A new double-labelling procedure for determination of amino acid composition: application to bacteriorhodopsin

A new double-labelling procedure for amino acid analysis which requires only routine chromatographic equipment is described. When 1-fluoro-2,4-dinitro[3H]benzene is reacted with a mixture of 14C-labelled amino acids followed by reaction with the same 14C-labelled amino acid mixture diluted with an unlabelled sample of amino acids, the 3H:14C ratio in the resulting 2,4-dinitrophenyl (DNP) amino acid derivatives of the diluted sample will be increased in proportion to the quantity of unlabelled amino acid in the diluted sample. This procedure gave reliable results when applied to the known proteins insulin and lysozyme. The procedure is most advantageous when applied to amino acids which are unstable during acid hydrolysis or present in low molar fractions. When applied to the analysis of the bacteriorhodopsin in Halobacterium cutirubrum, this procedure showed the presence of one histidine residue and four tryptophan residues per mole protein but no cystine or cysteine; in general, the analyses obtained were consistent with those originally reported by Oesterhelt, D. and Stoeckenius, W. (1971) (Nature (London) New Biol. 233, 149-152) for bacteriorhodopsin of H. halobium.     

Effects of alpha-glycerophosphate and of palmityl-coenzyme A on lipid synthesis in yeast extracts

White, David (Ames Research Center, Moffett Field, Calif.), and Harold P. Klein. Effects of alpha-glycerophosphate and of palmityl-coenzyme A on lipid synthesis in yeast extracts. J. Bacteriol. 91:1218-1223. 1966.-The incorporation of acetate into fatty acids, but not into nonsaponifiable lipids, was stimulated by alpha-glycerophosphate in a supernatant fraction of Saccharomyces cerevisiae, obtained after centrifugation at 86,000 x g for 60 min. There was a pronounced effect at concentrations below 2 mm, but at concentrations above 5 mm alpha-glycerophosphate was relatively less stimulatory. alpha-Glycerophosphate markedly increased the percentage of esterified fatty acids among the products, and the formation of both saturated and unsaturated fatty acids was stimulated. Palmityl-coenzyme A inhibited fatty acid synthesis, affecting the formation of unsaturated acids more severely than saturated acids. In the presence of sufficient alpha-glycerophosphate to alleviate these inhibitions, palmityl-coenzyme A still reduced the formation of certain unsaturated fatty acids.     

Isolation of a gene that encodes a new retinal protein, archaerhodopsin, from Halobacterium sp. aus-1

We have cloned and sequenced the gene that encodes archaerhodopsin, a light-driven H+ pump in Halobacterium sp. aus-1 (Mukohata, Y., Sugiyama, Y., Ihara, K., and Yoshida, M. (1988) Biochem. Biophys. Res. Commun. 151, 1339-1345). The nucleotide sequence of this gene contained an open reading frame which corresponded to a protein of 260 amino acids with a molecular mass of 27,851 daltons, including a precursor sequence of 6 amino acids at the amino terminus and 2 amino acids at the carboxyl terminus. The deduced amino acid sequence of archaerhodopsin exhibited 59 and 32% homology to the sequences of bacteriorhodopsin and halorhodopsin, respectively, from Halobacterium halobium. Three charged residues (Asp-121, Asp-218, and Lys-222) are conserved in the transmembrane segments among the three retinal proteins. Residues Asp-91 and Asp-102 which, it has been suggested, may be essential for the pumping of protons (Mogi, T., Stern, L. J., Marti, T., Chao, B. H., and Khorana, H. G. (1988) Proc. Natl. Acad. Sci. U. S. A. 85,4148-4152) are conserved between archaerhodopsin and bacteriorhodopsin.     

Purification and chemical characterization of the rodlet layer of Neurospora crassa conidia.

