Analysis of the proteins in thymocyte plasma membrane and smooth endoplasmic reticulum by sodium dodecylsulfate-gel electrophoresis

We have purified the plasma membranes and membranes of endoplasmic reticulum from calf and rabbit thymocytes and from calf mediastinal lymph node lymphocytes. We disrupted the cells by the “nitrogen cavitation method” and prepared a microsomal isolate by differential centrifugation. We fractionated this by isopycnic ultracentrifugation in dextran gradients into membrane vesicles, PM₁ and PM₂, most likely derived from plasma membrane and a fraction, ER, most likely originating from endoplasmic reticulum. More than 80% of the microsomal 5′-nucleotidase and acid p-nitrophenylphosphatase concentrates in the PM₁ and PM₂ fractions; alkaline p-nitrophenylphosphatase, another presumptive PM marker, is concentrated in the PM₁ fraction. These data are confirmed by the lacroperoxidase radioiodination of intact rabbit thymocytes followed by subcellular fractionation. The specific content of phospholipids (822 nmoles/mg protein) and cholesterol (1032 nmoles/mg protein) is highest in PM₁ and PM₂ plasma membrane fractions. NADH-oxidoreductase, our endoplasmic reticulum marker, is clearly enriched in gradient pellet.The membrane proteins were separated by electrophoretic molecular sieving in sodium dodecylsulfate-polyacrylamide gel electrophoresis, containing dithiothreitol (sodium dodecylsulfate-polyacrylamide gel electrophoresis). We numbered the 10 major protein components of the “microsomal fraction” (apparent molecular weights between 280000 and 15000) from 1–10 according to their decreasing molecular weights. Of these proteins, those with higher molecular weight, predominantly glycoproteins, appear in the PM₁ fraction, while the endoplasmic reticulum fraction contains mainly low molecular weight components.     

Interaction of muscle glycogen phosphorylase with pyridoxal 5'-methylenephosphonate

Rabbit muscle glycogen phosphorylase (EC 2.4.1.1) was reconstituted with pyridoxal 5′-methylenephosphonate with ca. 25% restoration of enzymatic activity. The modified enzyme has very similar chemical and physical properties to native phosphorylase including UV and fluorescence spectra, quaternary structure, high energy of activation in the reconstitution reaction, optimum pH and susceptibility to phosphorylase kinase in the b to a conversion. While V_max is reduced to ca. one-fifth, affinities for the substrate glucose 1-P and the effector AMP are increased. This is the first analog of pyridoxal 5′-P modified in the 5′-position found to restore catalytic activity to apophosphorylase.     

Synthèse, à partir du D-galactose,d'un dérivé protégé de la célestosamine (6-amino -6,8-didésoxy-7-0-méthyl-D-érythro-D-galact o-octose), sucre de certains antibiotiques du groupe de la lincomycine

Treatment of 1,2:3,4-di-O-isopropylidene-α-D-galactopyranuronyl chloride with diazoethane gave a diazoketone that was converted with methanol in the presence of boron trifluoride into a mixture of keto-ethers. One of these keto-ethers was transformed by reduction of its oxime and N-acetylation into a blocked derivative of celestosamine, which was found to be identical with 5 prepared from a natural source, i.e. lincomycine. The properties of the other two keto-ethers are in agreement with a (D or L)-glycero-L-altro configuration (14) for one of them, and with the presence of a 3-oxetanone ring in the structure (18) of the other one.     

Interrelationships between polyamines and nucleic acids

Summary The distribution of radioactivity from ³H-putrescine was studied in intact and degenerated sciatic nerves, and spinal ganglia of rats by means of high resolution autoradiography. During the first three days after the administration of the labeled putrescine, the main proportion of radioactive material in the nerves was represented by spermidine and putrescine. Both, in intact and degenerating nerves, developed silver grains were deposited in all cellular components of the nervous tissue, the myelin sheath being markedly tagged. Perineural tissue was also labeled considerably, however, there was no significant amount of label in the extracellular space and in the collagen fibrils. The possible physiological significance of putrescine and spermidine in myelin and in other cellular components of nerves is discussed.Herrn Prof. Dr. W. Krücke zum 60. Geburtstag gewidmet.     

