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K W Koch

Publications and source records attributed to K W Koch.

36 records · Page 2Linked to original sources
PubMed1995

Control of photoreceptor proteins by Ca2+.

A decrease of cytoplasmic Ca(2+)-concentration in vertebrate photoreceptor cells after illumination is necessary for light adaptation. Although the mechanisms of adaptation is not completely understood, several Ca(2+)-dependent cellular processes have been discovered. Some involve calcium-binding proteins like recoverin, guanylyl cyclase-activating protein and calmodulin, and their target proteins rhodopsin kinase, guanylyl cyclase, the cGMP-gated channel, and NO synthase. The activity of several enzymes or channels is directly controlled by Ca2+ and does not involve calcium-binding proteins. These proteins are pyrophosphatase, protein kinase C and the cGMP-gated channel.

Animals↗
PubMed1994

Functional coupling of a Ca2+/calmodulin-dependent nitric oxide synthase and a soluble guanylyl cyclase in vertebrate photoreceptor cells.

Electrophysiological recordings on retinal rod cells, horizontal cells and on-bipolar cells indicate that exogenous nitric oxide (NO) has neuromodulatory effects in the vertebrate retina. We report here endogenous NO formation in mammalian photoreceptor cells. Photoreceptor NO synthase resembled the neuronal NOS type I from mammalian brain. NOS activity utilized the substrate L-arginine (Km = 4 microM) and the cofactors NADPH, FAD, FMN and tetrahydrobiopterin. The activity showed a complete dependence on the free calcium concentration ([Ca2+]) and was mediated by calmodulin. NO synthase activity was sufficient to activate an endogenous soluble guanylyl cyclase that copurified in photoreceptor preparations. This functional coupling was strictly controlled by the free [Ca2+] (EC50 = 0.84 microM). Activation of the soluble guanylyl cyclase by endogenous NO was up to 100% of the maximal activation of this enzyme observed with the exogenous NO donor compound sodium nitroprusside. This NO/cGMP pathway was predominantly localized in inner and not in outer segments of photoreceptors. Immunocytochemically, we localized NO synthase type I mainly in the ellipsoid region of the inner segments and a soluble guanylyl cyclase in cell bodies of cone photoreceptor cells. We conclude that in photoreceptors endogenous NO is functionally coupled to a soluble guanylyl cyclase and suggest that it has a neuromodulatory role in visual transduction and in synaptic transmission in the outer retina.

Amino Acid Oxidoreductases↗
PubMed1994

Bovine retinal rod guanyl cyclase represents a new N-glycosylated subtype of membrane-bound guanyl cyclases.

The molecular properties of retinal rod guanyl cyclase were investigated. Peptides were derived from a 112-kDa protein previously identified as the particulate bovine retinal rod guanyl cyclase. The peptides showed 100% identity to the deduced amino acid sequence of the cloned human retina-specific membrane guanyl cyclase, whereas identity to the members of the natriuretic peptide receptor guanyl cyclases was 14-59%. The 112-kDa protein was further purified by a new approach using wheat-germ agglutinin chromatography. This indicated N-linked glycosylation in retinal rod guanyl cyclase. N-glycosylation was unexpected from the sequence of the human retina-specific membrane guanyl cyclase, although it is a common property of natriuretic peptide receptors. Therefore, we further analyzed the carbohydrate composition of bovine retinal rod guanyl cyclase by lectin binding using the lectins Galanthus nivalis agglutinin, Sambucus nigra agglutinin, Maackia amurensis agglutinin, Ricinus communis agglutinin, Datura stramonium agglutinin, peanut agglutinin and by chromatography of the purified enzyme using concanavalin-A-Sepharose. The oligosaccharide side chains were of the high-mannose type or hybrid type, probably with mannose, N-acetylglucosamine and sialic acid as terminal sugars. Enzymic deglycosylation by N-glycosidase F was achieved after proteolytic digestion with endoproteinase Glu-C. Lectins neither influenced the basal nor the stimulated guanyl-cyclase activity at low calcium concentrations. Our results indicate that the particulate rod guanyl cyclase represents an unusual new subtype of membrane-bound guanyl cyclases.

Amino Acid Sequence↗
PubMed1993

Frequenin--a novel calcium-binding protein that modulates synaptic efficacy in the Drosophila nervous system.

