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R Uhl

Publications and source records attributed to R Uhl.

At least 37 records · Page 2Linked to original sources
PubMed1989

Optical probes of intradiskal processes in rod photoreceptors. II: Light-scattering study of ATP-dependent light reactions.

Rod outer segment (ROS) disks, either stacked or freely floating, respond to flash illumination to yield a specific, ATP-dependent, light-scattering signal AL. In broken ROS AL signals occur only when AD signals have preceded them. The degree to which the preceding AD signal has been completed determines the amplitude of the following AL signal. However, in freshly detached ROS from dark-adapted frogs Al signals with maximal size can be obtained without pre-incubation with exogenous ATP. The energized state, which is restored in broken ROS with the help of ATP, appears to prevail in the living retina and must therefore be considered to be "physiological". AL signals require structurally intact disks. Neither peripheral ROS proteins nor connecting filaments between adjacent disks are necessary. Their structural origin is the same as that of the preceding AD signal, i.e. osmotic disk swelling. AL signals consist of a single slow kinetic component (half-life 10 s at room temperature) and multiphase fast kinetic component (70 ms). The slow phase corresponds to a light-stimulated resumption of ATPase activity (this has been dealt with in a previous paper) whereas the fast component reflects an immediate response of the energized disk to the metarhodopsin I to metarhodopsin II transition. The latter effect is the subject of this paper. A variety of experiments, using different ATPase inhibitors, ionophores and membrane-permeable salts, have been carried out; they are all consistent with notion that AL originates in the disk interior and probes the existence of a proton electrochemical potential difference delta mu (H+) across the disk membrane. A model is presented which can explain all given properties of AL satisfactorily. According to this model the photolysis of rhodopsin causes a proton release in the disk lumen. This, in turn, results in osmotic swelling of the disks, provided that the internal buffer sites have been (at least partially) titrated with protons prior to the flash. Such conditions, i.e. a low internal pH, are provided by the proton transport across the disk membrane, which presumably takes place during the course of the preceding AD signal.

Adenosine Triphosphate↗
PubMed1989

Biosynthesis of the two halobacterial light sensors P480 and sensory rhodopsin and variation in gain of their signal transduction chains.

The two retinal-containing photoreceptors of halobacteria, P480 and sensory rhodopsin, are formed constitutively and inducibly, respectively. Both photoreceptors are synthesized as apoproteins in cells with nicotine-inhibited retinal synthesis and are reconstituted as chromoproteins by the addition of all-trans retinal to cell membrane preparations. The decrease in photoreceptor-mediated photophobic response at the stationary growth phase of cells is not due to photoreceptor degradation but due to a deficiency of the signal transduction chain in the cell.

Bacterial Proteins↗
PubMed1989

Team physician #6. Surgical treatment of Achilles tendon rupture.

The ideal treatment of Achilles tendon ruptures has always been controversial. Nonoperative treatment has been criticized for its high rerupture rate and surgical treatment has been criticized for its high complication rate. This paper describes a surgical method of repair that can be used for acute or late ruptures, and has been associated with a low complication and rerupture rate.

Achilles Tendon↗
PubMed1988

Rapid transducin deactivation in intact stacks of bovine rod outer segment disks as studied by light scattering techniques. Arrestin requires additional soluble proteins for rapid quenching of rhodopsin catalytic activity.

In photoreceptors of the living retina both activation and deactivation of transducin must occur in less than 1 s. In ROS preparations used for in vitro studies, however, deactivation takes minutes. This is due to the fact that activated transducin is released into the free aqueous space, whereby GTPase activity and consequent deactivation of the protein are slowed down, and due to the dilution of soluble ROS proteins involved in the quenching of rhodopsin activity. In this paper, using a convenient, non-invasive light scattering assay, we demonstrate that in an intact stack of disks, where active transducin stays membrane associated and is rapidly deactivated, the activity of rhodopsin can also be quenched in the time range of seconds when soluble ROS proteins are supplemented. Arrestin, the 48 kDa protein of the photoreceptor, is one of the proteins required for rapid recovery, however, it requires the synergistic action of other soluble proteins (besides rhodopsin kinase) in order to exert its effect: When arrestin is included in the reaction mixture without the 'helper protein(s)', it cannot speed recovery, and when a mixture of soluble proteins is added which lacks arrestin, there is also no effect. The nature and identity of this (these) helper protein(s) are still unclear.

