PubMed Health⌕ Search

Biomedical subjects

N J Cook

Publications and source records attributed to N J Cook.

At least 55 records · Page 3Linked to original sources
PubMed1988

Solubilization, purification, and reconstitution of the sodium-calcium exchanger from bovine retinal rod outer segments.

We have previously described a method for the solubilization and reconstitution of the cGMP-gated cation channel from the membranes of bovine rod outer segments (Cook, N. J., Zeilinger, C., Koch, K.-W., and Kaupp, U. B. (1986) J. Biol. Chem. 261, 17033-17039). Here we report that not only cGMP but also sodium is capable of releasing entrapped calcium from liposomes reconstituted with total rod outer segment membrane proteins. Other alkali cations tested were unable to induce calcium efflux; therefore, we concluded that the sodium-induced calcium efflux was due to the sodium-calcium exchanger. Sodium was found to activate calcium efflux from these liposomes with an EC50 of approximately equal to 35 mM, comparable to values reported for the sodium-calcium exchanger in native rod outer segment membranes. We found that reconstitution of the sodium-calcium exchanger is quantitative and used this method to assay the exchange protein during purification using conventional protein chromatographic techniques. In this way, we were able to purify and identify as the rod outer segment sodium-calcium exchanger a glycoprotein of apparent Mr = 220,000 to greater than 90% homogeneity. The specific activity of the purified protein at room temperature was 8.2 mumol of Ca2+ exchanged min-1 mg-1 of protein at 50 mM Na+, corresponding to a turnover number of approximately equal to 30 Ca2+ (or 90 Na+) s-1 exchanger-1. The Mr = 220,000 protein reported here appears to be distinct from another protein ("rim protein") with an identical Mr known to exist in these membranes.

Algorithms↗
PubMed1988

cGMP-dependent channel protein from photoreceptor membranes: single-channel activity of the purified and reconstituted protein.

The cGMP-dependent channel protein has been purified from bovine rod photoreceptor membranes and incorporated into planar lipid membranes. At low divalent cation concentrations, cGMP stimulated single-channel current fluctuations. The probability Po of the channel being open strongly depended on the cGMP concentration (EC50 = 31 microM; Hill coefficient, n = 2.3); whereas the single-channel conductance (lambda = 26 pS) was independent of the agonist concentration. The agonist-stimulated increase in the probability of an open channel was largely due to shorter closed times and, to a lesser extent, due to the channel staying open for a longer time. The current-voltage relationship of the single open channel deviated from ohmic behavior, and the open probability decreased at more negative membrane potentials. The rectification of the macroscopic cGMP-induced current in artificial bilayers that contained many channel copies can be accounted for by the voltage dependence of channel gating together with the nonlinearity of the current-voltage curve of an open channel. Current fluctuations exhibited a variety of sublevels, indicating that the channel may exist in more than one conductive state.

Animals↗
PubMed1987

The cGMP-dependent channel of vertebrate rod photoreceptors exists in two forms of different cGMP sensitivity and pharmacological behavior.

cGMP cooperatively activates a cation-selective channel in vertebrate photoreceptors probably by direct binding to a membrane receptor (Fesenko, E. E., Kolesnikov, S. S., and Lyubarsky, A.L. (1985) Nature 313, 310-313). We have recently described the existence of a similar channel in photoreceptor disc membranes (Koch, K. W., and Kaupp, U. B. (1985) J. Biol. Chem. 260, 6788-6800). In this paper we report that the cGMP-dependent channel in the disc membrane of outer segments exists in at least two forms of different cGMP sensitivity and pharmacological behavior. One form has a low Km value of activation by cGMP (25 microM), and it is not blocked by l-cis-diltiazem. The other form has a high Km value (170 microM) and is blocked by this drug. Both channel forms are cooperatively activated by cGMP (Hill coefficient n = 2.0-3.5), suggesting that the channel may have a cGMP receptor that can bind at least three, and possibly four, molecules of cGMP. In the presence of cGMP, the open state of both channel forms inactivates to a weakly or nonconductive state (cf. Puckett, K. L., and Goldin, S. M. (1986) Biochemistry 25, 1739-1746). A mixed population of different forms of the cGMP-dependent channel may explain the variable numbers reported for the Michaelis constant Km for the channel activation by cGMP and the cooperativity n.

Animals↗
PubMed1987

Identification, purification, and functional reconstitution of the cyclic GMP-dependent channel from rod photoreceptors.

The cyclic GMP-dependent cation channel from bovine rod outer segments has been purified to greater than 90% homogeneity by a rapid two-step chromatographic procedure. The purified channel has an apparent molecular mass of 63 kDa as determined by NaDodSO4/gel electrophoresis. When incorporated into the membrane of liposomes, the purified protein mediates the cyclic GMP-dependent efflux of entrapped Ca2+. The reconstituted channel protein exhibits properties similar to the cyclic GMP-dependent channel observed in excised patches of the plasma membrane and in disk membranes. Cyclic GMP activated the channel cooperatively (Hill coefficient n = 3.1) with an apparent Michaelis constant of approximately 11 microM. After reconstitution of the purified protein into a planar lipid bilayer, we recorded cyclic GMP-stimulated single-channel activity. The single-channel conductance at physiological salt concentrations and in the absence of divalent cations was 26 pS. The drug l-cis-diltiazem, shown to block the cyclic GMP-dependent channel in excised patches of the plasma membrane and in isolated disks of rod outer segments, was ineffective against the purified channel.

