Different binding of human interferon alpha 1 and alpha 2 to common receptors on human and bovine cells. Studies with recombination interferons produced in Escherichia coli

Minicells from Escherichia coli DS410 harboring cDNA for human interferon (IFN) alpha 1 or alpha 2 were metabolically labeled with [3H]leucine and the radioactive IFN was purified to homogeneity by immune precipitation with anti-IFN-alpha serum. These preparations of radioactive IFN-alpha 1 and -alpha 2 were used to study the binding on two human (FL and Daudi) and one bovine (MDBK) cell lines. IFN-alpha 2 specifically bound well to both human and bovine cells, while IFN-alpha 1 bound very poorly to human cells but well to bovine cells. Specific binding of radioactive IFN-alpha 2 to these cell lines was completely inhibited by not only nonradioactive IFN-alpha 2 but also IFN-alpha 1, and binding of IFN-alpha 1 to bovine cell was also competed by IFN-alpha 2 as well as IFN-alpha 1, indicating that the receptors for both IFNs are identical. However, 50-100-fold (on human cells) or 4-fold (on bovine cell) more nonradioactive IFN-alpha 1 than -alpha 2 was required to inhibit the binding of radioactive IFN-alpha 2 to the receptors. Scatchard analysis showed that IFN-alpha 1 and -alpha 2 bind to the receptors on human cells with an apparent Kd of greater than 6 X 10(-10) and 3 X 10(-11) M, respectively, while on bovine cells with a Kd of 4.2 X 10(-11) and 1.6 X 10(-11) M, respectively. These results show that the different target cell specificity of IFN-alpha 1 and -alpha 2 in regard to antiviral activity (Streuli, M., Hall, A., Boll, W., Stewart, W. E., II, Nagata, S., and Weissmann, C. (1981) Proc. Natl. Acad. Sci. U. S. A. 78, 2848-2852) is due to the different binding activity of IFN-alpha molecules to their common receptors.     

Binding of Triton X-100 to purified cytochrome P-450scc and enhancement of the cholesterol side chain cleavage activity.

The cholesterol side chain cleavage activity of highly purified adrenal cytochrome P-450scc was enhanced 6-fold by the addition of Triton X-100 in the assay solution in final concentrations of 0.03 to 0.05%, while the same detergent was much less effective in the higher concentrations and Tween 80 was not stimulative to the enzyme in various concentrations. It was shown by gel-filtration chromatography of the P-450 with 0.05% Triton X-100 that the detergent was bound to the P-450 in an amount greater than 0.5 mg per mg of protein. By the addition of the detergent, 415-nm light absorption of the P-450 was intensified and the isoelectric point was shifted to the alkaline side. Furthermore, the P-450 showed a sedimentation coefficient of 5.1 S in the presence of 0.05% Triton X-100, whereas it showed a sedimentation coefficient of 8.2S in the absence of the detergent. These results suggest that the observed enhancement of the enzyme activity is largely due to the direct effect of the detergent to the P-450 molecule itself. During these experiments, it was also noted that the P-450 was not resolved into more than one species.     

Binding of the structural protein soc to the head shell of bacteriophage T4

Qβ plus strands with a 70 S ribosome bound to the coat cistron initiation site were used as template for Qβ replicase. Minus strand synthesis proceeded until the replicase reached the ribosome. The ribosome was removed and elongation was continued in a substrate-controlled, stepwise fashion. The nucleotide analog N⁴-hydroxyCMP was introduced into the positions complementary to the third and fourth nucleotides of the coat cistron. The minus strands were elongated to completion, purified and used as template for Qβ replicase. The final plus strand preparation consisted of four species, with the sequences -A-U-G-G- (wild type), -A-U-A-G- (mutant C₃), -A-U-G-A- (mutant C₄) and -A-U-A-A- (mutant $\text{C}{}_3\text{C}{}_4$) at the coat initiation site. The ribosome binding capacity of the mutant RNAs relative to wild type was <0.1 (C₃), 3.2 (C₄) and 0.3 ($\text{C}{}_3\text{C}{}_4$). The finding that mutant C₃ no longer formed an initiation complex suggests that the interaction of the ribosome binding site with fMet-tRNA plays an essential role in the formation of the 70 S initiation complex. The fact that mutant C₄ RNA bound more efficiently than wild type, and that mutant $\text{C}{}_3\text{C}{}_4$ RNA showed substantial ribosome binding capacity whereas the single mutant C₃ did not, can be explained by assuming that an A residue following the A-U-G triplet interacts with a complementary U residue in the anticodon loop sequence. In the case of $\text{C}{}_3\text{C}{}_4$ this additional base-pair may offset the reduced codon-anticodon interaction resulting from the modification of the A-U-G codon.     

