Über die Einwirkung Hypotonischer Medien auf das Zwischenhirn-Hypophysensystem von Mugil und Gobius

Zusammenfassung Bei den Teleostiern Mugil und Gobius wurden die Veränderungen des Zwischenhirn-Hypophysensystems in veränderten Außenmedien (hypotonisches Seewasser) histologisch untersucht.Beim langsamen Überführen aus Seewasser von 38,4 S in Seewasser von 25,4 und 20,8 S nahm das Sekret nicht, wie erwartet, zu. Im Nucleus praeopticus und im Hinterlappen der Hypophyse erfolgt eine starke Verringerung des Sekretbestandes (Mugil). Erst in Seewasser von weit niedrigerem Salzgehalt (6,3 und 1, 4) nimmt das Sekret im Hypophysenhinterlappen wieder zu (Gobius). Bei 6 S ist eine intensive Sekretableitung im N.p. und Tractus praeopticohypophyseus zu beobachten. Eine Deutung der Ergebnisse wird versucht, indem das weitgehend konstante Innenmedium mit den wechselnden Außenfaktoren in Beziehung gebracht wird.Die Befunde weisen darauf hin, daß das Zwischenhirn-Hypophysensystem auch bei Teleostiern an der Regulation des Wasserhaushaltes beteiligt ist.Der Deutschen Forschungsgemeinschaft danke ich für die Gewährung eines Stipendiums, das mir den Aufenthalt in Neapel ermöglichte. Mein besonderer Dank gilt auch den Herren Prof. Dr. Remane, Prof. Dr. Bargmann und Dozent Dr. Schiebler für ihre Unterstützung.     

Utilization of exogenous sugars by excised maize embryos in culture

Sucrose was markedly superior to fructose and glucose in promoting growth of plantlets from immature maize embryos. The elongation of roots is shown to be more sucrose dependent than that of shoots. On the other hand, the exogenous sucrose was less effective than fructose as substrate for carbohydrate catabolism and for the synthesis of alcohol-insoluble compounds at the beginning of embryo cultivation. The absorbed fructose was found to be rapidly converted to sucrose and the level of endogenous sucrose derived from sugar supplied to the medium was higher in fructosethan in sucrose-fed embryos. The preferential utilization of fructose over sucrose, however, declined with the progress of germination which may be related to the decrease in proportion of scutellum in total mass and physiological activity of the embryo.     

The interaction of monomeric actin with two binding sites on Acanthamoeba actobindin

Actobindin was previously shown to be an 88-residue polypeptide (Mr 9761) with an internal tandem repeat of 33-34 amino acids. Sedimentation equilibrium experiments have confirmed this Mr for native actobindin. Pyreneglyoxal-labeled actobindin had a similar Mr by sedimentation equilibrium analysis and bound to actin in a manner qualitatively similar to unmodified actobindin as determined by gel electrophoretic analysis of covalently cross-linked products. The stoichiometry of the actin-actobindin interaction was determined from the change in apparent Mr of pyrene-glyoxal-labeled actobindin in the presence of actin, as determined by scanning the ultracentrifuge cell at a wavelength that detected only the labeled protein. These data were consistent with the formation of a complex containing two actin and one actobindin molecules. The overall KD describing the binding of the first actin to either of the two sites on actobindin was 3.3 microM. The binding constant for the second actin suggested either negative cooperativity or inequality of the two actin-binding sites. Similar binding constants were obtained by analysis of the fluorescence enhancement that occurred when actobindin bound to actin labeled with either pyrene iodoacetamide or 4-(N-iodoacetoxyethyl-N-methyl)-7-nitrobenz-2-oxa-1,3-diazole. Cross-linking experiments with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide and N-hydroxy-sulfosuccinimide qualitatively agreed with predictions made from a two-binding site model. Additionally, both the fluorescence and cross-linking experiments suggested that the interaction of the two actin molecules may contribute to the stability of the heterotrimeric complex.     

A [Na+]o-independent, pHo-dependent mechanism for reduction of intracellular [Ca2+] after influx through Ca2+ channels in mouse pituitary cells

The effect of extracellular pH (pHo) on the duration of calcium-dependent chloride currents (ICl(Ca] was studied in voltage clamped AtT-20 pituitary cells. ICl(Ca) was activated by Ca2+ influx through plasma membrane Ca2+ channels, which were opened by step depolarization to voltages between -20 and +60 mV. Increasing pHo from 7.3 to 8.0 reversibly prolonged ICl(Ca) tail currents in perforated patch recordings from cells bathed in both Na(+)-containing and Na(+)-free solutions. This prolongation was prevented in standard whole cell recordings when the pipette solution contained 0.5 mM EGTA. The effects of raised pHo were not due to alteration of intracellular pH, since tail current prolongation still occurred when intracellular pH was buffered at 7.3 with 80 mM HEPES. The prolongation of ICl(Ca) at pHo 8 could not be accounted for by a direct action on Ca2+ channels, since tail currents were prolonged when pHo was changed rapidly during the tail current, after all Ca2+ channels were closed. The effects of increasing pHo on ICl(Ca) also could not be explained by a direct action on Cl- channels, since changing to pHo 8 did not prolong Cl- tail currents when intracellular Ca2+ concentration [( Ca2+]i) was fixed by EGTA in whole cell recordings. Raising pHo did, however, prolong depolarization-evoked [Ca2+]i transients, measured directly with the Ca2+ indicator dye, fura-2. Taken together, these data demonstrate the presence of a Na(+)-independent, pHo-sensitive mechanism for reduction of [Ca2+]i after influx through Ca2+ channels. This mechanism is associated with the plasma membrane, and is active on a time scale that is relevant to the duration of single action potentials in these cells. We suggest that this mechanism is the plasma membrane Ca2+ ATPase.     