The rodlet layer of Neurospora crassa macroconidia has been purified and chemically characterized. Sheets of rodlets were released from the conidial surface by vigorously shaking conidia in water. Conidia were removed by filtration and low-speed centrifugation, and the rodlets were recovered from the supernatant by high-speed centrifugation. The rodlet pellet comprised 1.9% of the initial dry weight. Chemical analysis was hampered by the insolubility of the rodlets. They were not solubilized by heating in various protein-denaturing buffers and were only partially dissolved by heating in 1 M NaOH at 100 degrees C for 5 min. Nevertheless, they were found to be largely composed of protein (91%, based on total nitrogen). The major amino acids in acid hydrolysates were aspartic acid, glycine, serine, alanine, half-cystine, and valine. Glucosamine was not detected in acid hydrolysates. The sulfur content was 2.5%, and this could be accounted for in half-cystine and methionine. Carbohydrate comprised just over 2%. The phosphorus content was 0.21%, of which less than one-third was accounted for in phospholipid. The total fatty acid content was 1.0%, most of which could be accounted for by the fatty acids of the phospholipids.     

Endogenous suppression of neutral-active and calcium-dependent phospholipase A2 in human polymorphonuclear leukocytes

Phospholipase A2 activity was measured in homogenized and acid-extracted human polymorphonuclear leukocytes using [1-14C]oleate-labelled autoclaved Escherichia coli as substrate. In whole homogenate and in the supernatant and particular fractions separated by centrifugation at 150,000 X g, phospholipase activity was barely detectable (1-4 pmol/h per 10(6) cell equivalents). By contrast, acid extracts of these fractions contained over 10-times as much phospholipase activity in the dialyzed supernatants (20-300 pmol/h per 10(6) cell equivalents), whereas phospholipase inhibitor(s) were found in the sediment. The acid-solubilized phospholipase A2 activity was absolutely Ca2+-dependent and optimal at pH 7.0-7.5 with 1.0 mM added Ca2+. Addition of the resuspended sediment of the acid extract dose-dependently suppressed phospholipase activity in the supernatant; less than equivalent amounts were sufficient to inhibit 95%. Suppressor activity was lipid-extractable. After thin layer chromatography of lipid extracts, the bulk of inhibitory activity was recovered from the free fatty acid region. Analysis of the fatty acids by gas liquid chromatography showed that 63% were unsaturated. All unsaturated fatty acids tested were potent inhibitors of phospholipase A2 activity (IC50 3-10 microM). Oleoyl-CoA, hydroxyeicosatetraenoic acids and leukotriene D4 were also inhibitory, while methyl oleate, saturated fatty acids and the prostaglandins E2 and F2 alpha had no effect. These in vitro data indicate that neutral-active and calcium-dependent phospholipase A2 in human polymorphonuclear leukocytes is largely suppressed by endogenous inhibitors and suggest that unsaturated fatty acids and some of their metabolites may partly account for this suppressor activity.     

Primary and secondary chloride transport in Halobacterium halobium.

Chloride uptake in intact cells of Halobacterium halobium was characterized by rates of influx and efflux of 36Cl- under conditions of light, respiration, or both. Halobacterial mutant strains with and without retinal transport proteins allowed study of the effects of halorhodopsin and bacteriorhodopsin under illumination. Two structurally independent chloride transport systems could be distinguished: halorhodopsin, the already known light-driven chloride pump, and a newly described secondary uptake system, which was energized by respiration or by light via bacteriorhodopsin.     

Large scale preparation of homogeneous bacteriorhodopsin

Homogeneous bacteriorhodopsin was obtained preparatively (100 mg batches) from purple membrane of Halobacterium halobium cells. The homogeneity of the protein was considerably affected by variations in the growth conditions of the bacteria. Fully matured bacteriorhodopsin having a blocked N-terminus and a homogeneous C-terminus, was reproducibly obtained when cells were grown in a sufficiently aerated medium.     

