S.L. Bonting

Studies on (Na+-K+)-activated ATPase XXIV. Localization and properties of ATPase in the inner ear of the guinea pig

1. (1) The properties and distribution of an ouabain-sensitive (Na+-K+)-ATPase system in the inner ear structures of the guinea pig were investigated with ultramicrochemical techniques. The properties of this enzyme system are in good agreement with those found in other tissues. 2. (2) The enzyme was present in high activity in the stria vascularis (7.95 moles/kg dry wt. per h). There was a clear decrease in enzyme activity from the base of the cochlea to the apex. 3. (3) The enzyme activity in the other cochlear structures was rather low (< 0.5 mole/kg dry wt. per h), with the exception of the highly vascularized part of the spiral ligament (1.60 mg moles/kg dry wt. per h). 4. (4) These results strongly suggest that the (Na+-K+)-ATPase system in the stria vascularis plays the primary role in the maintenance of the cochlear cation gradients, while the contribution of Reissners membrane to this process can only be very minor. 5. (5) The good agreement between the half maximal inhibition concentration of ouabain for the enzyme system and the cochlear potentials, indicates moreover, a dependence of these potentials on the functioning of the (Na+-K+)-ATPase system.

Biochemical aspects of the visual process: XXVII. Stereospecificity of ocular retinol dehydrogenases and the visual cycle

A comparative study is made of the stereospecificity of two particulate retinol dehydrogenases from bovine eyes and of horse liver alcohol dehydrogenase. The particulate retinol dehydrogenase of outer segments reacts with the all-trans isomers of retinaldehyde and retinol but not with the 11-cis compounds. In contrast, a particulate retinol dehydrogenase present in pigment epithelium reacts preferentially with the 11-cis compounds. Horse liver alcohol dehydrogenase (EC 1.1.1.1.) can convert both isomers, but the all-trans isomers are clearly preferred. Differences with regard to cofactor preference and stability are also noted. The outer segment enzyme clearly functions in the rhodopsin cycle. It is unlikely that the 11-cis retinol dehydrogenase from pigment epithelium is directly involved in providing 11-cis retinaldehyde from rhodopsin regeneration, but it may serve to make available 11-cis retinaldehyde from rhodopdsin, digested in phagocytized rod sacs, for the synthesis of visual pigment by the visual cells.

Enrichment of rhodopsin in rod outer segment membrane preparations: Biochemical aspects of the visual process—XVIII

Abstract Substantial, seasonally varying amounts of opsin are present in cattle rhodopsin preparations, which cannot be converted to rhodopsin by prior dark adaptation of the excised eyes. A method is described for the quantitative conversion of the opsin to rhodopsin by treatment of a rod outer segment preparation with 11- cis retinaldehyde, followed by removal of excess retinaldehyde through reduction by endogenous retinol dehydrogenase and added NADPH and elution of the resulting retinol by sucrose gradient centrifugation. The resulting rhodopsin preparation has a high pigment content of 8 nMol/mg dry weight, which is constant within 2.5 per cent over the entire year. The preparation is of high purity, as indicated by theA 278 /A 500 ratio of 2.0–2.5 and theA 400 /A 500 ratio of 0.20–0.26. The yield is high, representing 50 per cent of the pigment present in the retina. The preparation is free of detergent, has a full regenerating capacity, and can be stored for long periods of time.

Biochemical aspects of the visual process: VI. The lipid composition of native and hexane-extracted cattle rod outer segments

Abstract Cattle rod outer segments were isolated by means of a continuous sucrose gradient technique. These preparations contained 39% total lipid and 31.5% phospholipid on a dry weight basis. The fatty acid composition of the total rod outer segment lipids was determined by means of gas-liquid chromatography. The three predominant fatty acids were: palmitic acid (19.4% (w/w) of total fatty acids), stearic acid (23.1%) and docosahexaenoic acid (34.3%). Quantitative analysis of the phospholipids by two-dimensional thin-layer chromatography showed that the three major phospholipids are: phosphatidyl ethanolamine (35.4%), phosphatidyl choline (34.7%) and phosphatidyl serine (11.2%). Half of the total amount of phospholipids was extractable with n hexane without changing the spectral and photolytic properties of the rhodopsin. Relatively more phosphatidyl ethanolamine than phosphatidyl choline was extracted by hexane, reducing the molar phosphatidyl ethanolamine: retinaldehyde ratio from 29 to 9.

