Interspecific variation in the visual pigments of deep-sea fishes.

Visual pigments in the rods of 38 species of deep-sea fish were examined by microspectrophotometry. 33 species were found to have a single rhodopsin with a wavelength of maximum absorbance (lambda max) in the range 470-495 nm. Such visual pigments have absorbance maxima close to the wavelengths of maximum spectral transmission of oceanic water. 5 species, however, did not conform to this pattern and visual pigments were found with lambda max values ranging from 451 nm to 539 nm. In 4 of these species two visual pigments were found located in two types of rod. Some 2-pigment species which have unusual red sensitivity, also have red-emitting photophores. These species have both rhodopsin and porphyropsin pigments in their retinae, which was confirmed by HPLC, and the two pigments are apparently located in separate rods in the same retinal area. In deep-sea fishes the occurrence of 'unusual' visual pigments seems to be correlated with aspects of the species' depth ranges. In addition to ecological influences we present evidence, in the form of lambda max spectral clustering, that indicates the degree of molecular constraint imposed on the evolution of visual pigments in the deep-sea.

Synthesis of functional bovine opsin in insect cells under control of the baculovirus polyhedrin promoter.

In vitro expression of cDNA encoding bovine opsin is accomplished using the baculovirus expression vector system. Full-length opsin was synthesized which was recognized by poly- and monoclonal antisera raised against bovine rhodopsin. Upon infection with a recombinant virus, 1 x 10(6) insect cells produced up to 3 micrograms opsin. Incubation of the in vitro synthesized opsin with 11-cis retinal produced a hydroxylamine-stable, photosensitive pigment.

Studies on (K+ + H+)-ATPase. VI. Determination on the molecular size by radiation inactivation analysis.

(1) A (K+ + H+)-ATPase containing membrane fraction, isolated from pig gastric mucosa, has been further purified by means of zonal electrophoresis, leading to a 20% increase in specific activity and an increase in ratio of (K+ + H+)-ATPase to basal Mg2+-ATPase activity from 9 to 20. (2) The target size of (Na+ + K+)-ATPase, determined by radiation inactivation analysis, is 332 kDa, in excellent agreement with the earlier value of 327 kDa obtained from the subunit composition and subunit molecular weights. This shows that the Kepner-Macey factor of 6.4 X 10(11) is valid for membrane-bound ATPases. (3) The target size of (K+ + H+)-ATPase is 444 kDa, which, in connection with a subunit molecular weight of 110000, suggests a tetrameric assembly of the native enzyme. The ouabain-insensitive K+-stimulated p-nitrophenylphosphatase activity has a target size of 295 kDa. (4) In the presence of added Mg2+ the target sizes of the (K+ + H+)-ATPase and its phosphatase activity are decreased by about 15%, while that for the (Na+ + K+)-ATPase is not significantly changed. This observation is discussed in terms of a Mg2+-induced tightening of the subunits composing the (K+ + H+)-ATPase molecule.

Studies on (K+ + H+)-ATPase. IV. Effects of phospholipase C treatment.

(1) The total phospholipid content of a gradient purified (K+ + H+)-ATPase preparation from pig gastric mucosa is 105 mumol per 100 mg protein, and consists of 29% sphingomyelin, 29% phosphatidylcholine, 28% phosphatidylethanolamine, 10% phosphatidylserine and 4% phosphatidylinositol. The cholesterol content corresponds to 50 mumol per 100 mg protein. (2) Treatment with phospholipase C (from Clostridium welchii and Bacillus cereus) results in an immediate decrease of the phosphate content. Up to 50% of the phospholipids are hydrolyzed by each phospholipase C preparation alone, without further hydrolysis by increased phospholipase concentration or prolonged incubation time. Combined treatment with the two phospholipase C preparations, sequentially or simultaneously, hydrolyzes up to 65% of the phospholipids. (3) The (K+ + H+)-ATPase and K+ stimulated p-nitrophenylphosphatase activities are decreased proportionally with the total phospholipid content, indicating that these enzyme activities are dependent on phospholipids. (4) Phospholipase C treatment does not change optimal pH, Km value for ATP and temperature dependence of the gastric (K+ + H+)-ATPase, but slightly decreases the Ka value for K+. (5) Phospholipase C treatment lowers the AdoPP[NH]P binding and phosphorylation capacities, suggesting that inactivation occurs primarily on the substrate binding level. (6) Most of the results can be understood by assuming that hydrolysis of the phospholipids by phospholipase C leads to aggregation of the membrane protein molecules and complete inactivation of the aggregated ATPase molecules.