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.     

Selective inhibition of thymidine transport at low doses of the alkylating agents triethyleneiminobenzoquinone (Trenimon)

The alkylating antitumor agent triethyleneiminobenzoquinone (Trenimon) causes a rapid decrease in the incorporation of labeled thymidine into the DNA of Yoshida or Ehrlich ascites tumor cells. The effect is expressed 4 h after administration of 6 × 10⁻⁸ moles/kg of the drug to mice bearing Yoshida ascites tumors or of 6 × 10⁻⁷ moles/kg to Ehrlich ascites tumor-bearing animals, respectively. The reduced incorporation of labeled thymidine which is observed under these conditions is not due to an inhibition of DNA synthesis. DNA synthesis was measured by an isotope dilution assay after pulse-labeling with ³H-thymidine and by monitoring the increase in the total amount of DNA of the cell populations. The data demonstrate that DNA synthesis is not affected during the first 8 h after exposure to the drug. This conclusion is supported by cell kinetic measurements which indicate that the alkylating agent does not interfere with the progression of cells into the S phase, but exerts a block at the G 2 stage of the cell cycle. The reduced incorporation of thymidine into DNA is explained by a decreased transport of the nucleoside into the cells.