BLEACHING OF BACTERIORHODOPSIN IN PURPLE MEMBRANE, BROWN HOLO-MEMBRANE ANDL–1690-SOLUBILIZED MONOMERS WITH HYDROXYLAMINE IN LIGHT AND THE DARK

Abstract— The reactions of hydroxylamine with bacteriorhodopsin in the states of the purple membrane, the brown holo-membrane andL–1690-solubilized monomers were studied in light and the dark. The bleaching rate was strongly dependent on the state of the bacteriorhodopsin and largely enhanced by light. Analysis of the isomeric composition of the resultant retinaloximes by high performance liquid chromatography (HPLC) showed that more all- trans retinaloximes were present in the dark than 13- cis retinaloximes. However, under illumination, the relative amounts of 13- cis retinaloximes increased greatly, indicating the occurrence of trans to cis isomerization during the photochemical cycle of bacteriorhodopsin. Molar extinction coefficients of 13- cis retinaloximes were determined to estimate the isomeric composition of retinaloximes by HPLC analysis.     

ELECTRIC DICHROISM OF PURPLE MEMBRANE SUSPENSIONS

Abstract— Suspensions of purple membranes were exposed to reversing rectangular pulses of relatively low electric field (less than 100V/cm) at various frequencies. Orientation of the membranes was estimated by measuring linear dichroism at 565nm. In the electric field of frequency lower than about 10Hz the purple membranes tended to align perpendicular to the electric field. This orientation was induced mostly by permanent dipole which was perpendicular to the membrane surface. The value of permanent dipole moment was determined to be 140D per protein at pH 7.0, 25°C, in the presence of 5m M phosphate buffer.
In an electric field of frequency more than 100Hz the membrane tended to align parallel to the electric field. Electric polarizability parallel to the membrane surface was estimated to be 1 ± 10⁻¹² cm³ under the same conditions.
Electric dichroism of light-adapted and dark-adapted purple membranes was found to coincide with each other at high frequency. From this result the angle of retinal to the membrane surface was concluded to be the same between light-adapted and dark-adapted bacteriorhodopsins.     

INTERACTION OF DIBUCAINEHCl LOCAL ANESTHETICS WITH BACTERIORHODOPSIN IN PURPLE MEMBRANE: A SPECTROSCOPIC STUDY

Several spectroscopic techniques (absorption, emission, transient absorption and differential scanning calorimetry--DSC) were used to investigate the deprotonation of dibucaine.HCl in a hydrophobic environment, and the interaction sites and mechanisms of the local anesthetic dibucaine.HCl on bacteriorhodopsin (bR) in purple membrane. The important results are summarized as follows: (1) the visible absorption features of native (lambda max = 568 nm) and deionized (lambda max = 608 nm) bR are sensitive to the amount of dibucaine.HCl added; (2) the emission spectrum of dibucaine.HCl embedded in the retinal-free mutant bR is similar to that of dibucaine free base in Triton X-100 micellar solutions; (3) the phosphorescence emission of dibucaine at 77 K is completely quenched by bR and the fluorescence quenching rate for the incorporated dibucaine.HCl in bR was determined as kq = 4.09 x 10(13) M-1 s-1; (4) the incorporation of dibucaine.HCl in bR inhibits the slow component rate of formation of M412 and decreases the amount of M412 formation in the photochemical cycle of bR; and (5) the thermal stability of native bR was measured by DSC in the presence and absence of dibucaine and yielded an endothermic transition at 95.9 +/- 1.0 degrees C with 13.6 J/g (3.25 +/- 0.12 cal/g) of enthalpy changes. All observations suggest that the action site of the local anesthetic, dibucaine.HCl, is near or at the chromophore, i.e. the retinal Schiff base of bR. The anesthetic action on bR purple membrane is probably via a specific site binding, but not a conformational mechanism.     

