Halide binding by the purified halorhodopsin chromoprotein. I. Effects on the chromophore

The halorhodopsin chromoprotein, a retinal-protein complex with an apparent molecular mass of 20 kilo-daltons, exhibits all of the halide-dependent effects found for the chromophore of functional halorhodopsin in cell envelope vesicles. With increasing halide concentration (a) an alkali-dependent 580/410 nm chromophore equilibrium (attributed to reversible deprotonation of the retinal Schiff's base) is shifted toward the 580-nm chromophore and (b) the flash-induced photocycle proceeds increasingly via P520, rather than via P660. The halide-binding site(s) responsible for these effects must reside, therefore, in the chromoprotein. Chloride and bromide are about equivalent, but iodide is much less effective in these effects and in being transported. Several other anions, i.e. thiocyanate, nitrate, phosphate, and acetate, affect the absorption maximum of the chromophore but do not allow the production of P520 upon flash illumination and are not transported. However, these ions appear to compete with chloride in the flash experiments. These observations suggest that binding of anions to a relatively nonspecific site affects the protonation state of the Schiff's base in the chromophore. Either this site directly or a more specific site, connected to the first one by a sequential pathway, is involved with the photocycle intermediates and with chloride transport by halorhodopsin.     

Translational control of insulin biosynthesis. Evidence for regulation of elongation, initiation and signal-recognition-particle-mediated translational arrest by glucose.

The biosynthesis of insulin in the islets of Langerhans is strongly controlled at the translational level by glucose. We have used a variety of experimental approaches in efforts to dissect the mechanisms underlying the stimulatory effect of glucose. To assess its effects on rates of peptide-chain elongation, isolated rat islets were labelled with [3H]leucine at different glucose concentrations in the presence or absence of low concentrations of cycloheximide. Under these conditions, at glucose concentrations up to 5.6 mM, endogenous insulin mRNA did not become rate-limiting for the synthesis of insulin, whereas stimulation of non-insulin protein synthesis was abolished by cycloheximide at all glucose concentrations, indicating either that insulin synthesis is selectively regulated at the level of elongation at glucose concentrations up to 5.6 mM, or that at these concentrations inactive insulin mRNA is transferred to an actively translating pool. Glucose-induced changes in the intracellular distribution of insulin mRNA in cultured islets were assessed by subcellular fractionation and blot-hybridization using insulin cDNA probes. At glucose concentrations above 3.3 mM, cytoplasmic insulin mRNA was increasingly transferred to fractions co-sedimenting with ribosomes, and relatively more of the ribosome-associated insulin mRNA became membrane-associated, consistent with effects of glucose above 3.3 mM on both the initiation of insulin mRNA and SRP (signal recognition particle)-mediated transfer of cytosolic nascent preproinsulin to the endoplasmic reticulum. When freshly isolated islets were homogenized and incubated with 125I-Tyr-tRNA, run-off incorporation of 125I into preproinsulin was increased by prior incubation of the islets at 16.7 mM-glucose. The addition of purified SRP receptor increased the run-off incorporation of [125I]iodotyrosine into preproinsulin, especially when the islets had been preincubated at 16.7 mM-glucose. These findings taken together suggest that glucose may stimulate elongation rates of nascent preproinsulin at concentrations up to 5.6 mM, stimulates initiation of protein synthesis involving both insulin and non-insulin mRNA at concentrations above 3.3 mM, and increases the transfer of initiated insulin mRNA molecules from the cytoplasm to microsomal membranes by an SRP-mediated mechanism that involves the modification of interactions between SRP and its receptor.     