Animals↗
PubMed1987

Salivary cortisol for monitoring adrenal activity during marathon runs.

In non-elite male runners (n = 8), changes in adrenal activity were monitored by measurement of salivary cortisol in samples collected at 4-mile intervals during marathon runs. These changes were compared with those in similarly timed samples collected on rest days. Immediately prior to the Cardiff marathon, at 09.00 h, mean salivary cortisol concentrations (21.5 nmol/l) were higher than those in similarly timed rest day samples (14.9 nmol/l). Cortisol concentrations increased during the marathon, and although values at 25 miles were high (79.4 nmol/l), maximum values (87.9 nmol/l) were observed in samples collected 30 min after completion of the run. Some Cardiff marathon runners also participated in the Bristol marathon (n = 4) and a non-competitive event (n = 3). The changing pattern in secretory activity was similar in all events. The easy collection of saliva without cessation of exercise is ideal for monitoring the hormonal response to exercise.

Adrenal Cortex↗
PubMed1986

Solubilization and functional reconstitution of the cGMP-dependent cation channel from bovine rod outer segments.

The protein(s) that constitute(s) the cGMP-regulated channel in vertebrate photoreceptors has been solubilized from rod outer segment membranes and reincorporated into the membrane of calcium-containing liposomes. The properties of the reconstituted channel protein were determined by studying the cGMP-stimulated efflux of Ca2+ from these liposomes. Among several detergents tested the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) proved to be the most suitable. Solubilization of channel activity was found to be optimal at a detergent concentration of about 18 mM. The presence of Ca2+ ions and phospholipids during solubilization greatly increased the channel stability. The reconstituted channel shared most but not all properties with the channel in situ. It is cooperatively activated by cGMP with an EC50 of 19 microM. The cooperativity as determined from Hill plots was n = 2.7. Unlike the cGMP-sensitive channel in the native membrane of isolated discs and excised patches of plasma membrane it is not blocked by l-cis-diltiazem. Reconstitution of this channel protein(s) may serve as a valuable tool for identifying the polypeptide composition and to study structural and functional aspects of the purified protein(s).

Animals↗
PubMed1986

Interplay between calcium and activated cGMP phosphodiesterase from retinal rod outer segments.

Hypotonic extraction of bovine retinal rod outer segments after bleaching in isotonic buffer yielded an extract exhibiting activated cGMP phosphodiesterase properties. Since this extract was virtually devoid of other proteins involved in the rod outer segment cGMP enzymatic cascade, it was used to study phosphodiesterase catalytic activity. The hypotonic extract required Mg2+ in the range 0.1-1.0 mM for optimal cGMP hydrolysis. At these Mg2+ concentrations hydrolysis could be effectively inhibited by Ca2+ at concentrations which might be attainable in rod outer segments. Since higher Ca2+ concentrations were required to give a chosen degree of inhibition at higher Mg2+ concentrations, this inhibition was probably due to competition by Ca2+ for Mg2+ binding site(s) on the phosphodiesterase catalytic unit. Other divalent cations were also able to inhibit cGMP hydrolysis, many of them (especially those with ionic radii close to that of magnesium) more effectively than calcium. It is suggested that Ca2+ may play a role in phototransduction by participating in the control of photoreceptor sensitivity, and that this is achieved by modulating rod outer segment cGMP hydrolysis.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1986

Changes in adrenal and testicular activity monitored by salivary sampling in males throughout marathon runs.

Measurement of cortisol and testosterone in saliva samples provided by marathon runners at 6.4 km (4-mile) intervals has been used for monitoring acute changes in adrenal and testicular activity, and the changes compared with mean values in timed samples on five rest days. The collection of mixed whole saliva was well accepted; the missed sample rate in the 8 runners in the Cardiff marathon was less than 10%. On rest days, salivary cortisol and testosterone were within the normal male range and showed a circadian rhythm; mean values at 08.00 h (23.5 nmol L-1; 258 pmol L-1, p less than 0.001, p less than 0.001 respectively) were higher than at 22.00 h (2.8 nmol L-1; 130 pmol L-1). In samples collected at 09.00 h, immediately prior to the Cardiff marathon, cortisol (25.1 nmol L-1) and testosterone (304 pmol L-1) were higher than the mean values (14.9 nmol L-1; 209 pmol L-1) on non-run days. Concentrations of both steroids increased during the marathon; testosterone peaked (442 pmol L-1) at 21 miles, whereas cortisol continued to increase, being maximal (87.9 nmol L-1) at 30 min after completion of the run. Four of the runners in the Cardiff marathon also participated in the Bristol marathon and the changing patterns in salivary hormones were strictly comparable. Salivary sampling would appear to be of value in monitoring acute and rhythmic changes in endocrine function in marathon runners. The temporal relationship between changes in salivary cortisol and testosterone are consistent with direct inhibition of testicular secretion by high cortisol concentrations.