The T(X;Y)V7 rearrangement in Drosophila has originally been recognized as a Shaker-like mutant because of its behavioral and electrophysiological phenotype. The gene whose expression is altered by the V7 rearrangement has been characterized. It encodes a novel Ca(2+)-binding protein named frequenin, which is related to recoverin and visinin. In vitro, the frequenin protein functions like recoverin as a Ca(2+)-sensitive guanylyl cyclase activator. Anti-frequenin antibodies stain the central and peripheral nervous system in Drosophila embryos and in larval and adult tissue sections. Frequenin appears to be particularly enriched in synapses, such as the motor nerve endings at neuromuscular junctions. Neuromuscular junctions of transgenic flies, which overexpress frequenin upon heat shock, exhibit an extraordinarily enhanced, frequency-dependent facilitation of neurotransmitter release, with properties identical to those observed in V7 junctions. We propose that frequenin represents a new element for the Ca(2+)-dependent modulation of synaptic efficacy.

Amino Acid Sequence↗
PubMed1992

Recoverin, a novel calcium-binding protein from vertebrate photoreceptors.

Photoreceptor guanylyl cyclase activity is modulated by an endogenous calcium-binding protein called recoverin. A modified isolation procedure for recoverin using gel-filtration chromatography instead of a heat denaturation step is presented. The elution volume of recoverin corresponds to a monomer. Recoverin exhibits a calcium-dependent mobility shift in a native gel electrophoresis. Isoelectric focusing revealed a pI of 5.25. No subspecies of recoverin were detected.

Animals↗
PubMed1992

Biochemical mechanism of light adaptation in vertebrate photoreceptors.

Vertebrate photoreceptors can adjust their sensitivity to a wide range of light intensities spanning several orders of magnitude, the phenomenon of which is called light adaptation. Electrophysiological and biochemical studies have revealed that calcium can serve as an intracellular transmitter of light adaptation under the control of cGMP metabolism. After illumination, the cytoplasmic calcium concentration of a photoreceptor decreases, which in turn strongly activates photoreceptor guanylate cyclase. This calcium-dependent effect is mediated by a novel calcium-binding protein (recoverin) and leads to the restoration of the depleted cGMP pool after illumination.

Adaptation, Ocular↗
PubMed1991

Phosphorylation of recoverin, the calcium-sensitive activator of photoreceptor guanylyl cyclase.

Recoverin, a new calcium binding protein from bovine rod photoreceptor cells, activates guanylyl cyclase below a free calcium concentration of 200 nM. We show here that recoverin is phosphorylated by an endogenous kinase and Mg-ATP at the same decreased calcium concentration. The calcium-dependent activation of guanylyl cyclase is enhanced in the presence of ATP. We suggest that phosphorylation of recoverin reinforces the stimulation of guanylyl cyclase at decreased calcium concentrations.

Adenosine Triphosphate↗
PubMed1991

Purification and identification of photoreceptor guanylate cyclase.

Photoreceptor guanylate cyclase was solubilized and purified from bovine rod outer segments with 50-150-fold increase in specific activity using the nonionic detergent n-dodecyl-beta-D-maltoside. Guanylate cyclase activities correlated with the enrichment of a protein with an apparent Mr = 112,000. The purified enzyme showed specific activities of 100-700 nmol of cGMP produced/min/mg protein and exhibited positive cooperativity with respect to MnGTP (Hill coefficient n = 1.6 +/- 0.1). The apparent Km was 274 +/- 67 microM, and the turnover number was determined to be 0.2-1.3 cGMP produced/s. The molar ratio of the 112-kDa protein to rhodopsin corresponds to 1:104. This indicates that the amount of guanylate cyclase in rod photoreceptors is nearly equimolar to the amount of the phosphodiesterase.

Animals↗
PubMed1991

A 26 kd calcium binding protein from bovine rod outer segments as modulator of photoreceptor guanylate cyclase.