Animals↗
PubMed1988

Sub-second turnover of transducin GTPase in bovine rod outer segments. A light scattering study.

A fast, regenerative light scattering signal from bovine ROS, the PA-signal, reflects the light-induced, transient activation of transducin. Its rate of recovery depends on the number of photolysed rhodopsin molecules, indicating that rhodopsin deactivation and not GTPase activity is rate limiting in our in vitro system. When rhodopsin deactivation is accelerated (in the presence of NH2OH), PA-signal recovery is also accelerated. A GTPase turnover number of more than 2 s-1 (at 37 degrees C) can be derived from these experiments. This is more than one order of magnitude faster than the GTPase rates so far described in the literature and is rapid enough for a physiological shut-off mechanism. The fast GTPase is attributed to a highly intact disk stack, which never releases transducin into the free aqueous space.

Adenosine Triphosphate↗
PubMed1987

A simple and rapid procedure for the isolation of intact bovine rod outer segments (ROS).

A method is described which allows the rapid isolation and purification of intact rod outer segments (ROS) from cattle eyes. It requires very fresh retinal material and can be completed within less than 2 h of the death of the animals. Cattle eyes are dissected in the usual manner, the retinae are isolated and the ROS are separated from the rest of the retina by gentle vortexing and filtration through a nylon mesh. The resulting crude ROS suspension is purified on a discontinuous sucrose density gradient. Two fractions are obtained, the major one consisting of mostly intact ROS, the minor one of RIS-ROS, i.e. of ROS which are still connected to part of their inner segment. The ROS are washed once and can be stored on ice for several days without loosing their intact plasma membrane. They can be transformed to leaky ROS by a quick freeze/thawing cycle or, if one wants unobstructed access to the interdiskal space, they can be subjected to a mild lysis treatment. The resulting ROS material is characterised using light microscopy, electron microscopy, light scattering, gel electrophoresis and absorption spectroscopy. It contains unusually low levels of 48k-protein and very high levels of G-protein. The latter cannot be washed out in the presence of GTP-gamma-S, even in the case of leaky ROS.

Animals↗
PubMed1985

Lipid-protein interactions in frog rod outer segment disc membranes. Characterization by spin labels.

Freely-diffusing phospholipid spin labels have been employed to study rhodopsin-lipid interactions in frog rod outer segment disc membranes. Examination of the ESR spectra leads us to the conclusion that there are two motionally distinguishable populations of lipid existing in frog rod outer segment membranes over a wide physiological temperature range. Each of the spin probes used shows a two-component electron spin resonance (ESR) spectrum, one component of which is motionally restricted on the ESR timescale, and represents between 33 and 40% of the total integrated spectral intensity. The second spectral component which accounts for the remainder of the spectral intensity possesses a lineshape characteristic of anisotropic motion in a lipid bilayer, very similar in shape to that observed from the same spin labels in dispersions of whole extracted frog rod outer segment lipid. The motionally restricted spectral component is attributed to those spin labels in contact with the surface of rhodospin, while the major component is believed to originate from spin labels in the fluid lipid bilayer region of the membranes. Calculations indicate that the motionally restricted lipid is sufficient to cover the protein surface. This population of lipids is shown here and elsewhere (Watts, A., Volotovski, I.D. and Marsh, D. (1979) Biochemistry 18, 5006-5013) to be by no means rigidly immobilized, having motion in the 20 ns time regime as opposed to motions in the one nanosecond time regime found in the fluid bilayer. Little selectivity for the motionally restricted population is observed between the different spin-labelled phospholipid classes nor with a spin-labelled fatty acid or sterol.

Animals↗
PubMed1985

Separation and characterisation of light scattering transients from rod outer segments of vertebrate photoreceptors: design and performance of a Multi Angle Flash Photolysis Apparatus (MAFPA).