Factors influencing the frequency of mitomycin C-induced sister-chromatid exchanges in 5-bromodeoxyuridine-substituted human lymphocytes in culture.

Newly developed techniques for the detection of sister-chromatid exchanges (SCE) require the substitution of 5-bromodeoxyuridine (BrdU) for thymidine in DNA. We investigated the possibility of interactions between BrdU and one mutagen--carcinogen, mitomycin C (MMC) for the induction of both chromosomal aberrations and SCE in human peripheral lymphocytes in culture. No effect on aberration yield was found. Neither comparisons between the yields of SCE by BrdU substitution and differential staining and those detected by tritiated thymidine incorporation and autoradiography nor between the yields of SCE for different levels of BrdU incorporation provided any evidence of synergism. It was found, however, that MMC persists in cultures and continues to increase SCE frequencies for about 30 h. It was also observed that some MMC-induced DNA lesions persist long enough so that some of those present prior to S phase of the first cell cycle cause additional SCE in the third cycle.     

Anhydrotrypsin and trypsin: subtle difference in the active-site conformations detected by chemical modification and CD spectroscopy.

The reactivities of the active-site histidine residue in bovine trypsin and its anhydro-derivative, as well as in Streptomyces griseus trypsin and its anhydro-derivative have been compared. The reactivity with TLCK was found to be lost in both of the anhydrotrypsins. On the other hand, alkylation by iodoacetamide either in the presence or absence of 1-methylguanidine proceeded faster in anhydrotrypsins than in trypsins. These differential responses to alkylating reagents are discussed in terms of a subtle change in the active-site conformation which occurs during the conversion of trypsin into anhydrotrypsin. The examination of difference CD spectra, produced by interaction with benzamidine or beta-naphthamidine, also suggested a conformational difference of the active-site between the proteins of bovine origin.     

Epidemiological studies on Japanese encephalitis in Kyoto City area, Japan. IV. Natural infection in sentinel pigs.

Natural infection with Japanese encephalitis virus in three sentinel pigs held in separate experimental huts was examined daily by virus recovery from blood samples of the pigs and from mosquitoes after incubation for about 7 days from their blood feeding and by HI antibody titration of the blood samples. After a period of low infection rates, below 6%, for about two weeks in engorged Culex tritaeniorhynchus summorosus, high mosquito infections of over 30% from each viremic pig occurred for two to three days. The pigs may be probably have been bitten by many infected but not infective mosquitoes in a period of about 10 days before infection.     

Bacteriolytic enzyme induced from pyocinogenic Pseudomonas aeruginosa. Purification and characterization of PR1-lysozyme.

