Carbon Balance Studies of Glucose Metabolism in Rat Cerebral Cortical Synaptosomes

Synaptosomes were isolated from rat cerebral cortex and incubated with [U-14C]-, [1-14C]- or [6-14C]glucose. Glucose utilization and the metabolic partitioning of glucose carbon in products were determined by isotopic methods. From the data obtained a carbon balance was constructed, showing lactate to be the main product of glucose metabolism, followed by CO2, amino acids and pyruvate. Measuring the release of 14CO2 from glucose labelled in three different positions allowed the construction of a flow diagram of glucose carbon atoms in synaptosomes, which provides information about the contribution of the various pathways of glucose metabolism. Some 2% of glucose utilized was calculated to be degraded via the pentose phosphate pathway. Addition of chlorpromazine, imipramine or haloperidol at concentrations of 10(-5) M reduced glucose utilisation by 30% without changing the distribution pattern of radioactivity in the various products.     

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.     

Chymotryptic-like hydrolysis of luliberin (LH-RF) by an adenohypophyseal enzyme of high molecular weight

An enzyme that hydrolyzes the fluorogenic chymotrypsin substrate glutaryl-Gly-Gly-Phe-β-naphthylamide has been partially purified from extracts of bovine anterior pituitaries. Like chymotrypsin, this enzyme hydrolyzes the neuropeptide Luliberin (LH-RF, <Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH₂) at the carboxyl-side of Trp and Tyr, but it differs from the pancreatic protease by its high molecular weight, insensitivity towards OH-reactive agents and other enzymechemical parameters. It seems, however, to be identical to the “cation-sensitive neutral endopeptidase”. In the course of this study evidence has also been obtained that LH-RF is not degraded by the cystinyl-arylamidase.     

Characterization of "thyroliberin-deamidating enzyme" as a post-proline-cleaving enzyme. Partial purification and enzyme-chemical analysis of the enzyme from anterior pituitary tissue.

An enzyme which catalyzes the deamidation of thyroliberin (TRF; less than Glu-His-Pro-NH2) has been purified 110-fold from extracts of bovine anterior pituitary by ammonium sulfate fractionation, ion exchange chromatography on DEAE-cellulose, and gel filtration. This enzyme of 76,000 molecular weight (as estimated by gel filtration) exhibits maximal activity at neutral pH (optimum pH 7.4 to 7.6) in buffers of high ionic strength supplemented with thiol-protecting agents. As indicated by the strong inhibition of the enzymatic activity by N-ethylmaleimide and Hg2+, as well as by the extreme sensitivity toward diisopropyl fluorophosphate, -SH, and -OH residues apparently represent essential functional groups of the enzyme. The stereospecific deamidation of TRF (Km = 4.1 . 10(-4) M) is inhibited competitively by TRF analogues which contain proline or by the proline containing biologically active peptides luliberin (LH-RF), oxytocin, vasopressin, angiotensin II, and Substance P. TRF analogues without proline or peptide amides without proline are ineffective. This enzyme cleaves the appropriate Pro-X bonds in luliberin, angiotensin II, pyroGlu-His-Pro-Gly-NH2, and the collagenase substrate Z-Gly-Pro-Leu-Gly-Pro. Thus, it may be characterized as a post-proline-cleaving enzyme.     

Early molecular events in the interaction of enveloped viruses with cells

The fluorescence depolarization of 1,6-diphenyl-hexatriene was used to study the dynamic properties of the hydrophobic regions of the lipid envelopes of ortho- and paramyxoviruses as well as of the Rous sarcoma virus and of the membrane lipids of susceptible and nonsusceptible cells.The systems investigated where active and inactive influenza viruses, and NDV virus acting on chick embryo fibroblasts and Rous sarcoma virus acting on susceptible (C/E) and nonsusceptible (C/B) chicken-cell.Polarization degrees and mean rotational correlation times of DPH embedded in viral lipids were significantly higher than those of DPH in the cell membranes, due to a higher rigidity of the virus envelopes. When suspensions of labelled viruses and unlabelled cells or unlabelled viruses and labelled cells were mixed, a characteristic change of the fluorescence polarization degrees with time was observed. This behaviour was ascribed to a label transfer from virus to cell membranes or vice versa.While the rate constants of label transfer from virus to cells and cells to virus were about the same for the penetrating viruses the rate constants of label release from inactive virus to cells were much larger than for the migration in the opposite direction.     

The mode of action of primycin

Primycin, an antibiotic active against Gram-positive microorganisms increased the permeability ofBacillus subtilis cell membranes when used in bacteriostatic concentrations. On addition of the antibiotic to the washed cell suspension, a dose-dependent increase in the conductivity was observed. Furthermore, an enhanced leakage of the nucleotides (measured by the³²P-ATP release from the³²P-labelled culture) could be detected.To get more information about the mechanism of the primycin-membrane interaction, the effect of the antibiotic on the ATPase activity of membrane vesicles prepared from bothBacillus subtilis andEscherichia coli B was studied. Activation was found at about 0.5 nmol antibiotic/g protein and its extent was approximately the same as with sonicated membranes used as controls. Stimulation of ATPase activity was also achieved with vesicles prewashed with 3 mM Tris-HCl buffer.Purified membrane ATPase fromBacillus subtilis could not be activated by primycin at all; above 0.3 nmol/g protein concentration the enzyme was inhibited. When acting on membrane vesicles isolated fromEscherichia coli B, inhibition without previous activation was observed, although sonication caused a substantial activation on the ATPase of these membranes.These observations confirmed our suggestion that the primary target of primycin action is the cell membrane in Gram-positive microorganisms.Abbreviations OD Optical density     

Role of lectins in plant--microorganism interactions. IV. Ultrastructural localization of soybean lectin binding sites of Rhizobium japonicum

The binding of purified, ferritin-labeled soybean seed lectin to the cell surfaces of Rhizobium japonicum 31 lb 138 has been examined by whole mount, thin section, and freeze-etch electron microscopy. The ferritin-labeled lectin binds in a biochemically specific manner to the capsular material of this bacterium. The lectin does not bind to the outer membranes of the cells or to flagella. Labeled lectin binds to sites throughout the capsular structure, although the density of labeling is somewhat greater on the outer surface of the capsule. Some cells appear to be partially encapsulated. Preservation of the capsular material proved difficult, and methods for retaining most of the capsular material were developed.     

Propagation of mouse mammary tumor cell lines and production of mouse mammary tumor virus in a serum-free medium.

Five different mouse mammary tumor cell lines were propagated in a serum free medium. Evaluation of growth characteristics, including logarithmic growth, cell population increase, protein production and days to confluency, showed serum-free medium comparable to serum-containing medium. Mouse mammary tumor virus expression and production, in C3H and GR tumor cell lines, as determined by virus particle counting and RNA dependent DNA polymerase assays, subsequent to dexamethasone stimulation revealed equivalent to higher levels of virus in serum-free medium as compared to serum-containing medium.