A device was built for the simple computer-controlled routine determination of the angular dependence of light scattering transients obtained from biological material. It was called Multi Angle Flash Photolysis Apparatus (MAFPA). The MAFPA allows the simultaneous registration of rapid, light-induced light scattering transients at eight scattering angles between 0 degree and 28 degrees. In typical applications changes in scattered light intensity as small as delta I/I = 4 X 10(-5) can be resolved at scattering angles less than 24 degrees, while at 28 degrees the resolution drops to delta I/I = 2 X 10(-4). The time resolution is 32 microseconds. The MAFPA was designed for high accuracy, ease of use and ruggedness. It is made from relatively inexpensive parts and can be copied fairly easily by a good machine/electronics shop. In this communication we describe the design of the MAFPA and how it was used for the characterisation of four structurally distinct light-induced light scattering signals from photoreceptor rod outer segments. These signals are known as P (or binding) signal, G- (or dissociation) signal, N (or rhodopsin) signal and as the ATP-dependent signal AL. The signals have been separated by means of their different angular dependence, their different saturation behavior and nucleotide requirement. A great number of detailed studies will have to be carried out before one can fully understand the physical and biochemical origin of these signals. At this point, however, it can be stated that the so-called 'dissociation signal', showing an angular dependence indicative of a change in refractive index or scattering mass, is not merely an inversion of the preceding 'binding signal', the latter clearly reflecting a gross structural change, i.e. a shrinkage of the disks. Moreover, there are conditions where P signals are observed to persist even after the completion of the subsequent dissociation signals. The two remaining signals N and AL show a pronounced angular dependence which is not easily interpreted. The fact that both exhibit a maximal amplitude at relatively small angles seems to indicate the participation of rather large structural domains.

Adenosine Triphosphate↗
PubMed1984

A polychromatic flash photolysis apparatus (PFPA).

A wide variety of biologically relevant chemical intermediates have been identified and characterised by their spectral properties. When rapid kinetics, i.e. rapid changes in these spectral properties are studied, the equipment designed for these studies (flash photolysis-, T-jump apparatus) usually allows only the registration of intensity changes of the monitoring light beam at one particular wavelength. Quite frequently, however, particularly in biological systems, the reactions of interest are too complex to be fully understood using single wavelength techniques. We have therefore designed and built a flash photolysis apparatus which permits the simultaneous recording of absorbance changes at 32 wavelengths, freely selectable between 300 and 1000 nm, as well as changes in fluorescence, luminescence, birefringence and light scattering. The apparatus, which we have called Polychromatic Flash Photolysis Apparatus (PFPA), acquires up to 8000 difference spectra per second with an amplitude resolution of better than 0.0001 absorbance unit. Data acquisition and activation of an actinic xenon flash unit occurs under computer control. The same computer is responsible for data storage, handling and graphic display. This communication describes the PFPA, its performance, and, as a demonstration of its potential usefulness, its application to the measurement of the light driven photocycle of bacterial rhodopsin, the proton pumping protein of Halobacterium halobium.

Photolysis↗
PubMed1982

ESR spin-label studies of lipid-protein interactions in membranes.

Lipid spin labels have been used to study lipid-protein interactions in bovine and frog rod outer segment disc membranes, in (Na+, K+)-ATPase membranes from shark rectal gland, and in yeast cytochrome oxidase-dimyristoyl phosphatidylcholine complexes. These systems all display a two component ESR spectrum from 14-doxyl lipid spin-labels. One component corresponds to the normal fluid bilayer lipids. The second component has a greater degree of motional restriction and arises from lipids interacting with the protein. For the phosphatidylcholine spin label there are effectively 55 +/- 5 lipids/200,000-dalton cytochrome oxidase, 58 +/- 4 mol lipid/265,000 dalton (Na+, K+)-ATPase, and 24 +/- 3 and 22 +/- 2 mol lipid/37,000 dalton rhodopsin for the bovine and frog preparations, respectively. These values correlate roughly with the intramembrane protein perimeter and scale with the square root of the molecular weight of the protein. For cytochrome oxidase the motionally restricted component bears a fixed stoichiometry to the protein at high lipid:protein ratios, and is reduced at low lipid:protein ratios to an extent which can be quantitatively accounted for by random protein-protein contacts. Experiments with spin labels of different headgroups indicate a marked selectivity of cytochrome oxidase and the (Na+, K+)-ATPase for stearic acid and for cardiolipin, relative to phosphatidylcholine. The motionally restricted component from the cardiolipin spin label is 80% greater than from the phosphatidylcholine spin label for cytochrome oxidase (at lipid:protein = 90.1), and 160% greater for the (Na+, K+)-ATPase. The corresponding increases for the stearic acid label are 20% for cytochrome oxidase and 40% for (Na+, K+)-ATPase. The effective association constant for cardiolipin is approximately 4.5 times greater than for phosphatidylcholine, and that for stearic acid is 1.5 times greater, in both systems. Almost no specificity is found in the interaction of spin-labeled lipids (including cardiolipin) with rhodopsin in the rod outer segment disc membrane. The linewidths of the fluid spin-label component in bovine rod outer segment membranes are consistently higher than those in bilayers of the extracted membrane lipids and provide valuable information on the rate of exchange between the two lipid components, which is suggested to be in the range of 10(6)-10(7) s-1.

