Nicholas Ryba

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°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.

Calcium regulates the rate of rhodopsin disactivation and the primary amplification step in visual transduction.

The kinetics of the light‐induced activation of transducin as well as the subsequent disactivation process can be monitored by means of a specific light scattering transient PA. In this communication it is demonstrated that the rate of transducin disactivation is calcium dependent, increasing when the calcium concentration is decreased. As a consequence of the accelerated recovery in low calcium, the time to the peak of the transducin activation process is shortened and the gain of the primary amplification step, i.e. the number of transducin molecules activated per bleached rhodopsin, is reduced. Experiments using hydroxylamine as an artificial quencher of rhodopsin activity suggest that calcium acts upon rhodopsin kinase and not upon the rate of the GTPase. This would indicate that calcium may control visual adaptation not only by regulating guanine cyclase activity, but also by affecting the primary step in the transduction cascade, the rhodopsin‐transducin coupling

Phase transition from a gel to a fluid phase of cubic symmetry in dimyristoylphosphatidylcholine/myristic acid (1"2, mol/mol) bilayers

Aqueous dispersions (pH 4.0) of a 2:1 (mol/mol) mixture of myristic acid with dimyristoylphosphatidylcholine undergo a sharp transition at 45-47 degrees C from a lamellar gel phase to a fluid phase which is optically isotropic. This fluid phase gives rise to 31P-NMR spectra, and 2H-NMR spectra of the chain-deuterated components, which are also isotropic. X-ray diffraction studies of the fluid phase at 49 degrees C, reveal reflections with spacings in the ratio square root of 2: (square root of 3): square root of 4: square root of 6: square root of 8, accompanied by a strong diffuse scatter. These reflections index on a cubic lattice of primitive space group Pn3 or Pn3m, or possibly the body-centered group Im3m, with a lattice constant of 21.2 nm. The dimensions of the phase are consistent with a structure composed of two systems of tetrahedrally (octahedrally) oriented inverted lipid cylinders, found for other cubic lipid phases with Pn3m (Im3m) symmetry. At higher temperatures the cubic phase gradually converts, with increasing temperature, to a coexisting inverted hexagonal phase.

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.

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.

The amplified P-signal, an extremely photosensitive light scattering signal from rod outer segments, which is not affected by pre-activation of phosphodiesterase with Gα-GTP-γ-S

A light scattering signal from bovine rod outer segments in the presence of GTP is described. It has the same angular dependence as the P‐signal but is 3 orders of magnitude more sensitive to light and therefore we have called in the amplified P‐signal. Adding Gα‐GTP‐γ‐S has no significant effect on the light scattering signal despite the activation of PDE. cGMP affects the amplified P‐signal, but subsequent addition of Gα‐GTP‐γ‐S restores the normal signal character. All these facts strongly support the view that the amplified P‐signal reflects G‐protein activation rather than that of PDE. This is in striking contrast to an interpretation of a very similar light scattering signal previously described by other groups.

The kinetics and thermodynamics of bleaching of rhodopsin in dimyristoylphosphatidylcholine. Identification of meta-I, meta-II, and meta-III intermediates.

The effects of light on rhodopsin reconstituted into dimyristoylphosphatidylcholine at a molar ratio of 1:70 have been studied as a function of temperature and time. The lipid phase behavior and thermal stability of rhodopsin in the system used to measure the photolytic reactions were also determined. Thus, it was shown that the gel-to-fluid phase transition of the reconstituted membrane had a marked influence on the bleaching kinetics and thermodynamics of rhodopsin-bleaching equilibria, whereas lipid-protein interactions were also directly involved. Rhodopsin photolysis resulted in temperature-sensitive equilibria between three main photoproducts, with absorption maximal of approximately 480, 380, and 465 nm. Below the lipid phase transition temperature, the main photoproduct had an absorption maximum at 480 nm. With increasing temperature progressively more of the 380 nm-absorbing species was formed. The photoproduct with a spectral-maximum at 465 nm absorption was formed more slowly. Increasing temperatures decreased the ratio of the 465:380 nm-absorbing species. The thermal reactions were reversible: on cooling the higher-temperature products were converted back to the lower-temperature products. The results indicate that rhodopsin has extensive photochemical activity when reconstituted in dimyristoylphosphatidylcholine. The equilibria that we have measured resemble those of rhodopsin in the disk membrane. However, the kinetics of meta-II and meta-III formation appear to be considerably faster in the reconstituted membranes and the meta-I-to-meta-II equilibrium is displaced in the direction of the meta-I state relative to native rod outer segment disk membranes. The displacement of the meta-rhodopsin equilibrium from its position in the rod outer segment is attributed mainly to the effects of lipid-lipid interactions in the membrane bilayer and correlates with the difference in gel-to-fluid phase transition temperature of the different lipids.