Separate metabolic pathways for Leu-enkephalin and Met-enkephalin-Arg6-Phe7 degradation by rat striatal synaptosomal membranes

Metabolism of Leu-enkephalin and Met-enkephalin-Arg⁶-Phe⁷ was studied using synaptosomal plasma membranes prepared from rat corpus striatum and whole brain. Cleavage of the pentapeptide was mediated largely by an aminopeptidase leading to the release of Tyr and Gly-Gly-Phe-Leu. Bestatin, an aminopeptidase inhibitor, prevented the release of Tyr and the tetrapeptide, but not secondary cleavage at the Gly Phe site leading to the release of Tyr-Gly-Gly and Phe-Leu. Cleavage at the latter site was inhibited by low concentrations of Thiorphan, an inhibitor of a non-aminopeptidase enkephalinase. MK-421, an inhibitor of the angiotensin converting enzyme, acted only at high substrate concentrations of Leu-enkephalin, indicating that the converting enzyme has a relatively low affinity for the pentapeptide. In contrast to the pentapeptide the major products found upon incubation of heptapeptide with synaptosomal plasma membrane were Arg-Phe and Met-enkephalin. Product release was inhibited by low concentrations of MK-421 but not by Thiorphan, indicating that the cleavage of the heptapeptide was mediated by the angiotensin converting enzyme. This pathway may represent a mechanism for the formation of Met-enkephalin from larger precursors present in striatum and other regions of the central nervous system.     

Modulation of collagen production by fibroblasts. Effects of chronic exposure to agonists that increase intracellular cyclic AMP.

Cultured human lung fibroblasts were evaluated for their responsiveness to isoprenaline (isoproterenol) or prostaglandin E2 before and after chronic incubation with the agonist. Cells incubated for 6 h with either agonist were suppressed in terms of collagen production and exhibited increased intracellular cyclic AMP. Cells incubated for 72 h with the agonist and then re-challenged for 6 h with the same agonist did not demonstrate suppressed collagen production or increased cyclic AMP. Cells incubated for 72 h with isoprenaline still responded to prostaglandin E2 when challenged for 6 h; however, when the order of agonist exposure was reversed, cells incubated with prostaglandin E2 did not respond to a challenge by isoprenaline. If cells were allowed to recover for 48 h without the agonist after a 72 h chronic incubation, they recovered their responsiveness to the agonist. The results indicate that, although cultured fibroblasts may become desensitized to one agonist, they may retain their sensitivity to a second agonist and chronic suppression of collagen production may be achieved by alternate exposure to isoprenaline and prostaglandin E2.     

Effect of leupeptin on the intracellular degradation of asialofetuin in isolated rat hepatocytes

The effect of the protease inhibitor leupeptin on the intracellular distribution of [14C]-sucrose-asialofetuin in isolated rat hepatocytes was investigated. Leupeptin had no effect on the uptake but reduced the degradation of asialofetuin. Fractionation of hepatocytes by isopycnic centrifugation in sucrose gradients indicated that prolonged treatment with leupeptin inhibited the uptake of asialofetuin into the lysosomes. Therefore, leupeptin inhibits degradation of asialofetuin both by inhibiting intralysosomal proteolysis and transport of endocytosed asialofetuin to the lysosomes.     

Fermentative Conversion of Cellulose to Acetic Acid and Cellulolytic Enzyme Production by a Bacterial Mixed Culture Obtained from Sewage Sludge

A simple procedure that uses a cellulose-enriched culture started from sewage sludge was developed for producing cellulolytic enzymes and converting cellulose to acetic acid rather than CH₄ and CO₂. In this procedure, the culture which converts cellulose to CH₄ and CO₂ was mixed with a synthetic medium and cellulose and heated to 80°C for 15 min before incubation. The end products formed were acetic acid, propionic acid, CO₂, and traces of ethanol and H₂. Supernatants from 6- to 10-day-old cultures contained 16 to 36 mM acetic acid. Cellulolytic enzymes in the supernatant were stable at 2°C under aerobic conditions for up to 4 weeks and had the ability to hydrolyze carboxymethyl cellulose, a microcystalline cellulose, cellobiose, xylan, and filter paper to reducing sugars.     