The resynthesis of cGMP in vertebrate photoreceptors by guanylate cyclase is one of the key events leading to the reopening of cGMP-gated channels after photoexcitation. Guanylate cyclase activity in vertebrate rod outer segments is dependent on the free calcium concentration. The basal activity of the enzyme observed at high concentrations of free calcium (greater than 0.5 microM) increases when the free calcium concentration is lowered into the nanomolar range (less than 0.1 microM). This effect of calcium is known to be mediated by a soluble calcium-sensitive protein in a highly cooperative way. We here show that this soluble protein, i.e. the modulator of photoreceptor guanylate cyclase, is a 26 kd protein. Reconstitution of the purified 26 kd protein with washed rod outer segment membranes containing guanylate cyclase revealed a 3- to 4-fold increase of cyclase activity when the free calcium concentration was lowered in the physiological range from 0.5 microM to 4 nM. Guanylate cyclase in whole rod outer segments was stimulated 10-fold in the same calcium range. The activation process in the reconstituted system was similar to the one in the native rod outer segment preparation, it showed a high cooperativity with a Hill coefficient n between 1.4 and 3.5. The half-maximal activation occurred between 110 and 220 nM free calcium. The molar ratio of the modulator to rhodopsin is 1:76 +/- 32. The protein is a calcium binding protein as detected with 45Ca autoradiography. Partial amino acid sequence analysis revealed a 60% homology to visinin from chicken cones.

Amino Acid Sequence↗
PubMed1990

Stereochemical course of the reaction catalyzed by guanylate cyclase from bovine retinal rod outer segments.

The stereochemical course of the reaction catalyzed by guanylate cyclase from bovine retinal rod outer segments was investigated using phosphorothioate analogs of GTP as chiral probes. (Sp)-Guanosine 5'-O-(1-thiotriphosphate) (Sp-GTP alpha S) is a substrate, whereas (Rp)-GTP alpha S is a competitive inhibitor (K1 = 0.1 mM), but not a substrate. (Sp)-GTP alpha S is converted into (Rp)-guanosine 3':5'-monophosphorothioate, showing that the reaction proceeds with inversion of configuration at the alpha-phosphorus atom. Km and Vmax for (Sp)-GTP alpha S (at low [Ca2+], 20 nM) are 3.7 mM and 1.1 nmol/min/mg of rhodopsin, respectively, compared with 1.1 mM and 23.1 nmol/min/mg of rhodopsin for GTP. Vmax for the cyclization of (Sp)-GTP alpha S, as for GTP, increases 10-20-fold when the calcium level is lowered. This activity change is centered at approximately 90 nM and has a Hill coefficient of 4.8. The configuration of the metal-substrate complex was determined by measuring the effectiveness of the Sp and Rp isomers of GTP alpha S and guanosine 5'-O-(2-thiotriphosphate) (GTP beta S) in the presence of Mg2+ or Mn2+. (Sp)-GTP alpha S is a substrate with either Mg2+ or Mn2+, whereas (Rp)-GTP beta S is a substrate with only Mn2+. These findings suggest that the substrate is a metal-beta, gamma-bidentate complex with delta screwsense. We also found that the cyclization reaction catalyzed by the membrane-bound guanylate cyclase from sea urchin sperm proceeds with inversion of configuration at the alpha-phosphorus atom. The stereochemical course of the reactions catalyzed by all prokaryotic and eukaryotic adenylate cyclases and guanylate cyclases studied thus far is the same.

Animals↗
PubMed1989

Unit activity in monkey parietal cortex related to haptic perception and temporary memory.

The neural responses of 456 single units were recorded in parietal cortex of behaving monkeys during a haptic delayed matching-to-sample task. (1) In areas 2 and 5 together, 22% of the neurons were activated by the auditory cue that signalled the beginning of a trial. Virtually all of these cells were also activated during the arm movements required by the task. These neurons, showing both auditory-related and movement-related responses, may function in sensorimotor integration. (2) Responses related to arm projection frequently began before movement onset, sometimes as much as 320 ms before. Such "premovement" responses were approximately equally common, and showed the same latency distribution, in areas 2, 5a, and 5b. (3) There was a topographic rostral-to-caudal gradient of decreasing neural responsiveness to the animal's manipulation of the cue (sample) objects. Eleven percent of manipulation-activated cells responded preferentially to one of the sample objects. (4) Many cells showed sustained (greater than 3 s) activation during the delay period (the time between handling of the sample object and palpation of the choice objects), even though at that time the monkey was sitting quietly and without stimulation. (5) Cells with sustained activation throughout most or all of the 18-s delay period were rare in all areas tested except area 5a. These cells, especially those that were preferentially activated depending on which sample object was palpated, may function in the temporary retention of haptic attributes. (6) The population of cells activated during sample manipulation was largely distinct from the population of cells showing sustained activation during the delay period. These two cell populations may represent different but complementary aspects of haptic perception. (7) The most common response during the delay period was sustained inhibition. This may be an expression of a nonspecific mechanism for decreasing background noise and enhancing neural responses to an anticipated perceptual event. (8) Relatively little evidence was found to support a functional distinction between the neural response properties of areas 2 and 5a. This suggests that area 2 may be at a higher level in the somatosensory hierarchy of the posterior parietal cortex than usually considered.