[36] Isolation and purification of bovine rhodopsin

Publisher Summary This chapter describes the isolation and purification of bovine rhodopsin. Rhodopsin is the general name for the visual pigments of both the rod cells in the vertebrate retina and the invertebrate photoreceptor cells. Rhodopsin is an intrinsic membrane glycoprotein, confined to the membranous structures (photoreceptor membranes) of a special part of the rod cell, the rod outer segment (ROS), which is highly specialized for light reception. Rhodopsin is determined by measuring the decrease in absorbance at 500 nm upon illumination, using a molar absorbance of 40,500. Under some conditions, the photoproducts also absorb at 500 nm, but addition of hydroxylamine will convert all free and bound retinaldehyde into retinal oxime, which has virtually no absorption beyond 450 nm. In detergents like the quarternary ammonium derivatives (DTAB, TTAB, CTAB) or the amine oxides (C10DAO, C12DAO), addition of hydroxylamine is not required, because under these conditions retinal is completely released and hardly interferes at 500 nm. Hydroxylamine should be handled with care, because it is highly mutagenic. The assay can be performed on suspensions of photoreceptor membranes, but scattering artifacts may interfere. Most accurate results are obtained after prior solubilization of the membrane in an appropriate detergent, followed by centrifugation or filtration through a membrane filter if some turbidity remains.

Adenylate cyclase in the rat pancreas properties and stimulation by hormones

Abstract 1. 1. In view of evidence for a role of cyclic AMP in the secretin-stimulated water and electrolyte secretion and the pancreozymin-stimulated enzyme secretion by the pancreas, the occurrence of adenylate cyclase in the pancreas has been investigated. 2. 2. The presence of the enzyme in a particulate fraction of rat pancreas has been demonstrated. Basal activity is very low under standard conditions (less than 3 pmoles/min per mg protein), but 10 mM NaF stimulates the enzyme to 15–40 pmoles/min per mg protein. 3. 3. Properties of the enzyme have been determined in the presence of 10 mM NaF. The apparent K m for ATP, derived from a Lineweaver-Burk plot is 0.3 mM. At ATP concentrations above 1.5 mM there is substrate inhibition. The optimal pH is 7.4. The enzyme requires Mg 2+ for its activity, concentrations of 5 mM and higher giving maximal activity. Ca 2+ in concentrations above 0.1 mM inhibits the enzyme. 4. 4. The enzyme is stimulated by a synthetic secretin preparation (half maximally activating concentration 1.5·10 −8 M) as well as by a purified pancreozymin preparation (half maximally activating concentration 1.5·10 −6 M). The latter activation is not due to a contamination of the pancreozymin preparation by secretin. The secretin-stimulated enzyme has the same optimal pH of 7.4 and shows the same Ca 2+ inhibition as the NaF-stimulated enzyme. 5. 5. The enzyme is not stimulated by adrenalin and its analogues, by acetylcholine and its analogues, and by the hormones glucagon and gastrin (synthetic). 6. 6. Specific enzyme activity (per mg protein) in enzyme preparations obtained from rats with free access to food and stimulated in vivo by secretin and pancreozymin is twice as high as that in enzyme preparations from starved animals. 7. 7. The physiological significance of these findings is discussed.

Role of negatively charged phospholipids in highly purified (Na+ + K+)-ATPase from rabbit kidney outer medulla. Studies on (Na+ + K+)-activated ATPase, XXXIX

