Tom Borys

Evidence for structural changes in the photoreceptor disk membrane, enabled by magnesium ATPase activity and triggered by light

To account for the immense amplification involved in visual transduction, Wald, in 1954, [l] proposed the existence of a diffusable transmitter, which was either released from a compartment (compartment theory) or produced by an enzyme (enzyme theory). Until very recently the Ca’+ theory of visual transduction [2], i.e., a specific compartment theory, was widely favoured. However, as more and more evidence accumulated against Ca*+ being the transmitter [3,4] the enzymatic hydrolysis of cGMP attracted increasing attention and is now considered the most likely candidate for the transmitter process [5,6]. An enzymatic transmitter model, however, would pose the question as to the functional significance of the special form of the photoreceptor disk membrane, i.e., the fact that the rhodopsin-containing membranes form topologically closed compartments with strikingly low ion-permeabilities [7]. The currently discussed mechanisms of light-regulation of cGMP levels in the photoreceptor do not make use of this particular feature of the cell. Here we report the existence of a Mg-ATPase system in the rod outer segment (ROS) disk membrane, which, in many respects, resembles the ion translocating ATPases found in other systems. We

Evidence for a magnesium dependent ATPase in bovine rod outer segment disk membranes.

Abstract—In the presence of Mg2+ and adenosine triphosphate (ATP), a rapid. light‐induced, light‐scattering transient is observed from bovine rod outer segments (ROS). This light‐scattering transient we have labelled ‘A’. Ca2+ cannot replace Mg2+. nor can guanosine triphosphate (GTP) replace ATP. ‘A’ is observed at ATP concentrations as low as a few μM.

The presence of two major protein components in the bovine photoreceptor disc membrane.

Abstract Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of thoroughly washed rod outer segment membrane preparations from bovine retinae revealed two major membrane-bound components and not one as has been generally thought. The higher molecular weight peak (⋍38500 molecular weight) contains a carbohydrate component and is covalently bound to the retinylidene chromophore. Moreover, this material is extensively phosphorylated in vitro upon illumination. Therefore, this component (peak H) is rhodopsin. The nature and function of the other photoreceptor disc membrane component (peak L, ⋍34500 molecular weight) remains to be determined.

THE STRUCTURAL CHANGES IN BOVINE ROD OUTER SEGMENTS IN THE PRESENCE OF ATP

Abstract— We have, previously, described a light‐induced near infrared (700–850 nm) light scattering transient obtained in the presence of ATP from bovine rod outer segments suspensions in which the plasma but not the disk membranes were perforated (Uhl et al., 1979a). This transient was termed the ‘A’ signal. To elucidate its possible origin, we have analyzed their angular and wavelength dependencies. These data have been compared with osmotically controlled (non‐light) induced light scattering changes from identical control rod outer segments suspensions. It has been found that AD (the dark light scattering signal obtained in the presence of ATP) and ALS (the slow component of the actinic flash induced light scattering signal, AL) can be assigned to the swelling of the disk membranes while ALf (the fast component of this latter signal) can be attributed to the change in refractive index of the ROS caused by the hypsochromic spectral shift of photolyzed rhodopsin. The collective disk swelling associated with A, and ALS is consistent with the pumping of ions into the disk lumen by the action of a disk membrane bound ATPase.

LIGHT INDUCED INTERACTION BETWEEN RHODOPSIN AND GTP DEPENDENT PROCESSES IN ROD OUTER SEGMENTS—I. KINETIC ANALYSES OF LIGHT SCATTERING TRANSIENTS

Abstract— A fast light‐induced light scattering transient in the presence of Mg2+ and GTP is observed from bovine rod outer segment suspension. This transient is studied at various temperatures, degrees of photo‐bleaching and GTP concentration. The kinetic analysis of this signal at low photo‐bleaching (∼ 5%) reveals two different processes which are consecutive rather than simultaneous. The rate of both processes depends upon the extent of photo‐bleaching and indicates that each process requires a photolyzed rhodopsin intermediate. At low temperature, the rate constant of both the processes increases with the extent of photo‐bleaching while at higher temperatures it decreases. The calculated activation energy of both processes decreases with increasing photo‐bleaching. It has also been observed that with decreasing GTP concentration, the rates of both processes decrease indicating that the two processes though first order are most likely bimolecular, first order in both GTP and the photolytic rhodopsin intermediate (metarhodopsin II)

[67] Assays and characterization of Mg2+-ATPase in the rod outer segment of vertebrate photoreceptors

Publisher Summary This chapter discusses the assays and characterization of Mg 2+ - adenosine triphosphatase (ATPase) in the rod outer segment (ROS) of vertebrate photoreceptors. It reports the existence of a specific ATPase system that undoubtedly resides in the photoreceptor outer segment and whose presence can be assayed by three distinct techniques: (1) conventional assay of ATPase activity measuring the amount of inorganic phosphate liberated, (2) light-scattering: “A D .” A large decrease in turbidity occurs when rod outer segments (ROS) are incubated in the presence of Mg 2+ and ATP in the dark, and (3) light-scattering: “A L .” A rapid further decrease in turditity occurs on flash illumination when ROS had been preincubated in the presence of Mg 2+ and ATP. ATP hydrolysis by ROS membranes was measured as release of inorganic phosphate, employing the method of Taussky and Schorr. When ROS fragments are incubated with both Mg 2+ and ATP in the dark, their turbidity is rapidly reduced.

Correlation of Cellular and Molecular Changes in Visual Photoreceptors by Light Scattering Relaxation Photometry

The correlation of molecular events with structural changes within a cell requires a non-destructive relaxation technique that can be adapted to measure such cellular changes in a time range of milliseconds to minutes. Light scattering relaxation techniques have proved useful for such studies as they can often le measured simultaneously or in parallel with spectral changes characterizing molecular or macromolecular processes. Such techniques are proving useful in the study of photobiological processes particularly in cases where specific cytological changes produced photo chemically can be effected by alternate controlled perturbations such as osmotic shrinking or swelling of cell organelles and/or whole cells.

Three independent assays of a specific magnesium ATPase in the rod outer segments of vertebrate photoreceptors

Reports on ATPase activity in rod outer segments (ROS) have been conflicting, mainly because it has been very difficult to establish that the ATPase activity measured from ROS material actually resides in the ROS and not in small amounts Of contaminating material. Here we present evidence for the existence of a very specific Mg-ATPase system in ROS of both bovine and frog retinae. Its activity can be monitored in three ways:

On the Protein Composition of Bovine Rod Outer Segment Disk Membranes

The pigment molecule rhodopsin is generally believed to account for 90% of the total disk membrane protein1. This figure has recently been questioned by Siebert et al.2, who report the existence of three major protein peaks on SDS-PAGE scans, all in the molecular weight region of 30 000 to 42 000. One of the proteins can be washed out, the remaining two, however, are intrinsic. If this peak separation were not artefactual and if only one of the peaks were rhodopsin, it certainly could not constitute 90% of the total disk membrane protein. We have therefore attempted to confirm Siebert’s data and to unequivocally identify rhodopsin among the disk protein components.

Structural Consequences of Magnesium ATP-ase Activity in Bovine Rod Outer Segment Disk Membranes

Fresh bovine rod outer segment (ROS) preparations exhibit relatively high Mg-ATPase activity (initial rate: 400 nmole ATP hydrolysed min−1 mg rhodopsin−1). The enzyme activity is accompanied by a dramatic change in light-scattering from the ROS: in 5 min the total light-scattering decreases by about 50%. The angular dependence of this dark light-scattering increment suggests that the underlying event is a change in refractive index.