The molecular organization of nerve membranes

Summary Plasma membranes were isolated from two types of squid nerves which have morphologically, different ratios of axolemma/Schwannlemma (A/S). These membranes were studied by means of differential and density gradient centrifugation.Thoroughly dissected giant axons were used as membrane source having low A/S ratio. Retinal fibers were used as membrane source with high A/S ratio. A similar procedure for the isolation of the plasma membranes was used for both types of squid axons.Differential centrifugation showed that at 1,500×g, the yield of membrane enzymes (Na, K-ATPase and NADH-ferricyanide oxidoreductase) from giant axon homogenates was 2 to 5 times greater than from retinal nerve homogenates, but at 105,000×g the opposite was the case, the yield from retinal axons being about two times greater. Thus, the major part of the membrane material from the retinal nerve seems to be less dense than the membrane material from giant axons.The behavior of the 105,000×g fraction from both types of fibers was studied by determining protein Na, K-ATPase, and NADH-oxidoreductase along a lineal sucrose gradient (10 to 40%; centrifuged at 40,600×g for 90 min). By any of the three measurements, retinal axons yielded a greater amount (2:1) of plasma membranes sedimenting at low sucrose concentration (16 to 25%) as compared to that observed at high sucrose concentration (35 to 38%). Giant axons, on the contrary, yielded a higher proportion of membranes (2.5:1) sedimenting at high sucrose concentrations (over 40%).The experimental data indicate that a different cellular origin can account for the behavior of nerve membranes along lineal gradient centrifugation. The membranes floating at low sucrose concentration (light membranes) can be tentatively ascribed to the axolemma; the membranes found at high sucrose concentration (heavy membranes) to the Schwannlemma and basement membranes.In accord with their high A/S morphological ratio, squid retinal axons yielded 5 times more light membranes (axolemma) than dissected giant axons.     

Molecular sieving of red cell membranes during gradual osmotic hemolysis

Summary Rat red blood corpuscles were held stationary with respect to a continuously flowing solution in either a specially constructed centrifuge or in glass filters. The concentration of the solution was gradually decreased to cause the swelling and subsequent gradual osmotic hemolysis of the cells. The passage of the intracellular molecules —potassium, adenylate kinase, and hemoglobin—across the cell membranes and into the flowing solution was determined as a function of time. Ions and molecules begin passage across the membranes in the order of increasing molecular size. The initial flow of potassium is followed by the initial flows of hemoglobin and adenylate kinase. The flow of hemoglobin has been interpreted as the flows of hemoglobin monomers, dimers, and tetramers such that the time sequence is: potassium; hemoglobin monomer; adenylate kinase/hemoglobin dimer; and finally, hemoglobin tetramer. It is concluded that the stressed cell membrane has molecular sieving properties and that the exclusion limit (effective hole size) increases as a function of time during the initial stages of gradual osmotic hemolysis. The process of gradual osmotic hemolysis is discussed in terms of molecular sieving through stress-induced effective membrane holes. It is suggested that a portion of the membrane protein might form an elastic network which would account for the gradual increase in size and apparent homogeneity of the effective holes.This work was prepared under the auspices of the U.S. Atomic Energy Commission.     

Methodik und Ergebnisse der Erforschung des Schwarmverhaltens von Fischen mit der Tauchmethode

Zusammenfassung 1. Die marinen Schwarmfische kommen ausschließlich als echter Schwarmverband vor, solange sie sich im pelagischen Raum aufhalten. Bei der Nahrungsaufnahme und in der Nacht wird der Schwarmverband aufgelöst.2. Die Reaktionen innerhalb eines Schwarmes breiten sich nach bestimmten Gesetzmäßigkeiten aus und sind zeitlich meßbar.3. Eine Reaktion innerhalb eines Schwarmes kann von verschiedenen Stellen aus ausgelöst werden.4. Leitfische im Schwarm existieren nicht.5. Die echten Schwarmfische des Süßwassers entsprechen in ihren Reaktionen den Schwarmfischen des Meerwassers. Im Süßwasser treten außerdem noch andere Sozietätsformen auf.6. Die SüßwasserartAlburnus alburnus eignet sich als Modellfisch für Untersuchungen der Reaktionen von Schwärmen auf Fanggeräte und für die weitere Erforschung der Schwarmgesetzmäßigkeiten.

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Methods and results of studies on the schooling behaviour of fishes using the diving method

Knowledge of schooling behaviour is becoming increasingly important in deep-sea fishery. Observations and experiments were conducted to elucidate standard responses of some schooling fishes in situ and in net aquaria using diving apparatus, cine and photocameras. The 5 species of sea-water schooling fishes studied show mainly uniform behaviour. Using 4 measurable criteria it was possible to examine and time the pattern of behaviour within the school. The way a response spreads within a school differs, and 3 basic types can be differentiated. Of 6 freshwater fish species studied,Alburnus alburnus is the only obligatory school fish; it is the only one which shows values within the range recorded for the sea-water school fishes studied. The other freshwater fishes examined are obligatory school fishes only in their juvenile phases. With increasing age they conform with the other 4 types of social behaviour differentiated in the study. The recorded reaction times of schools and the responses to nets of varying mesh size (meshes up to 1 m length were avoided by schools) are important for fishery. Qualitative intensification gains in importance by the frequently observed unification of different schools. The freshwater-livingAlburnus alburnus is a suitable model fish for studies on the conformity of school behaviour, as well as for experiments with new fishing gear and methods.