THE UV PROTEIN FLUORESCENCE OF PURPLE MEMBRANE AND ITS APOMEMBRANE

The ultraviolet fluorescence of the purple membrane of H. halobium and its apomembrane was characterized by measuring emission spectra, polarization, decay lifetimes and the changes induced by pH and temperature. The fluorescence quantum yields of the two membranes are 0.024 × 0.003 and 0.17 × 0.03, respectively. The emission, which shows lifetimes in the 0.4 to 4 ns range, was assigned to heterogeneous populations of emitters, consisting, probably, of two tryptophans in the purple membrane and seven or eight residues in the apomembrane. Acrylamide quenching experiments showed that the accessibility of this neutral quencher to the fluorophors is reduced greatly in both membranes. Fluorimetric methods were also used in an attempt to monitor the purple complex reconstitution process. It was concluded that the fluorescence quantum yields of any monomers, dimers and trimers present in the partially reconstituted membranes should be very similar.
Finally, based on the spectroscopic results and on specific folding patterns of the seven α-helical regions of bacteriorhodopsin (Stoeckenius and Bogomolni, 1982), it is proposed that Trp 137, Trp 138 (and perhaps Trp 10) of the protein molecule are the most plausible fluorophors in the purple membrane. It is also suggested that the protein in the apomembrane takes a more open configuration which is permeable to small ions and molecules.     

A SPIN LABELED RETINAL PIGMENT ANALOGUE OF THE PURPLE MEMBRANE

Abstract— A spin labeled derivative of retinal (4-[2, 2, 5,5-tetramethyl-l-pyrrolidinyloxy-3-carboxyl]-retinal) has been synthesized. The all- trans -isomer combined with the apoprotein of the purple membrane Halobacterium halobium to form a pigment which absorbs at 480 nm and which is unstable to hydroxyl-amine. The electron paramagnetic resonance (EPR) spectrum of the pigment showed an anisotropic signal indicating immobilization of the nitroxide on the chromophore. The ester linkage on the chromo-phore hydrolyzed in situ yielding the 4-hydroxypigment analogue, absorbing at 540 nm, and the free 2, 2, 5,5-tetramethyl-l-pyrrolidinyloxy-3-carboxylic acid.     

SALT AND pH-DEPENDENT CHANGES OF THE PURPLE MEMBRANE ABSORPTION SPECTRUM

Abstract —Purple membrane suspensions change their color to blue and the absorption maximum shifts to 608 nm when the membrane is deionized on a cation exchange column or when it is washed first with < 2N NaCl followed by deionized water. The deionized chromophore is essentially identical with the chromophore produced by lowering the pH of the native membrane to < 4.0 (p K < 3.0). However, the deionized membrane does not aggregate and can be obtained in the pure state. The original purple color of the membrane is restored by addition of around 1 m M Na⁺, K⁺ or 10 μ M Mg²⁺, Ca²⁺, Sr²⁺, Mn²⁺, Pb²⁺ or La²⁺ when the protein concentration is 5μ M . The required salt concentrations decrease with decreasing pH. Direct measurement of bound Ca²⁺ by atomic absorption spectroscopy yields a ratio of Ca²⁺ to protein of <2 and a binding constant of 1.4 × 10⁶. Titration of the spectral change with salts at different pH values shows a linear relation between the pH and the logarithm of the salt concentration, with a 1:1 ratio for Na⁺ and 1:2 ratio for Ca²⁺. These relations are well predicted by Gouy-Chapman theory; however, the accompanying release of protons, changes of the CD spectrum, the complex kinetics of the spectral change during reconstitution with salt and preliminary X-ray diffraction results all suggest that conformational changes may be occurring in the protein.     

