Search

The effect of red light and far-red light on the appearance of mRNA for the small and large subunit of ribulose-1,5-bisphosphate carboxylase in rye seedlings has been analysed. Irradiation of etiolated seedlings with a pulse of red light increased the mRNA level of the small subunit by a factor of 7-10 and that of the large subunit by a factor of 3. A pulse of far-red light resulted in a much lower, but measurable increase. The effect of red light is reverted by an immediate far-red pulse, demonstrating the classical phytochrome response.In vitro transcription experiments with nuclei isolated from red light-treated seedlings showed that the transcription rate of the small subunit mRNA was increased only by a factor of 1.5-2.5, indicating posttranscriptional as wells as transcriptional regulations of the small subunit of ribulose-1,5-bisphosphate carboxylase.

The orientation of the light-driven chloride pump, halorhodopsin, in the membrane was determined using antibodies directed against a synthetic peptide which represents the C-terminal segment of the protein. Antibodies against this decapeptide did not bind to right-side-out cell vesicles. Partial inversion by sonication or lysis under low salt conditions exposed this COOH-terminal antigenic site. Antibody binding was removed by preincubation with the decapeptide. The COOH terminus of the molecule is therefore located on the cytoplasmic surface of the membrane.

Based on quantumchemical MNDOC calculations it is shown that the ground-state properties of a retinal Schiff base depend sensitively on its protonation state and charge environment. This is exemplified for the equilibrium geometry, for the distribution of partial charges and, in particular, for the thermal isomerization barriers around the pi-bonds. It is demonstrated that a protein, by protonating the retinal Schiff base and by providing one or two negative ions in its environment, can reduce double-bond isomerization barriers from 50 kcal/mol for the unprotonated compound to approximately 5 kcal/mol and can increase single bond barriers from 5 kcal/mol to approximately 20 kcal/mol. Thereby, the specific location of the ions relative to the polyene chain of the protonated retinal Schiff base determines the barrier heights. The results explain the ground-state isomerization reactions of retinal observed in bacteriorhodopsin and in squid retinochrome.

Thermoplasma acidophilum and Sulfolobus acidocaldarius contain coenzyme A-acylating 2-oxoacid:ferredoxin oxidoreductases similar to those found in halophilic archaebacteria. A common feature of these enzymes is the formation of a free radical intermediate in the course of the catalytic cycle. The electron-accepting ferredoxins and a similar protein from Desulfurococcus mobilis have been purified and characterized. In contrast to the [2Fe-2S] ferredoxin of Halobacterium halobium, the ferredoxins of thermoacidophilic archaebacteria most likely contain two [4Fe-4S]2 + (2 + .1 +) clusters per molecule. Properties of these proteins are compared with respect to the evolution of archaebacteria.

Retinal analogues in which the 13-methyl group is replaced by H, C(2)H(5), CF(3), and OCH(3) residues are studied by means of quantumchemical modified neglect of diatomic overlap-correlated version (MNDOC) calculations. The analogues are suitable to test the stereochemical mechanism of proton pumping in bacteriorhodopsin. The results explain the proton-pumping activities of bacterio-opsin reconstituted with these analogues and elucidate the decisive role of retinal's ground-state intramolecular properties in the pump cycle of bacteriorhodopsin.

Using three different techniques, isopentenyladenosine was identified and quantitated in an anise cell line (Pimpinella anisum L.), growing on a medium without cytokinin. A method to quantitate cytokinins was developed which utilizes extraction of cytokinins in the presence of a deuterated reference compound and subsequent quantitation of the cytokinins by single-ion monitoring on a gas chromatography-mass spectrometry system. The results were compared with those obtained by radioimmunoassay as well as a bioassay. Quantitations by gas chromatography-mass spectrometry and radioimmunoassay correlated well, whereas the estimates by the bioassay gave considerably lower values.

