-V9 cells.     

Isolation, purification, and properties of mouse heavy-chain immunoglobulin mRNAs.

A procedure is described for the isolation of highly purified heavy-chain immunoglobulin mRNAs from a variety of mouse plasmacytomas (IgA, IgG, and IgM producers). The use of fresh tissue and the rapid isolation and direct extraction of membrane-bound polyribosomes were found to be essential in obtaining large quantities of undegraded heavy-chain mRNAs. The individual mRNAs were purified by two cycles of oligo(dT)-cellulose chromatography, sodium dodecyl sulfate--sucrose gradient centrifugation, and electrophoresis on 98% formamide containing polyacrylamide gels. When added to a cell-free protein-synthesizing system from wheat germ, the MPC-11 gamma2b and H2020 alpha heavy-chain mRNAs efficiently directed the synthesis of a predominant product of 55 000 molecular weight, while the synthesis of a 70 000 dalton protein in addition to other lower molecular weight polypeptides were observed with MOPC 3741 mu mRNA. All of these proteins were immunoprecipitable with class-specific heavy-chain antisera, and in the case of the gamma2b in vitro products good correspondence in a comparative trypsin--chymotrypsin fingerpring with in vivo labeled gamma2b heavy chain was observed. The gamma2b and a alpha heavy-chain mRNAs possessed a chain length of approximately 1800 nucleotides and the mu mRNA a size of approximately 2150 nucleotides when examined under stringent denaturation conditions. The purities of the alpha, gamma2b, and mu mRNAs were estimated to be 60--80%, 50--70%, and 50--83%, respectively, on the basis of their hybridization rates with cDNA probes in comparison to mRNA standards of known complexity. Heavy-chain mRNAs of the same class isolated from different mouse strains (Balb/C or NZB) display no detectable sequence differences in cross hybridization experiments, even though the cDNA--mRNA hybrids are submitted to stringent S1 nuclease digestion. These results indicate that allotypic determinants represent only a minor fraction of the heavy-chain constant region sequence in the mouse.     

Amine dehydrogenase of Pseudomonas putida: properties of the heme-prosthetic group.

There was approximately five times more hemoprotein (amine dehydrogenase) in crude extracts obtained from Pseudomonas putida grown on benzylamine than present in extracts from succinate-grown cells. The difference (reduced minus oxidized) spectrum of the purified enzyme possessed alpha,beta, and gamma bands at 550, 523, and 416 nm, respectively. The difference spectrum of the pyridine hemochrome derivative had absorption maxima at 416, 520, and 550 nm. These results, together with the fact that the heme group was covalently bound to the enzyme, indicated that the amine dehydrogenase from P. putida was a hemoprotein which contained heme c. The heme content was calculated at 2.01 mol/mol of enzyme. The enzyme was composed of two nonidentical subunits, but heme was present solely in the heavier unit. Carbon monoxide did not inhibit enzymatic activity, nor would it combine with the reduced or oxidized form of the enzyme. Amine dehydrogenase activity was inhibited by carbonyl agents with semicarbazide and cuprizone acting noncompetitively, whereas KCN and isoniazid inhibited by competitive and uncompetitive mechanisms, respectively. Spectral observations suggested that inhibition by these reagents was not due to an interaction with the heme moiety.     

Effect of partial hepatectomy on removal of O6-methylguanine from alkylated DNA by rat liver extracts.

1. The activity of an enzyme catalysing the loss of O6-methylguanine from methylated DNA was increasing during liver regeneration after partial hepatectomy. Activity was increased 3-fold by 24h and was maximal (6-fold increase) over the period 48-72h after operation. 2. This activity could also be induced by chronic treatment with dimethylnitrosamine, but the maximal response amounted to a 2-3-fold change (with the greater effect in male rats) after 4-6 weeks of exposure to daily doses of 2 mg of dimethylnitrosamine/kg. 3. Neither partial hepatectomy nor treatment with dimethylnitrosamine increased the activities of two other enzymes repairing alkylated DNA, DNA (7-methylguanine-)glycosylase and DNA (3-methyladenine-)glycosylase. 4. These results therefore indicate that there is a selective induction of the O6-methylguanine removal system during hepatocyte proliferation. Since this product is known to lead to mutations and its persistence in DNA throughout cell replication has been implicated in tumour initiation, this induction may play a role in resistance to carcinogenesis by alkylating agents.     

Freeze-drying of solutions of serum albumin containing dimethylsulfoxide.

Although several publications have emphasized the inadvisability of drying biologic materials containing dimethylsulfoxide (DMSO) by sublimation of ice in vacuo, our studies showed a relationship to exist between the relative concentrations of serum albumin, human, and dimethylsulfoxide for successful or unsuccessful freeze-drying of albumin-DMSO solutions. The cycle of freeze-drying for the successful drying of an albumin-DMSO solution was a modification of the cycle used for the successful drying of suspensions of measles virus by sublimation of ice in vacuo. Using nuclear magnetic resonance spectroscopy, a strong, sharp signal for DMSO was obtained in preparations of freeze-dried albumin-DMSO solutions rehydrated with deuterium oxide, D₂O.