温度和离子对紫膜LB膜光循环的影响

用闪光动力学光谱仪测量了水平拉制的紫膜LB膜中菌紫质中间体M₄₁₂的衰减过程,观察了温度和离子对M₄₁₂衰减过程的影响。实验结果表明:在一定的温度范围内(10℃-60℃),随着温度的升高,M₄₁₂的衰减速率加快。对M₄₁₂s的衰减的抑制作用,La³⁺在低浓度时就很明显,而K⁺则在较高浓度时才表现出来,Ca²⁺的影响不明显;La³⁺对M₄₁₂f的衰减无明显影响,K⁺和Ca²⁺则稍微加快了其速率,pH的变化(H⁺浓度)明显影响到M₄₁₂的衰减速率,尤其在高pH情况,M₄₁₂s的衰减比正常pH值时要慢一个数量级。     

SOME OBSERVATIONS ON THE MORPHOGENESIS OF LATTICE STRUCTURE IN THE PURPLE MEMBRANE

Abstract— The formation process of purple membrane was studied morphologically by freeze-fracture and etching methods. The bacteria, H. halobium R₁, cultured in the medium containing 2m M nicotine, did not show the hexagonal structure of purple membrane, but a particle free area was frequently observed in the plasma membrane. However, within 24h after returning to the normal medium, hexagonal and triclinic lattices with spacings of 6 to 8nm occurred as small patches on the P face of the plasma membrane. Furthermore, it was revealed that the retinal was not required for the formation of the two-dimensional hexagonal lattice structure of purple membrane, since the mutant bacteria, H. halobium R₁mW, lacking the ability of retinal synthesis, displayed a similar lattice. The other mutant, H. halobium R₁mR, lacking the ability of bacterio-opsin synthesis, never showed any highly ordered arrangement. This evidence suggests that membrane proteins other than bacterio-opsin or bacteriorhodopsin do not form highly ordered lattices in the membrane.     

THE pH DEPENDENCE OF TRANSIENT CHANGES IN THE CURVATURE OF THE PURPLE MEMBRANE*

Abstract– We have observed transient changes in the curvature of purple membrane fragments upon photoexcitation as a function of the pH of the suspending medium. The room temperature suspensions have low ionic strengths, and the bending is observed by changes in the scattered light at 320 nm. The photoexcitation is from a 20 ns laser pulse at 532 nm. We use a simple first-order approximation to subtract any changes in the scattered light associated with transient absorption changes during the photocycle. The resultant scattering curves are then fitted to the sum of three fundamental bending processes. Each process has an exponential rise, an exponential decay and an amplitude. We model two of the processes as transient forces correlated with the charge motion during the photocycle. The third process is probably cuased by local changes in the pH as the protein pumps protons. However, this is not proved rigorously. Additional experiments are proposed.     

THE USE OF NEUTRAL RED TO MONITOR THE SURFACE POTENTIAL OF THE PURPLE MEMBRANE

Abstract— The binding of neutral red to purple membrane has been studied. The intrinsic p K _a and the apparent p K _a, of bound neutral red were determined by titration and by measuring the binding ratio of neutral red to purple membrane as a function of pH. The surface potential of purple membrane was inferred from the difference between these two p K _as. The H⁺/M412 ratio at different ionic strengths was also measured and compared with the surface potential. The results show that the H⁺/M412 decreased as the surface potential increased due to decreased salt concentrations. However, this correlation holds only for KCl concentrations higher than 30 m M . At lower salt concentrations, the change in surface potential is always less than the variation in the H⁺/M412 ratio.     

THE PHOTOREACTION OF THE DEIONIZED FORM OF THE PURPLE MEMBRANE INVESTIGATED BY FTIR DIFFERENCE SPECTROSCOPY

Abstract— Deionization of the purple membrane of Halobacterium halobium shifts the visible absorption maximum from 570 to 605 nm and inhibits proton transport. FTIR-difference-spectra of this blue membrane at 280 K reveal that the retinal chromophore adopts a 13 -cis and all -trans geometry in a light dependent ratio. In contrast to purple membrane the 13-cu isomer forms much faster in the dark. The all- trans component produces an L-intermediate which can be stabilized at 170 K. Spectral characteristics are similar to normal L. including comparable changes of internal aspartic acids of the opsin. However, stronger changes in the amide-I absorption are observed. IR bands of the chromo-protein states are assigned to retinal normal modes by the use of bacteriorhodopsin regenerated with'C-labeled retinals.     