Spring migration of some anadromous freshwater fish species in the northern Bothnian Sea

Quantitative estimations of spring migrating fish have been made in the mouth part of the small coastal river Ängerån which flows into the northern Bothnian Sea (63°35N, 19°50E). In 1981 nearly 3 000 fish were counted ascending to the spawning grounds in the lower reaches of the Ängerån. These species, such as pike, perch, roach and ide, adapted to the oligohaline environment in the Bothnian Sea for most of the year, migrate to spawn in the coastal stream. The reason for these migrations can be interpreted to indicate that the Ängerån offers more favourable water temperature conditions at spawning time compared with the Bothnian Sea, which is ice-covered up to the beginning of May. The most important result of the investigation in the Ängerån is that these fish species, in the same way as the salmonids, return to theirhome-stream every year as adults.Andreasson & Petersson (1982) listed 69 species of fish in the oligohaline Gulf of Bothnia (Table 1) where salinity varies from 2 near the mouth of the River Torneäly to 6 in the vicinity of the Åland Islands (Fig. 1). The fish fauna comprises freshwater and marine species, fish migrating between brackish and freshwater rivers and streams, and recently introduced non-endogenous species.Andreasson & Petersson (1982) designated only five species as anadromous migrators, whereas our studies show that 11 species migrate from the sea to spawn in the Ängerån, a small river discharging into the northern Bothnian Sea (Fig. 2). Earlier reports on these migrations have been given by Berglund (1978) and Johnson (1978, 1982) and for another small stream in the area by Berg (1982).The present paper describes the annual spring migrations of pike, perch, roach and ide between the northern Bothnian Sea and the Ängerån, for the year 1981.     

Genetics of the peroxidase isoenzymes in Petunia

Summary Three electrophoretic variants of the peroxidase b isoenzymes in Petunia have been found. The encoding gene prxB is shown to be located on chromosome I by its linkage with the gene Hfl. Analysis of prxB heterozygotes showed a gradual increase of the electrophoretic mobility of all three PRXb allozymes during development and differential expression in enzyme activity of three prxB alleles. The location of prxB on chromosome I was confirmed by an allelic dosage effect in trisomies I, trisomie segregation and the construction of trisomies I with triple-banded PRXb phenotype. From telotrisomic analysis it was concluded that prxB and Hfl are located on the same arm of chromosome I. The unexpected linkage of prxB and Hfl with the gene Fl in one of the crosses was suggested to be caused by a translocation in line SI, involving the gene Fl.     

IS50-mediated inverse transposition. Discrimination between the two ends of an IS element

The crystal and molecular structure of l-pyroglutamyl-β-(2-thienyl)-l-alanyl-l-prolinamide, < Glu-Thi-Pro-NH₂(Thi²-TRH), C₁₇H₂₂N₄O₄S, has been determined from X-ray diffraction data. Thi²-TRH is a highly active analogue of thyroliberin, a thyrotropin-releasing hormone (TRH), in which the imidazole ring of the central histidine moiety in the natural hormone has been replaced by a 2-thienyl group. Thi²-TRH crystallizes from water in the monoclinic space group P2₁, $\text{a = 9.340(1)}\text{A}\text{?}\text{, b = 21.961(3)}\text{A}\text{?}\text{, c = 9.449(1)}\text{A}\text{?}$ and β = 109.58(1) °, with two molecules per asymmetric unit. These independent molecules, A and B, have the same general backbone conformation with the φ₂, ψ₂ and ψ₃ torsional angles close to ?90 °, +120 ° and +150 °, respectively, but they show different magnitudes of rotational disorder in the thiophene ring as well as a certain disorder in the pyrrolidine ring. A and B are cross-linked by four interchain hydrogen bonds, forming a two-stranded antiparallel β-pleated sheet structure. The molecules in these dimer fragments are further hydrogen-bonded to successive translated molecules along the a and c axes, forming a pronounced two-dimensional predominantly hydrophobic layer structure. These layers, in which the atoms are almost equally arranged on both sides, are separated by ordinary van der Waals' distances. A close correlation between the molecular conformation in the solid state and the preferential conformation in solution is found. It is concluded that the crystalline structure of Thi²-TRH possesses structural features which may be of relevance in the hormone-receptor interaction process.     