Adrenal Glands↗
PubMed1985

Effects of fluoride on retinal rod outer segment cGMP phosphodiesterase and G-protein.

The effects of fluoride on ROS phosphodiesterase and G-protein have been studied using membrane-free extracts. When G-protein was present NaF, at millimolar concentrations, stimulated PDE activity however, in a G-protein free extract, cGMP hydrolysis was inhibited by high fluoride concentrations. Fluoride was also found to profoundly inhibit the ability of G-protein to bind a GTP analogue, GTP gamma S, both in the presence and absence of rhodopsin. Aluminium greatly modified these effects of fluoride on PDE and G-protein. The possibility that fluoride activates PDE through its effect on G-protein is discussed.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1985

Light-induced conformational changes in the extradiscal regions of bovine rhodopsin.

Light-induced conformational changes occurring at the cytosolic surface of rhodopsin were investigated by performing limited digestions of native and illuminated visual pigment with thermolysin, Arg-C endoproteinase, papain and proteinase K. A higher susceptibility of the extradiscal regions of the bleached pigment to the proteases were observed together with altered capacities of the digested bleached rhodopsins to activate the cGMP phosphodiesterase. The overall results strongly suggest that light induces conformational changes not only in the C-terminal end but also in the second and the third extradiscal loop of rhodopsin.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1985

Light-induced conformational change in rhodopsin detected by modification of G-protein binding, GTP gamma S binding and cGMP phosphodiesterase activation.

CNBr treatment of rod outer segments was performed in dark and in light conditions. With the subsequent modified rhodopsin and opsin the cGMP phosphodiesterase activation system was reconstituted. The recombination systems exhibited greatly reduced G-protein binding, GTP gamma S binding and cGMP phosphodiesterase activation. The reduction in activity of these three steps of the PDE activation cascade is most significant with modified opsin and is shown to be due to its inability to bind the G alpha subunit. The correlation between the localization of CNBr cleavage in dark and light conditions and these results is strongly indicative that a light-induced conformational change occurs in two extradiscal regions of rhodopsin.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1985

Investigation of rhodopsin catalyzed G-protein GTP-binding using [35S] GTP gamma S--effects of regeneration and hydroxylamine.

A simple reconstitution system for studying rhodopsin-catalyzed G-protein GTP-binding is described. Purified rhodopsin is recombined with a G-protein containing extract in the presence of [35S]GTP gamma S and after incubation the reaction is stopped by rapid cooling and filtration. The system has been used to study the effects of 11-cis-retinal and hydroxylamine on R*-catalyzed G-protein GTP-binding. Since both these compounds greatly reduced binding, our results provide further evidence that it is the rhodopsin intermediate metarhodopsin II which interacts with G-protein.

Animals↗
PubMed1984

Postnatal variations of endo-beta-D-N-acetyl-glucosaminidase in the developing rat cerebellum.

Some properties and the level of endo-beta-N-acetylglucosaminidase were studied during the postnatal development of the rat cerebellum. No isoenzymes were detected. The enzyme (essentially cytosolic) showed a peak of total and specific activity at the 24th postnatal day. The hypothesis was formulated that glycoprotein glycans could be degraded in two steps: one lysosomal, the other cytosolic.

Age Factors↗
PubMed1983

Urinary screening for abnormalities of amino acid or mucopolysaccharide metabolism in patients in a hospital for the mentally handicapped in Wessex.

Urine from 348 patients of a hospital for the mentally handicapped in Wessex was screened for abnormalities of aminoacid and mucopolysaccharide excretion. Persistent aminoacid abnormalities were found in 16 patients: two had phenylketonuria and one a severe variant form of hyperphenylalaninaemia (dihydropteridine reductase deficiency). In at least nine of the other patients, the observed abnormality was probably a secondary phenomenon. Two patients excreted excessive amounts of a glycosaminoglycan identified tentatively as hyaluronic acid. The study also uncovered one undiagnosed diabetic and three patients with urinary tract infection.

Adult↗
PubMed1981

Ethanol-amphetamine interaction effects on spontaneous motor activity and fixed-interval responding.

The spontaneous motor activity (SMA) of rats was recorded after injections of saline, d-amphetamine sulfate (0.8 mg/kg), and ethanol (400, 800, 1200, and 1600 mg/kg). Each drug treatment was given separately, and the amphetamine treatment was also combined with each ethanol dose. Ethanol, when injected without amphetamine, produced a dose-related decrement in SMA. Amphetamine, injected without ethanol, produced an increase in SMA. The combination of ethanol at 400 mg/kg with amphetamine potentiated the amphetamine-stimulant effect, but higher doses of ethanol counteracted amphetamine-produced increment in SMA. In a second experiment, similar combinations of ethanol and amphetamine were administered to rats lever-pressing for food pellets under a fixed-interval reinforcement schedule. The effect of amphetamine alone depended on baseline rate and varied among individual rats. Ethanol had a depressant effect on response rates, but combinations of the two drug treatments produced rates that, in most rats, were higher than after any single drug or saline treatment.

Animals↗