THE QUANTUM EFFICIENCY FOR THE INTERPHOTOCONVERSION OF THE BLUE and PINK FORMS OF PURPLE MEMBRANE

When the cations bound to purple membrane are removed it turns blue, and when this blue membrane is irradiated its color changes to pink. Irradiation of pink membrane leads to the reformation of blue membrane. We have determined that the quantum efficiency for the formation of pink membrane from deionized blue membrane is 1.6 ± 0.6 ± 10 ⁴ at 0^oC, pH 5.0. We also found that the quantum efficiency for the back photoconversion, i.e. the formation of blue membrane from pink membrane, is 8.8 ± 1.6 ± 10^-3 at 0^oC, 55 times greater than that of the forward photoconversion reaction. The extinction coefficients of the pink membrane and blue membrane were determined to be 44 500 ± 670 cm^-1 M^-1 at 491 nm and 54 760 ± 830 cm^-1 M ^-1 at 603 nm, respectively, assuming light-adapted purple membrane is 63 000 cm^-1 M ^-1 at 568 nm. The quantum efficiency for forming pink membrane from blue membrane is much lower than that for forming the photointermediate of the blue membrane's photocycle. Their relationship is similar to that of light-adaptation and photocycle of the dark-adapted purple membrane.     

BIOSYNTHESIS AND ASSEMBLY OF PURPLE MEMBRANE OF HALOBACTERIUM HALOBIUM S9: LIGHT STIMULATION OF BACTERIORHODOPSIN FORMATION

Abstract— The effect of light on purple membrane biogenesis in Halobacterium halobium S9 strain was investigated. When bacteria were grown in the dark, the 570nm absorption due to bacteriorhodopsin increased more slowly than under illumination, but eventually after longer incubation, reached the same level as that seen in the illuminated culture.
Analysis of membrane fractions by sucrose density gradient centrifugation revealed that two different membrane fractions, containing purple and brown membrane could be detected in the exponential growth phase. Another fraction whose density was higher than that of purple membrane, disappeared concomitantly with the increase in purple membrane and brown membrane, indicating that it may be related to purple membrane formation.
HPLC analysis of membrane proteins showed that there was no significant difference in de novo synthesis of bacterio–opsin between dark and illuminated cultures. This led us to conclude that light stimulated retinal binding to bacterio–opsin and/or retinal biosynthesis rather than bacterio–opsin synthesis. Bacteriorhodopsin seemed to form the brown membrane fraction first, which then spontaneously reorganized into purple membrane.
When incorporated in liposomes, bacteriorhodopsin in brown membrane was found to have rather higher proton pump activity than that in purple membrane. The H⁺ pumping activity was quite heat labile. This and the CD spectra indicated that bacteriorhodopsin in brown membrane might exist without forming normal timer unit.     

EFFECT OF MELITTIN ON PHOTOCYCLE AND PHOTOPOTENTIAL OF PURPLE MEMBRANE: SITES OF INTERACTION BETWEEN BACTERIORHODOPSIN AND MELITTIN

Abstract— Purple membrane (PM) suspension and artificial bilayer lipid membranes (BLM) containing PM sheets were treated with melittin. Both the decaying of the photocycle intermediate M412 and proton translocation were inhibited by melittin: The yields and rate of the slow-decaying component of M412 (M412s) together with the proton release and its uptake rate were significantly decreased, but the rate of the fast-decaying component of M412 (M4120 had only slight changes. Relatively high concentrations of melittin could cause aggregation in PM suspensions. Addition of melittin to a BLM solution increased the continuous photopotential signal but decreased the transient signal. We suggest that there might exist strong interactions between melittin and bacteriorhodopsin in addition to the melittin–lipid action. On the other hand, the results also indicate that proton translocation was more likely to be coupled with M412s and both were more sensitive to the changes caused by the melittin–PM interaction than was M412f.     