Partition of cytoplasmic lipides

By means of differential centrifugation three fractions: large granules (L₂), microsomes (M₁), and supernatant fluid (MS), have been isolated from the cytoplasm of mouse liver cells and the contained lipides have been extracted and characterized.The particulate fractions, large granules and microsomes, make up approximately 38% of the total solids of the cytoplasm, but they contain 62% of the total lipide and 85% of the phospholipide. The phospholipide of the particulates has an N:P ratio of 1 while the supernatant phospholipide has an N:P ratio of 1.3, suggesting that the supernatant fluid contains lipides of higher nitrogen content than that of either lecithins or cephalins.The fatty acids of the large granule fraction are highly unsaturated and contain 20% of fatty acids with four double bonds. The unsaturation of the fatty acids of microsomes and the supernatant fluid is comparable, as indicated by the iodine values, but the supernatant fluid contains much more of the fatty acids with one double bond and less of the fatty acids with four double bonds than do the microsomes.     

稻曲病菌厚垣孢子脂肪酸组成的研究

【目的】为探明细胞壁和细胞内脂肪酸成分及含量与细胞抗逆性的关系,【方法】采用酸热法、索氏提取法、有机溶剂法对稻曲病菌的厚垣孢子壁进行脂肪酸提取,并采用气相色谱检测其脂肪酸的组成和含量。【结果】采用酸热法提取脂肪酸效果最好,以该方法提取测定稻曲病菌黄色、黄绿色、黑色厚垣孢子壁饱和脂肪酸相对含量分别为26.92%、17.23%、23.71%,其不饱和脂肪酸相对含量分别为60.46%、61.52%、70.64%;厚垣孢子总(沉淀孢子壁和上清液)饱和脂肪酸相对含量分别为28.87%、21.00%、24.04%,厚垣孢子总不饱和脂肪酸相对含量分别为55.43%、55.87%、63.89%。硬脂酸在厚垣孢子壁中的含量:黄色>黄绿色>黑色;不饱和脂肪酸中顺式-5,8,11,14,17二十碳五烯酸(EPA)在厚垣孢子壁的含量:黑色>黄绿色>黄色。【结论】在3种颜色厚垣孢子中,黑色休眠型厚垣孢子在孢子壁、总不饱和脂肪酸含量均最高,表明不饱和脂肪酸的含量提高,有利于厚垣孢子的休眠越冬。     

2-Hydroxy-5-nitrobenzyl bromide as a specific reagent for tryptophan residues in membrane proteins: bacteriorhodopsin as an example

The use of 2-hydroxy-5-nitrobenzyl bromide for the modification of tryptophan residues in integral membrane proteins is exemplified by its application to bacteriorhodopsin from Halobacterium halobium. Complete elimination of the unreacted reagent requires delipidation of the sample with detergents and posterior chromatography. This method also allows separation of the modified from the unmodified bacteriorhodopsin molecules. Modified molecules have lost the retinal, and are thus bleached, whereas the unmodified molecules appear to retain all the characteristics of solubilized native bacteriorhodopsin.     

The interaction between halogenated anaesthetics and bacteriorhodopsin in purple membranes as examined by intrinsic ultraviolet fluorescence

In the presence of halogenated general anaesthetics such as enflurane and halothane, the spectral properties of the bacteriorhodopsin pigment contained in the purple membranes of Halobacterium halobium are strongly modified. It is reversibly transformed into a red-coloured species absorbing maximally at 480 nm, at the expense of its characteristic 570-nm absorption band. The ultraviolet fluorescence of bacteriorhodopsin has been used to probe the structural modifications that are reflected by this spectral change. Our results show that they are very small and do not perturb the energy transfer dynamics which take place between the aromatic amino acid residues and the retinyl chromophore. The fluorescence properties of anaesthetic-treated bacteriorhodopsin are dominated by the quenching properties of the halogenated hydrocarbon, which are obvious even at anaesthetic concentrations under those needed to induce a spectral change in the bacteriorhodopsin chromophore. This does not rule out direct interaction between anaesthetics and bacteriorhodopsin, but it indicates that the chromophoric site might well not be their primary target.     