Structure and stability of transposon 5-mediated cointegrates

We have determined the structure of a set of independently derived, Tn5-mediated cointegrates and examined the stability of several examples. A variety of cointegrate structures was found, including those mediated by the entire compound transposon, and those mediated by a single flanking IS50 element, which was always IS50-R, and never IS50-L. IS50-R but not IS50-L is reported to code for a protein(s) required for transposition. This finding confirms that IS50-L is relatively inactive and suggests that the active transposition protein(s) acts largely in cis on IS50-R. Another class of cointegrate was created by inverse transposition of Tn5 (using the inside ends of the flanking elements). In addition, we found an unexpectedly large set of cointegrates, in which the joint between the two plasmids was not adjacent to the transposon. All cointegrates analysed were found to be stable. This suggests that Tn5, unlike the transposon Tn3, does not transpose via an obligate cointegrate intermediate. This finding is compared to previous results with Tn5 and Tn9, and is discussed in terms of current models of transposition.     

The role of Ca2+ and cyclic AMP in the phosphorylation of rat-liver soluble proteins by endogenous protein kinases

Rat liver soluble proteins were phosphorylated by endogenous protein kinase with [gamma-32P]ATP. Proteins were separated in dodecyl sulphate slab gels and detected with the aid of autoradiography. The relative role of cAMP-dependent, cAMP-independent and Ca2+-activated protein kinases in the phosphorylation of soluble proteins was investigated. Heat-stable inhibitor of cAMP-dependent protein kinase inhibits nearly completed the phosphorylation of seven proteins, including L-type pyruvate kinase. The phosphorylation of eight proteins is not influenced by protein kinase inhibitor. The phosphorylation of six proteins, including phosphorylase, is partially inhibited by protein kinase inhibitor. These results indicate that phosphoproteins of rat liver can be subdivided into three groups: phosphoproteins that are phosphorylated by (a) cAMP-dependent protein kinase or (b) cAMP-independent protein kinase; (c) phosphoproteins in which both cAMP-dependent and cAMP-independent protein kinase play a role in the phosphorylation. The relative phosphorylation rate of substrates for cAMP-dependent protein kinase is about 15-fold the phosphorylation rate of substrates for cAMP-independent protein kinase. The Km for ATP of cAMP-dependent protein kinase and phosphorylase kinase is 8 microM and 38 microM, respectively. Ca2+ in the micromolare range stimulates the phosphorylation of (a) phosphorylase, (b) a protein with molecular weight of 130 000 and (c) a protein with molecular weight of 15 000. The phosphate incorporation into a protein with molecular weight of 115 000 is inhibited by Ca2+. Phosphorylation of phosphorylase and the 15 000-Mr protein in the presence of 100 microM Ca2+ could be completely inhibited by trifluoperazine. It can be concluded that calmodulin is involved in the phosphorylation of at least two soluble proteins. No evidence for Ca2+-stimulated phosphorylation of subunits of glycolytic or gluconeogenic enzymes, including pyruvate kinase, was found. This indicates that it is unlikely that direct phosphorylation by Ca2+-dependent protein kinases is involved in the stimulation of gluconeogenesis by hormones that act through a cAMP-independent, Ca2+-dependent mechanism.