Distribution and complementarity of hydropathy in multisubunit proteins

A survey of 40 multisubunit proteins and 2 protein-protein complexes was performed to assay quantitatively the distribution of hydropathy among the exterior surface, interior, contact surface, and noncontact exterior surface of the isolated subunits. We suggest a useful way to present this distribution by using a "hydropathy level diagram." Additionally, we have devised a function called "hydropathy complementarity" to quantitate the degree to which interacting surfaces have matching hydropathy distributions. Our survey revealed the following patterns: (1) The difference in hydropathy between the interior and exterior of subunits is a fairly invariant quantity. (2) On average, the hydropathy of the contact surface is higher than that of the exterior surface, but is not greater than that of the protein as a whole. There was variation, however, among the proteins. In some instances, the contact surface was more hydrophilic than the noncontact exterior, and in a few cases the contact surface was as hydrophobic as the protein interior. (3) The average interface manifests significant hydropathy complementarity, signifying that proteins interact by placing hydrophobic centers of one surface against hydrophobic centers of the other surface, and by similarly matching hydrophilic centers. As a measure of recognition and specificity, hydropathy complementarity could be a useful tool for predicting correct docking of interacting proteins. We suggest that high hydropathy complementarity is associated with static inflexible interactions. (4) We have found that some subunits that bind predominantly through hydrophilic forces, such as hydrogen bonds, ionic pairs, and water and metal bridges, are involved in dynamic quaternary organization and allostery.     

The covalent structure of Acanthamoeba actobindin

Actobindin is a protein from Acanthamoeba castellanii with bivalent affinity for monomeric actin. Because it can bind two molecules of actin, actobindin is a substantially more potent inhibitor of the early phase of actin polymerization than of F-actin elongation. The complete amino acid sequence of 88 residues has been deduced from the determined sequences of overlapping peptides obtained by cleavage with trypsin, Staphylococcus V8 protease, endoproteinase Asp-N, and CNBr. Actobindin contains 2 trimethyllysine residues and an acetylated NH2 terminus. About 76% of the actobindin molecule consists of two nearly identical repeated segments of approximately 33 residues each. This could explain actobindin's bivalent affinity for actin. The circular dichroism spectrum of actobindin is consistent with 15% alpha-helix and 22% beta-sheet structure. A hexapeptide with sequence LKHAET, which occurs at the beginning of each of the repeated segments of actobindin, is very similar to sequences found in tropomyosin, muscle myosin heavy chain, paramyosin, and Dictyostelium alpha-actinin. A longer stretch in each repeated segment is similar to sequences in mammalian and amoeba profilins. Interestingly, the sequences around the trimethyllysine residues in each of the repeats are similar to the sequences flanking the trimethyllysine residue of rabbit reticulocyte elongation factor 1 alpha, but not to the sequences around the trimethyllysine residues in Acanthamoeba actin and Acanthamoeba profilins I and II.     

The effects of cytochalasins on actin polymerization and actin ATPase provide insights into the mechanism of polymerization

Substoichiometric concentrations of cytochalasin D inhibited the rate of polymerization of actin in 0.5 mM MgCl2, increased its critical concentration and lowered its steady state viscosity. Stoichiometric concentrations of cytochalasin D in 0.5 mM MgCl2 and even substoichiometric concentrations of cytochalasin D in 30 mM KCl, however, accelerated the rate of actin polymerization, although still lowering the final steady state viscosity. Cytochalasin B, at all concentrations in 0.5 mM MgCl2 or in 30 mM KCl, accelerated the rate of polymerization and lowered the final steady state viscosity. In 0.5 mM MgCl2, cytochalasin D uncoupled the actin ATPase activity from actin polymerization, increasing the ATPase rate by at least 20 times while inhibiting polymerization. Cytochalasin B had a very much lower stimulating effect. Neither cytochalasin D nor B affected the actin ATPase activity in 30 mM KCl. The properties of cytochalasin E were intermediate between those of cytochalasin D and B. Cytochalasin D also stimulated the ATPase activity of monomeric actin in the absence of MgCl2 and KCl and, to a much greater extent, stimulated the ATPase activity of monomeric actin below its critical concentration in 0.5 mM MgCl2. Both above and below its critical concentration and in the presence and absence of cytochalasin D, the initial rate of actin ATPase activity, when little or no polymerization had occurred, was directly proportional to the actin concentration and, therefore, apparently was independent of actin-actin interactions. To rationalize all these data, a working model has been proposed in which the first step of actin polymerization is the conversion of monomeric actin-bound ATP, A . ATP, to monomeric actin-bound ADP and Pi, A* . ADP . Pi, which, like the preferred growing end of an actin filament, can bind cytochalasins.