The proton motive force across the cell membrane of halobacterial cells has been estimated and compared to intracellular values of ATP, ADP, and inorganic phosphate concentrations with respect to the chemiosmotic hypothesis. The accumulation of 14C-labeled indicator substances, triphenylmethylphosphonium for the membrane potential and 5,5-dimethyloxazolidine-2,4-dione for the pH difference between the cell interior and the medium, has been measured in the cells. Values up to 270 mV for the proton motive force have been found in cells pretreated with N,N'-dicyclohexylcarbodiimide (DCCD, 10(-4) M, 30 degrees C, 12 h). Upon illumination a high membrane potential is generated, which is then gradually replaced by a large pH difference. Cells treated with lower DCCD concentrations show only an enhancement of membrane potential upon illumination; the pH difference remains at a low level. Under anaerobic dark conditions, untreated cells maintain a proton motive force of 120-140 mV, which is equilibrated with the intracellular levels of ATP, ADP, and inorganic phosphate. The pH gradient is 1 unit at pH 6 but 0 at pH 8. The membrane potential is low (60-80 mV) at pH 6 and high (120-130 mV) at pH 8. We propose that the proton translocating ATPase compensates for the lowered pH difference at high external pH values by enhancing the membrane potential. The concentration difference of the potassium ions influences the proton motive force and the intracellular ATP levels, apparently via its action on the membrane potential. When the difference of the chemical potential of the potassium ion, expressed in millivolts, exceeds the preexisting membrane potential, the intracellular ATP level is enhanced. When the difference of the chemical potential of the potassium ion (millivolts) is smaller than the membrane potential, the ATP level is decreased.

Yeast fatty acid synthetase possesses very low malonyl-CoA decarboxylase activity. Treatment with iodoacetamide, while abolishing synthetase activity, induces a strong malonyl decarboxylase activity which, in turn, can be inhibited by N-ethylmaleimide. Kinetic analysis shows that the emergence of the decarboxylase activity is synchronized to the disappearance of the fatty-acid-synthesizing activity and thus, is due to carboxamidomethylation of the peripheral SH-groups of the multienzyme complex. Strong decarboxylase activity was also found after treatment of the synthetase with methylmalonyl-CoA. A hypothetical scheme is proposed which explains the origination of the decarboxylase activity as a consequence of conformational changes of the condensing enzyme component which happen when the peripheral SH-group is acylated or alkylated.

The gene encoding the alpha-subunit of the Na+ pump oxalacetate decarboxylase of Klebsiella pneumoniae was cloned and sequenced. The deduced primary structure of the protein was confirmed by protein sequencing of about 30% of the polypeptide chain. The gene has a GC content of 67% and codes for 596 amino acids. The N-terminal methionine is removed in the mature protein which has a calculated molecular mass of 63,600 daltons. The protein consists of two different domains that are connected by a stretch of amino acid residues susceptible to proteolytic cleavage. Limited proteolysis of the native enzyme with trypsin produced fragments of about 51 kDa and 10.2 kDa, the latter of which started with valine 491 and contained the biotin prosthetic group. Peptide sequencing indicated binding of the biotin prosthetic group to lysine 561, 35 residues from the C terminus. The decarboxylase contains an extended alanine- and proline-rich region (positions 502-532) on the N-terminal side of the 10.2-kDa biotin domain. This sequence includes a total of 16 alanine and 9 proline residues.

Halorhodopsin (HR), the light-driven chloride pump in halobacteria, was purified in the denatured as well as in the native state and chemically cleaved into peptide fragments. Isolation of peptide and liquid phase sequencing yielded approximately 20% of the halo-opsin (HO) structure in non-overlapping peptides. Chemically synthesized oligodeoxynucleotides corresponding to a peptide sequence obtained from both HR preparations were used to screen a cosmid gene bank of Halobacterium halobium strain L-33. A positive clone contained cosmid pAB H47 which by subcloning and nucleotide sequencing was shown to encode at least part of the HO gene.

The crystallization of purified fatty acid synthetase from yeast is facilitated by seeding techniques. The first seed crystals appeared as particles with almost negligible enzymatic activity in a solution that had been left at 4 degrees C for 15 months. Subsequently, the crystallization time has been reduced to two days, and the crystals now isolated have retained their full enzymatic activity. The loss of synthetase activity that can result from prolonged exposure during crystallization is attributable specifically to damage of the enzyme component responsible for condensation activity. Crystalline synthetase is identical to purified but noncrystalline synthetase by all criteria so far examined.

The purple membrane of Halobacterium halobium contains only one protein, bacteriorhodopsin, which closely resembles the visual pigments of animals. Light flashes cause a rapid transient shift of its absorption maximum from 560 to 415 nm. This shift is accompanied by release and uptake of protons. Respiring cells acidify the medium in the dark; if they contain purple membrane their O(2) consumption is reduced in the light. Starved or anaerobic cells containing purple membrane, in the absence of any apparent source of energy, generate and maintain a proton gradient across the cell membrane as long as they are exposed to light. We postulate that the light-generated proton gradient arises from a vectorial release and uptake of protons by bacteriorhodopsin, which is suitably oriented in the cell membrane and under continuous illumination oscillates rapidly between the long- and short-wavelength form. Preliminary results indicate that the gradient in H. halobium plays the central role in energy coupling attributed to such electrochemical gradients by Mitchell's chemiosmotic theory.