A bacteriolytic enzyme, PR1-lysozyme, has been purified from the lysate of mitomycin C-induced pyocinogenic Pseudomonas aeruginosa, by acrinol treatment, Amberlite CG-50 chromatography, ammonium sulfate fractionation, Sephadex G-100 gel filtration and two cycles of SP-Sephadex C-50 chromatography. Homogeneity of the preparation was demonstrated by three electrophoretic techniques. PR1-lysozyme is a basic protein (pI, 9.4) and consists of a single polypeptide chain having a molecular weight of 24,000. The amino acid composition of the protein was analyzed, and no cystein residue was found among more than 210 amino acid residues. The optimum pH for enzymatic activity was 6.4 and the enzyme exhibited about 50 to 70 times greater specific activity than hen egg-white lysozyme when assayed with chloroform-killed P. aeruginosa as a substrate. By analyzing the products of enzymatic action on purified peptidoglycan of P. aeruginosa, the enzyme was identified as an N-acetylmuramidase, i.e., the same classification as hen-egg-white lysozyme. PR1-lysozyme did not show any activity towards intact cells of gram-positive and gram-negative bacteria tested. However, the enzyme was able to lyse chloroform-killed gram-negative and gram-positive bacteria.     

Transmembrane permeability channels in vesicles reconstituted from single species of porins from Salmonella typhimurium.

Aggregates of the "major" outer membrane proteins, "porins," of Salmonella typhimurium form diffusion channels in reconstituted vesicle membranes. The aggregate consists of three species of porins with apparent molecular weights of 34,000, 35,000, and 36,000 when active aggregates are subjected to sodium dodecyl sulfate-acrylamide gel electrophoresis after heating in the presence of sodium dodecyl sulfate (Nakae, J. Biol. Chem. 251:2176-2178, 1976). Single species of porins were isolated by solubilization of membranes and subsequent gel filtration in the presence of sodium dodecyl sulfate from the mutant strains of Salmonella typhimurium that produced only single species of porin. The single species of porins of either 34,000, 35,000, or 36,000 daltons formed diffusion channels when assayed for sucrose permeability in the vesicle membranes reconstituted from porins, phospholipids, and lipopolysaccharides. The exclusion limits of the pores made of single species of porins were not distinguishable from each other and from the exclusion limits of the pores made of the porin aggregates from the wild-type strain, when the permeability of vesicle membranes to radioactive di-, tri-, and tetrasaccharides and to various sizes of radioactive polyethylene glycol was determined. Porin-deficient mutants produced residual amounts of porin amounting to 1 to 5% that produced by the parent strain. This residual porin made diffusion channels when the isolated porins were incorporated into the vesicle membrane and assayed for permeability of saccharides.     

Microdetermination of galactose and galactose 1-phosphate in dried blood spots

Newborn screening for galactosemia (galactose-1-phosphate uridyltransferase deficiency), as well as for other defects in galactose metabolism (galactokinase deficiency and uridine diphosphogalactose 4-epimerase deficiency), requires a method of determining both galactose and galactose 1-phosphate in dried blood. We have developed a sequential quantitative method for the microdetermination of galactose and galactose 1-phosphate that can be applied to 3-mm-diameter disks of dried blood and that can be used with a Technion Autoanalyser II equipped with a fluorometer.Galactose is determined by the fluorescence of NADH following treatment with β-galactose dehydrogenase and with the consequent reduction of NAD. The complete system includes alkaline phosphatase for the hydrolysis of galactose 1-phosphate, so that the total amounts of a galactose and galactose 1-phosphate are determined. For the measurement of galactose alone, alkaline phosphate is omitted from the system. The difference in fluorescence between that from the complete system and that from the alkaline phosphatase-omitted system yields the concentration of galactose 1-phosphate.     

Cellular metabolic control by chemical modification of cell membrane.

Various reagents used in the chemical modification of amino- and carboxy-groups of proteins, and of carbohydrates of glycoproteins and glycolipids, inhibit respiration in ascites tumor cells concomitant with release of potassium ion from those cells. The respiratory activity of washed ascites tumor cells is increased by exogenous addition of potassium ion. The lowered respiratory control index as well as oxidative phosphorylation of aged mitochondria are restored upon increasing the potassium concentration of the incubation mixture in the presence of respiratory substrates. The data suggest that the potassium ion level of cells is changed by modifying physicochemical properties of membrane components and that cellular energy metabolism is regulated by intracellular potassium ion concentration.