Nucleotide pools of growing,synchronized and stressed cultures of Saccharomyces cerevisiae

High pressure liquid chromatography has been used to study the acid soluble nucleotide pool of Saccharomyces cerevisiae under different conditions of growth. ATP, ADP, AMP, NAD, GTP, UTP, UDP, CTP, CDP, and UDP-sugars plus UMP could be separated and were found in concentrations higher than 0.1 mumol per g yeast cell dry weight (= detection limit). During glucose-limited continuous culture the levels of individual nucleotides depended on the growth rate, which was most pronounced with pyrimidine (uridine, cytidine) nucleotides. The energy charge (E.C.) remained high (0.9) at all growth rates (0.07-0.3 h-1). During synchronized growth at a constant growth rate (0.11 h-1) almost all nucleotide levels and the E.C. remained at constant values with the only exception of UDP-sugars and UMP of which increased levels were found during the phase of budding. Under conditions of metabolic stress (addition of antimycin A, deoxyglucose plus iodoacetate) pronounced changes in the levels of purine (adenine and guanine) nucleotides and the E.C. were observed. All other nucleotides were less influenced by these conditions. Only under these conditions IMP accumulation was observed. The results strongly argue against the significance of purine nucleotide or E.C. measurements under viable conditions. In contrast, changes in the levels of pyrimidine nucleotides seem to be indicative of changes in the flux through the metabolic pathways where they act as coenzymes.     

Periodate-induced lymphocyte transformation. 3. Effects of temperature and pH

The optimum conditions for sodium periodate induced lymphocyte transformation occur at low concentrations of NaIO₄, low temperature, pH between 6 and 7.5, and with short periods of exposure to the NaIO₄. Exposures for prolonged times at elevated temperatures or to NaIO₄ at pH above 7.5 result in diminished responses. The finding of maximum lymphocyte response under these conditions is compatible with the proposal that the triggering event in NaIO₄ transformation of lymphocytes is the oxidation and cleavage of the two terminal carbon atoms of sialic acid. Exposure of lymphocytes to NaIO₄ under conditions of higher temperature, higher pH or longer exposure times does not significantly reduce their ability to respond to phytohemagglutinin (PHA) or pokeweed mitogen (PWM). This finding suggests that these mitogens have different target sites from that of NaIO₄ or that they are acting on different populations of lymphocytes.     

Action spectrum for polarotropism of the germ tube of the liverwort Sphaerocarpos Donnellii aust.

Summary A series of dose response curves was worked out in the polarotropically active blue and UV spectral region. These dose response curves show changes in slope as a function of wavelength. In blue the slope values are higher than in UV. As a consequence, both the relative height of the peaks in blue and UV and the fine characteristics in blue of the action spectrum calculated on the basis of these dose response curves change decisively with different response levels taken for calculation. Therefore no decision can be made as to what photoreceptor(s) might be involved. Though at medium response levels the action spectra show similarity with action spectra of other blue-UV-mediated photoresponses, which generally are believed as being indicative of some flavin.     

Die submikroskopische Struktur der Assimilatleitbahnen von Polytrichum commune

Summary In the young part of the stem of Polytrichum commune the protoplasts of the two types of conducting cells, the leptoids and parenchyma cells, are nearly identically equipped with cell organelles and cytoplasmic structures. Both types contain a nucleus, chloroplasts, mitochondria, and dictyosomes. The endoplasmic reticulum builds characteristic cisterns in form of hollow cylinders extending from one end wall to the other. The cisterns are connected with many plasmodesmata, which occur only in the end walls. Leptoids have oblique end walls with 16 to 20 plasmodesmata per m², and parenchyma cells show cross walls perpendicular to the axis with 9 to 12 plasmodesmata per m².Since the leptoids are supposed to be the pathways for the longitudinal transport of assimilates (Eschrich and Steiner, 1967, 1968), it is of interest that early in their development these elements undergo a change in their protoplasmatic structure. Two to 3 cm below the apical cell the protoplasts degenerate and show lysosome-like structures. The endoplasmic reticulum and other structures are deformed or dissolved; the plasmodesmata are constricted by callose deposits. At the same level the parenchyma cells still retain the original structure of their protoplasts.Thus, assimilates moving upward in one row of leptoids may penetrate the whole lumen of the leptoids at lower levels, but they are restricted to the cisterns of the endoplasmic reticulum at higher levels of the stem.