Properties of a magnesium- or calcium-dependent adenosine triphosphatase from frog rod photoreceptor outer segment disks and its inhibition by illumination

Abstract

Isolated frog retinal rod photoreceptor disk membranes were found to possess an ouabain-insensitive adenosine triphosphatase (ATPase) which was dependent on the presence of Mg or Ca in the incubation medium. Several tests showed the activity was not of mitochondrial origin, although the level of mitochondrial contamination was about 5%. The level of ATPase activity was optimal at an Mg : ATP ratio of 1 :I. Ca supported a lower level of activity in the absence of Mg, showed no optimum Ca:ATP ratio in the range tested, and inhibited ATPase activity when added in the presence of Mg. The released inorganic phosphate was not due to an T he retinal photoreceptor outer segment is the site of the absorption of light in vision. The rod outer segment contains freely floating membranous organelles in the form of a stack of flattened disks. The membranes of these disks contain, as an integral part, the visual pigment rhodopsin which absorbs light and initiates vision. It has been shown previously that the disks contain a distinct internal space, separate from the intracellular space (Heller et a/., 1971) and that the membranes are semipermeable (Heller et a/., 1970).As a part of the biochemical study of these membranous organelles, we performed an electron microscopical and a chemical analysis of the isolated rod outer segment preparation, and then reinvestigated the known adenosine triphosphatase (ATPase)' activity of the preparation. This paper deals with the characteristics of the (Mg-Ca)-dependent, ouabain-insensitive ATPase first described by Bonting et al. (1964) and Frank and Goldsmith (1965).In most respects our results confirm and extend those already described. However, we found a distinct change in ATPase activity upon illumination, in contrast to the results of either Bonting et al. (1964) or Frank and Goldsmith (1965). Methods and MaterialsRod outer segments (ROS) were prepared fresh each day from Rana pipiens which were dark adapted overnight, as described previously (Heller et al., 1971). For experiments in imidazole buffer, the purified outer segments were washed three times, after the sucrose isolation procedure, in 60 mM

Electrical and adaptive properties of rod photoreceptors in Bufo marinus. II. Effects of cyclic nucleotides and prostaglandins.

“…One way this could be accomplished is through the stimulation of Ca2+-ATPase pumps by cGMP. It has been reported that Ca2+-ATPase pumps are located in both the plasma and disk membranes (Bownds et al, 1971;Hagins and Yoshikami, 1974;Mason et al, 1974;Ostwald and Heller, 1972); thus, when cGMP levels in the rod outer segment are increased, the activity of the Ca 2+ pumps is enhanced and cytosol Ca z+ levels are lowered. With lower intracellular Ca 2+ levels, more Na + channels are opened and available for blockade; the receptor depolarizes, and response amplitudes increase.…”

Molecular aspects of photoreceptor function

“…It has not been established if the active accumulation is due to a Ca 2+ pump with its associated ATPase. However, a Ca 2+ -or Mg 2+ -activated ATPase has recently been discovered by Ostwald & Heller (1972) in rod outer segments.…”

Visual Photoreceptors

“…In the Ca2+-transmitter model an active Ca2+ uptake mechanism must be present to return released Ca2+ to the disk lumen. Extending earlier work [104], Sack and Harris [116] have found a Ca'+-dependent ATPase, which increases in specific activity as ROS are purified, and may function in this capacity. ROS can easily be broken off from the retina yielding a suspension of light-sensitive organelles whose functioning can be studied independently of the inner segments.…”