Changes in the protonation state of bacterio-opsin during reconstitution of bacteriorhodopsin

Protonation changes of the protein occur during the reconstitution of bacteriorhodopsin from bacterio-opsin and all-trans retinal in the purple membrane of Halobacterium halobium. The protonation changes are conveniently determined from measures of the pH changes after photoisomerisation of 9-cis retinal in apomembrane preparations, which induces the reconstitution. In addition, to the omega-amino group of the lysine which is involved in the condensation of retinal and bacterio-opsin, the dissociation equilibria of at least two other amino acid residues are changed during the reconstitution. The results are consistent with a proposed model of chromophore structure in which an interaction of the Schiff's base occurs with two protonable amino acid residues.     

Time-resolved x-ray diffraction study of photostimulated purple membrane.

A nanosecond resolution laser-driven x-ray source has been used to perform a time-resolved, x-ray diffraction study of the purple membrane of the Halobacterium halobium. Alterations in diffraction patterns have been observed 1 ms after photostimulation, and are interpreted to show disorder of bacteriorhodopsin packing in the plane of the membrane with little bacteriorhodopsin structural change.     

The surface coat of chylomicrons: lipid chemistry

Chylomicrons from the thoracic duct lymph of dogs fed corn oil were isolated by centrifugation and disrupted by either freezing and thawing or rotary evaporation and rehydration. A pellet, representing the surface coat, was isolated by centrifugation. Pellets isolated by freezing and thawing contained a higher percentage of saturated triglycerides than pellets isolated by rotary evaporation; the presence of saturated triglyceride in the pellet was probably an artifact of the preparation of the surface coat material at low temperature. Exchange of free cholesterol between surface and core lipid of chylomicrons was complete within 1 hr. The percentage of cholesterol in pellets of surface material isolated by freezing and thawing was about twice that found for pellets after rotary evaporation at 25-40 degrees C. Cholesteryl ester was not present in the surface lipid and that present in the core lipid did not exchange with serum lipoprotein cholesteryl ester. For phosphatidyl choline, the percentage of linoleic acid in lymph chylomicrons was markedly higher than that in clear lymph or plasma, while the percentage of arachidonic acid was lower. Sphingomyelin of lymph chylomicrons was characterized by very high levels of 16:0 and relatively small percentages of very long-chain fatty acids as compared with clear lymph or plasma. The data are consistent with the view that in lymph chylomicrons: (a) cholesteryl esters are dissolved in a core of triglycerides which contain fatty acids derived primarily from dietary fatty acids, (b) free cholesterol is partitioned between core and surface and is freely exchangeable between the two, (c) the phospholipid fractions are present on the surface and are intracellular in origin.     

Chemosensory responses of Halobacterium halobium.

Responses of Halobacterium halobium cells to chemical stimuli have been shown by a capillary technique. Cells were attacted by D-glucose and several amino acids and repelled by phenol. Certain chemicals, such as acetate, benzoate, indole, and NiSO4, that are known to act as repellents of Escherichia coli cells served as attractants for Halobacterium. In the presence of ethionine, sensitivity to attractants was reduced. Arsenate prevented the attraction by glucose without lowering the cellular adenosine 5'-triphosphate level. The ability for chemo-accumulation toward glucose and histidine was interfered with by the formation of photosensory systems. Light-induced motor responses and chemosensory behavior toward glucose and histidine became detectable in the late stationary growth phase only. The behavior toward acetate and indole was not connected to photobehavior in that way: both substances acted as attractants already in the late log phase. Inhibition of bacteriorhodopsin synthesis by L-nicotine allowed chemo-accumulation toward glucose and histidine already in the late logarithmic phase.     

Substitution of amino acids in helix F of bacteriorhodopsin: effects on the photochemical cycle

The effects of amino acid substitutions in helix F of bacteriorhodopsin on the photocycle of this light-driven proton pump were studied. The photocycles of Ser-183----Ala and Glu-194----Gln mutants were qualitatively similar to that of wild-type bacteriorhodopsin produced in Escherichia coli and bacteriorhodopsin from Halobacterium halobium. The substitution of a Phe for either Trp-182 or Trp-189 significantly reduced the fraction of photocycling bacteriorhodopsin. The amino acid substitutions Tyr-185----Phe and Ser-193----Ala substantially increased the lifetime of the photocycle without substantially increasing the lifetime of the M photocycle intermediate. Similar results were also obtained with the Pro-186----Gly substitution. In contrast, replacing Pro-186 with the larger residue Leu inhibited the formation of the M photocycle intermediate. These results are consistent with a structural model of the retinal-binding pocket suggested by low-temperature UV/visible and Fourier transform infrared difference spectroscopies that has Trp-182, Tyr-185, Pro-186, and Trp-189 forming part of the binding pocket.     