Animals↗
PubMed1980

A light scattering study on the ion permeabilities of dark-adapted bovine rod outer segment disk membranes.

The ion permeability properties of dark adapted bovine rod outer segment disk membranes were studied using light scattering to monitor osmotic responses of disks to various salts and ionophores. A preparation procedure is presented which provides very fresh rod outer segment material with mostly intact stacked disks, but with perforated plasma membrane. It is shown that in this preparation the disks (or rod sacs) are the only osmotically responding compartments and that these responses can be readily monitored by means of light-scattering techniques. The disk membrane is found under the conditions tested, to possess no measurable permeability to cations Na+, Ca2+, Mg2+ nor the the anions Cl-, Br-, NO3-, SO4(2-), H2PO4- and HPO4(2-). There is a considerable K+ permeability, which can be completely abolished by millimolar amounts of divalent cations. The proton permeability of the disk membrane is found to depend dramatically upon the preparation procedure and duration. The fresher the material used the lower is the proton permeability measured. In our freshest preparations, even after freeze-thawing in liquid nitrogen, the disks exhibit an H+ permeability which is so low that it cannot be measured with the techniques used in this study. Even in mitochondrial or chloroplast membmranes, in which proton gradients and therefore a low proton conductance play an essential role, such low proton permeabilities have not been found. This would suggest that proton gradients across the disk membrane could play an important role in the physiological function of the photoreceptor cell. In summary it can be said that the disk membrane, apparently more than any other natural membrane system studied so far, is capable of retaining ion gradients for extended periods of time.

Animals↗
PubMed1978

On the light-stimulated coupling between rhodopsin and its disk membrane environment.

Disks from bovine ROS undergo a rapid shrinkage when flash illuminated with green light (Uhl, R., et al. (1977) Biochim. Biophys. Acta 469. 113-122). This can be monitored as a light scattering transient, referred to as the P signal. In this paper the P signal is studied at various temperatures and pH. The temperature dependence of the kinetics reveals that "P" consists of two sequential reaction steps. Both appear to occur within the receptor molecule rhodopsin. The actually observed event, the shrinkage of the disk, is therefore not rate limiting under the tested conditions. Both steps of "P" take place while there is only one spectroscopically detectable reaction of the rhodopsin molecule, the metarhodopsin I-metarhodopsin II transition. This implies that there are intermediates of the rhodopsin photolytic cycle which are not evident as spectroscopically separate species. The amplitude of "P", i.e., the extent of the disk shrinkage, is independent of the state of the equilibrium between the two photoproducts absorbing at 478 and 380 nm respectively and called MI and MII. A scheme is suggested in which the irreversible decay of MI (478) triggers the disk shrinkage (and maybe transduction), and in which there is an equilibrium between MII (380) and a proposed isochromic photoproduct MI' (478).

Animals↗
PubMed1976

Measurements on fast light-induced light-scattering and -absorption changes in outer segments of vertebrate light sensitive rod cells.

Flash-induced changes of light-absorption and of light-scattering of vertebrate rod outer segments (ROS) from frog and cattle in suspension were measured at 380 and 800 nm. The photometer used allows the observation of light intensity changes under well defined angles. We studied the successive decrease of the signal amplitude in series of flashes. One flash bleaches about 1% rhodopsin. The following results are discussed: 1. The signal at 380 nm is a superposition of the absorption change caused by formation of metarhodopsin II and of a biphasic additional signal. The latter exists only for the initial range of bleaching (15 to 25% rhodopsin). 2. At 800 nm three scattering signals are observed which are characterized by their successive amplitude decrease and time course: N: A small signal with time course and successive amplitude decrease comparable to the metarhodopsin II absorption change, probably arising from a structural change within the disc membrane. Ni: A slow signal, disappearing with the first flash, which may be understood as an outer membrane effect. P: A biphasic signal with a successive decrease rate, by a factor of 10 to 20 higher than that of the metarhodopsin II signal. The two kinetically different components are separated by variation of the observation angle. Two regions of different extension appear to change structurally with different time course. "P" may reflect an influence of the light-induced transmitter release on disc shape and/or mass.

Absorption↗