液膜法处理含氟废水

为了防止含氟废水的污染,人们研究了多种除氟方法,液膜技术是一项新的分离工艺,它操作简单,试剂用量少,有机相可重复利用。本文利用伯胺作迁移载体处理高含量的含氟废水,分离效率较好。 伯胺N_(1923)(简写RNH_2)为流动载体,上胺N_(205)(上海石油化工厂)为表面活性剂,煤油为溶剂。将内相CaCl_2,溶液与油相(含N_(1923)、N_(205)和煤油)按一定体积比例混合,高速搅拌,制成W/O     

PHOTOREACTION OF THE ACIDIFIED FORM OF BACTERIORHODOPSIN AND ITS 9-CIS DERIVATIVE IN PURPLE MEMBRANE AT LOW TEMPERATURES

Abstract— The photoreaction of the acidified form of bacteriorhodopsin and its 9-cis derivative was studied by low temperature spectroscopy.
A short exposure of the acidified form of bacteriorhodopsin, which was prepared by adding 2 m M HC1 to purple membrane suspension in 67% glycerol at 0°C, to red light at – 72°C resulted in the blue-shift of the spectrum. The feature of the shift was very similar to that accompanied by the formation of stable 9- cis acidified form of bacteriorhodopsin at 0°C, but only 13- cis - and all- trans -retinals were found in the extract from this product. No blue-shifted product was found on irradiation at – 190°C.
Irradiation of the 9- cis form of acidified bacteriorhodopsin at -72°C with blue light caused the isomerization of its 9- cis -retinylidene chromophore to 13- cis and all- trans forms without a significant spectral change. It became greater only after the sample was warmed above – 24°C. These results indicate the presence of the light-induced product which has trans configuration on the 9-10 double bond and exhibits the 9- cis type spectrum.     

顺铂和反铂与红细胞膜糖蛋白的相互作用

用凝胶电泳法、荧光分光度法和圆二色谱研究顺铂和反铂与人红细胞膜糖蛋白的作用结果表明，顺铂在糖蛋白上的结合量明显高于反铂，顺铂对糖蛋白构象的影响也大于反铂，这提示顺铂的抗癌作用可能与膜糖蛋白的作用有关。     

A ROLE FOR MENADIONE IN THE PURPLE MEMBRANE PROTON PUMP?

Abstract— It has been assumed that proton pumps such as purple membrane lack redox loops. However, purple membrane does contain an electron carrier. Kates et al. (Meth. Enzymol. 88,98–1 111, 1982) reported the presence of 1 mole of vitaminMK–8 to 6 mol of bacteriorhodopsin among the nonpolar lipids. Is this quinone functionally important in the proton pump mechanism? Proton pumping rates were measured with lipid-free bacteriorhodopsin reconstituted in vesicles to which varying amounts of vitamin K₁ were added. With soybean lipids, in the presence of tetraphenyl boron, the pump quantum yield was 0.04H⁺/photon. This result was independent of the amount of vitamin K, added over a range of 0 to a 100-fold mole ratio to bacteriorhodopsin. A similar result was obtained with H. halobium lipids. The pump quantum yield in vesicles is much less than reported for membrane sheets and whole cells. The results support the conclusion that a vitamin K Q-cycle is not involved in the purple membrane proton pump.     

FLASH-INDUCED FAST CHANGE IN SURFACE POTENTIAL AND FLUIDITY OF PURPLE MEMBRANE STUDIED BY SPIN LABEL METHOD

Abstract— Flash-induced changes in surface potential and fluidity of purple membrane were studied by a spin label method. Changes in surface potential and fluidity were monitored by observing the distribution of charged and uncharged spin labels between the purple membrane and the aqueous phase.
On flash illumination, a transient hyperpolarization of the surface potential and a transient fluidization of the membrane hydrophobic region are induced. The former may probably reflect the proton movement near the purple membrane surface, while the latter may result from photo-induced conformational changes of bacteriorhodopsin.
The two events are different in time course. The relationships between the two events, and the formation and decay of the intermediates of bacteriorhodopsin in the photoreaction cycle are discussed.