Morphine-like analgesic actions of enkephalin-like peptides containing the Tyr-Arg unit

The analgesic actions of some synthetically prepared peptides having the Tyr-D-Arg unit at the N terminal portion of met- and leu-enkephalin were measured by the intra-cisternal injection method in mice. Among them, Tyr-D-Arg-Gly-Phe (DR-4) induced the most potent naloxone-reversible analgesia and was also effective by s.c. injection. DR-4 showed the good affinity to mu-receptor, and the resistance to the enzymatic degradation.     

Crystal structure of the complex of subtilisin BPN' with its protein inhibitor Streptomyces subtilisin inhibitor. The structure at 4.3 Angstroms resolution

The crystal structure of the complex of subtilisin BPN′ (EC 3.4.21.14) with its protein inhibitor (Streptomyces subtilisin inhibitor) was solved at 4.3 Å resolution, thus establishing the following. (1) Two subtilisin BPN′ molecules (2E) associate with one dimeric inhibitor molecule (I₂) to form the complex molecule E₂I₂. (2) The conformation of neither the inhibitor nor subtilisin BPN′ undergoes any detectable change at this resolution upon complex formation. (3) The inhibitor binds to subtilisin to form an antiparallel β-sheet, as in the case of trypsin/ trypsin inhibitor complexes. (4) The scissible bond of the inhibitor is between Met73′ and Val74′, as proposed earlier (Ikenaka et al., 1974). (5) The protein inhibitor and the substrates bind to subtilisin BPN′ in essentially the same way.     

Formation of biologically active protein from the subunits of islets-activating protein (IAP), a new protein isolated from Bordetella pertussis.

Based on the finding reported in the preceding paper (Kanbayashi, et al.: J. Biochem) that subunits of islets-activating protein (IAP), a new protein purified from the culture media of Bordetella pertussis, were inactive as such, but regained the original biological activities when recombined, the conditions required for recovery of the biological activities were studied. Essentially the same biological activities as the native IAP were recovered when the smallest subunit, F-3, was incubated with one of the other subunits, F-1 and F-2, at a pH of around 7, at temperatures below 30 degrees C and for longer than 12 h. During the incubation, association products were formed which were isolated by gel filtration as homogenous proteins that consisted of two subunits probably in a molar ratio of 1 : 1. The native IAP (consisting of two IAP subunits including F-3) were equipotent in enhancing insulin secretory responses, in inhibiting epinephrine-induced hyperglycemia, in inducing leukocytosis and in increasing histamine sensitivity in experimental animals.     

Maintenance of a certain rumen protozoal population in a continuous in vitro fermentation system.

A continuous culture system suitable for maintaining certain rumen protozoa was developed by modifying the procedure of Walter and Pilgrim (R.A. Weller and A.F. Pilgrim, Br. J. Nutr. 32:341-351, 1974) to include a dialyzing system. The concentration of ciliate protozoa, the pH value, and concentrations of volatile fatty acid and ammonia-N could be maintained within normal rumen limits for more than 15 days by appropriate choice of mechanical agitation and of the amount of substrate and physical form of the substrate bags. The average concentration of protozoa in the free fluid around the substrate was about 10% that in the fluid squeezed from solid digesta residues. More than 10(6) protozoa per ml was present in the fresh substrates only 2 h after supplying the substrate. These facts suggest that sequestration of the protozoa among particulate digesta is an important factor in maintaining the concentration of protozoa.     

Flagellar adenosine triphosphatases from annelid spermatozoa: electrophoretic identification of dyneins

Axonemes of sperm flagella were prepared from the annelid, Tylorrhynchus heterochaetus. Dialysis of the axonemes against 1 mm Tris-HCl buffer (pH 8.3)-0.1 mm EDTA-0.1 mm dithiothreitol (Tris-EDTA solution) caused disintegration of typical 9 + 2 microtubules into each doublet, resulting in extraction of one-third of the protein and almost all ATPase activity. Agarose polyacrylamide gel electrophoresis of the extract showed the presence of three kinds of dyneins actively stained for ATPase (designated as bands I, II, and III) and two non-ATPase proteins (bands IV, V). The polypeptide components of each dynein molecule and intact axoneme were analyzed by subsequent sodium dodecyl sulfate-polyacrylamide gel electrophoresis to obtain the following results: (1) In the highmolecular-weight region, the intact axonemes yield two major polypeptides with molecular weights of 365,000 and 345,000 (designated as bands A and B, respectively) and three minor polypeptides, 310,000, 290,00, and 270,00 (C1, C2, C3). (2) All three dyneins contain A-band polypeptide as a common polypeptide component. In addition, band I dynein and band II dynein also contain B and C1 polypeptides, and C3 polypeptide, respectively, as high-molecular-weight components. (3) Band III dynein also contains four polypeptides in the lower molecular-weight region, which migrate similarly with those of 21 S dynein from sea urchin sperm flagella or 18 S dynein from Chlamydomonas.     

Two types of linkage between codon usage and gene-expression levels

The relation between codon usage and gene-expression levels is an intensively investigated and discussed topic in the field of molecular evolution. We statistically analyzed 25 Escherichia coli gene sequences by a new classification of synonymous codons and found that (i) there are two distinct types of linkage between codon usage and gene-expression levels in E. coli, and (ii) one of the two kinds of codon preferences (the codon preference concerned with interaction of GC/AT choice at three codon positions) is observed significantly in weakly expressed genes.     

Inhibition of actin-activated myosin Mg(2+)-ATPase in smooth muscle by ruthenium red.

Ruthenium red was found to inhibit actin-activated myosin Mg(2+)-ATPase in smooth muscle and to bind to myosin heavy chain, but not to F-actin. The inhibition by Ruthenium red of actin-activated Mg(2+)-ATPase was of the competitive type with respect to actin (Ki 4.4 microM) and of the non-competitive type with respect to ATP (Ki 6.6 microM). However, Ruthenium red scarcely dissociated the acto-heavy meromyosin complex during the ATPase reaction. These results suggest that Ruthenium red interacts directly with the binding site for F-actin on the myosin heavy chain. This site is considered to be necessary not for maintaining the binding affinity of myosin for F-actin, but for activation of the Mg(2+)-ATPase.