Coupling between the bacteriorhodopsin photocycle and the protonmotive force in Halobacterium halobium cell envelope vesicles. II. Quantitation and preliminary modeling of the M----bR reactions

The cell membrane of Halobacterium halobium (H. halobium) contains the proton-pump bacteriorhodopsin, which generates a light-driven transmembrane protonmotive force. The interaction of the bacteriorhodopsin photocycle with the electric potential component of the protonmotive force has been investigated. H. halobium cell envelope vesicles have been prepared by sonication and further purified by ultracentrifugation on Ficoll/NaCl/CsCl density gradients. Under continuous illumination (550 +/- 50 nm) varied from 0 to 40 mW cm-2, the vesicles maintain a membrane potential of 0 to -100 mV. The membrane potential was measured by flow dialysis of 3H-TPMP+ uptake and could be abolished by the uncoupler carbonylcyanide-m-chlorophenylhydrazone. Time-resolved absorption spectroscopy was used to measure the decay kinetics of the M photocycle intermediate, which was initiated by a weak laser flash (588 nm), while the vesicles were continuously illuminated as above. The M decay kinetics were fitted with two exponential decays by a computer deconvolution program. The faster decaying form decreases in amplitude (70 to 10% of the total) and the slower decaying form increases in amplitude and lifetime (23 to 42 ms) as the background light intensity increases. Although any correlation between the membrane potential and the bacteriorhodopsin photocycle M-forms is complex, the present data will allow specific tests of the physical mechanism for this interaction to be designed and conducted.     

Immunological probes for bacteriorhodopsin. Identification of three distinct antigenic sites on the cytoplasmic surface

We have prepared site-specific immunological reagents to study the orientation and surface topography of the integral membrane protein bacteriorhodopsin. Monoclonal and polyclonal antibodies with strong affinity for antigenic determinants on proteolytic and cyanogen bromide fragments of bacteriorhodopsin have been isolated and characterized. Three distinct antibody binding sites have been identified on the cytoplasmic surface of bacteriorhodopsin. The first due is readily accessible in native bacteriorhodopsin and lies close to the COOH terminus. This binding site is lost when only three amino acid residues are removed from the COOH terminus. The second site, which is also near the COOH terminus, is located approximately within the 17 COOH terminal amino acid residues. The third site is in the fragment that comprises Tyr-83 to Met-118 and is probably contained in the short loop connecting the third and fourth helices. The use of COOH terminus-specific antibodies in determination of the orientation of bacteriorhodopsin molecules in the Halobacterium halobium membrane confirms the earlier conclusion that the COOH terminus is on the cytoplasmic side.     

Displacement current on purple membrane fragments oriented in a suspension

The displacement current is measured in a suspension of electric field-oriented purple membranes isolated from Halobacterium halobium, the photocycle being driven by a light flash. A simple quantitative theory of the method is presented and used to evaluate the distances the protons move during their way through the bacteriorhodopsin molecules. A lower limit of the velocity of proton movement is also given.     

Effect of cross linkers on the bacteriorhodopsin photocycle

A general behavior of bacteriorhodopsin in purple membranes from Halobacterium halobium has been observed upon modification resulting in cross-linking of carboxyl and lysine groups. The rise of the M-intermediate contained two components with approximately 50-50% intensity; its decay showed three components with approximately 25-50-25% intensity respectively in a pH range of 5-9. The significance of these remarkably similar data with respect to the proton translocation mechanism in bacteriorhodopsin is that chemical modification allows us to conclude that disturbing parts of the hypothetical "proton conducting chain" does not inhibit proton translocation.