1. The requirement for specific polar head groups of phospholipids for activity of purified (Na+ + K+)ATPase from rabbit kidney outer medulla has been investigated. 2. Comparison of content and composition of phospholipids in microsomes and the purified enzyme indicates that purification leads to an increase in the phospholipid/protein ratio and in phosphatidylserine content. 3. The purified preparation contains 267 molecules phospholipid per molecule (Na+ + K+)-ATPase, viz. 95 phosphatidylcholine, 74 phosphatidylethanolamine, 48 spingomyelin, 35 phosphatidylserine and 15 phosphatidylinositol. 4. Complete conversion of phosphatidylserine into phosphatidylethanolamine by the enzyme phosphatidylserine decarboxylase has no effect on the (Na+ + K+)-ATPase activity of the purified preparation. 5. Complete hydrolysis of phosphatidylinositol by a phospholipase C from Staphylococcus aureus, which is specific for this phospholipid, has no effect on the (Na+ + K+)-ATPase activity. 6. Hydrolysis of 95% of the phosphatidylcholine and 60--70% of the spingomyelin and phosphatidylethanolamine by another phospholipase C (Clostridium welchii) lowers the (Na+ + K+)-ATPase activity by about 20%. 7. Combination of the phospholipid-converting enzymes has the same effect as can be calculated from the effects of the enzymes separately. Only complete conversion of both phosphatidylserine and phosphatidylinositol results in a loss of 44% of the (NA+ + K+)-ATPase activity and 36% of the potassium 4-nitrophenylphosphatase activity. 8. These experiments indicate that there is no absolute requirement for one of the polar head groups, although in the absence of negative charges the activity is lower than in their presence.

Biochemical aspects of the visual process. XXV. Light-induced calcium movements in isolated frog rod outer segments

Abstract Suspensions of isolated rod outer segments are shown to have a high calcium content of up to 11 moles calcium per mole rhodopsin. Osmotic lysis of the outer segments demonstrates the presence of two calcium fractions, a soluble one and a particulate one. The particulate fraction apparently coincides with the rod disks or with disk fragments. Illumination of intact rod outer segments in calcium-free ATP-containing Ringer solution has no measurable effect upon their total caclium content, but causes a significant shift of calcium from the particulate to the soluble fraction. This light effect is retained in lysed outer segments resuspended in calcium-free ATP-containing Ringer. These results support a function of calcium as a transmitter in light transduction in rod outer segments.

Studies on (Na+-Ka+)-activated ATPase. XX. Properties of (Na+-K+)-activated ATPase in rat liver

1. 1. The ouabain-sensitive (Na+-K+-activated ATPase enzyme system was present in rather low activity (0.37 mole/kg dry weight per h) in rat liver. 2. 2. Pretreatment with 1.5 M urea decreased the Mg2+-activated ATPase activity without significantly affecting the (Na+-K+-ATPase activity, thus causing the relative activity of the latter to rise from 13% to 37%. This permitted to determine the properties of the (Na+-K+)-ATPase system with greater accuracy. 3. 3. The (Na+-K+)-ATPase required for activation both Na+ (Km = 6 mM) and K+(Km = 0.9 mM). Rb+ could replace K+ in activating the enzyme (Km = 0.8 mM). Maximal activation of the (Na+-K+)-ATPase system required 2 mM Mg2+ at an ATP concentration of 2 mM. 4. 4. The pH optimum for (Na+-K+)-ATPase was 7.3, while the Mg2+-activated ATPase activity had a pH optimum of 8.7. 5. 5. The optimal temperature for (Na+-K+)-ATPase and for Mg2+-ATPase activity was 45°. 6. 6. The (Na+-K+)-ATPase was inhibited by the digitalis glycoside ouabain (pI50 = 3.9) and the Erythrophleum alkaloid erythropleine (pI50 = 5.1).

On the biochemical mechanism of the visual process

Evidence is presented supporting an ionic mechanism for the stimulation of the photoreceptor by light. Illumination of a rhodopsin monolayer at an air-water interface causes expansion of the layer, which can be interpreted as the result of penetration of the monolayer by retinal. Illumination of a rod outer segment suspension causes efflux of K ions and an equivalent influx of Na ions. The same effect, qualitatively and quantitatively, occurs upon addition of all-trans retinal to the suspension. All-trans retinal added to artificial phosphatidyl-ethanolamine membranes greatly increases cation leakage through these micelles, but has no effect on lecithin micelles. All-trans retinol has a small effect on both types of micelles. These findings are interpreted as suggesting that upon photolysis of rhodopsin all-trans retinal would be transferred from opsin to a Schiff base link with the amino group of phosphatidylethanolamine or phosphatidylserine present in the rod sac membrane. Blocking of the amino group would make the membrane more negatively charged and thus more cation-permeable. The resulting cation exchange would cause a depolarizing current stimulating the synapse with the bipolar cell.