Acoustic Stimulation↗
PubMed1988

Highly cooperative feedback control of retinal rod guanylate cyclase by calcium ions.

Visual excitation in retinal rod cells is mediated by a cascade that leads to the amplified hydrolysis of cyclic GMP (cGMP) and the consequent closure of cGMP-activated cation-specific channels in the plasma membrane. Recovery of the dark state requires the resynthesis of cGMP, which is catalysed by guanylate cyclase, an axoneme-associated enzyme. The lowering of the cytosolic calcium concentration (Cai) following illumination is thought to be important in stimulating cyclase activity. This hypothesis is supported by the finding that the cGMP content of rod outer segments increases several-fold when Cai is lowered to less than 10 nM. It is evident that cGMP and Cai levels are reciprocally controlled by negative feedback. Guanylate cyclase from toad ROS is strongly stimulated when the calcium level is lowered from 10 microM to 10 nM, but only if they are excited by light. We show here that the guanylate cyclase activity of unilluminated bovine rod outer segments increases markedly (5 to 20-fold) when the calcium level is lowered from 200 nM to 50 nM. This steep dependence of guanylate cyclase activity on the calcium level in the physiological range has a Hill coefficient of 3.9. Stimulation at low calcium levels is mediated by a protein that can be released from the outer segment membranes by washing with a low salt buffer. Calcium sensitivity is partially restored by adding the soluble extract back to the washed membranes. The highly cooperative activation of guanylate cyclase by the light-induced lowering of Cai is likely to be a key event in restoring the dark current after excitation.

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↗
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↗
PubMed1985

Cyclic GMP directly regulates a cation conductance in membranes of bovine rods by a cooperative mechanism.

Cyclic GMP causes the release of endogenous Ca2+ from rod outer segments, whose plasma membrane has been made permeable, or from isolated discs. Approximately 11,000 Ca2+ ions are released per disc at saturating concentrations of cyclic GMP. The velocity and the amplitude of the release of Ca2+ are dependent on the concentration of cyclic GMP. The maximal rate of the Ca2+ efflux is approximately 7 X 10(4) Ca2+ ions s-1 rod-1. The Ca2+ release by cyclic GMP is independent of light. The activation of the efflux occurred within a narrow range of the cyclic GMP concentration (30-80 microM) and does not obey a simple Michaelis-Menten scheme. Instead, the kinetic analysis of the Ca2+ efflux suggests that a minimum number of 2 molecules of cyclic GMP activates the ion conductance in a cooperative fashion. The release of Ca2+ by cyclic GMP requires a gradient of Ca2+ ions across the disc membrane. If the endogenous Ca2+ gradient is dissipated by means of the ionophore A23187, the release of Ca2+ by cyclic GMP is abolished. Ca2+ is released by analogues of cyclic GMP which are either modified at the 8-carbon position of the imidazole ring or by the deaza-analogue of cyclic GMP. Congeners of cyclic GMP which are modified at the ribose, phosphodiester, or pyrimidine portion of the molecule are ineffective. The hydrolysis of cyclic GMP by the light-regulated phosphodiesterase of rod outer segments is not a necessary condition for the Ca2+ release because 8-bromo-cyclic GMP, a congener resistant to hydrolysis, is a more powerful activator of the release than cyclic GMP itself. Ca2+ release by cyclic GMP is inhibited by organic and inorganic blockers of Ca2+ channels. The l-stereoisomer of cis-diltiazem blocks the release of Ca2+ at micromolar concentrations, whereas the d-form is much less effective. These results suggest that disc membranes contain a cationic conductance which is permeable to Ca2+ ions and which is regulated through the cooperative binding of at least 2 molecules of cyclic GMP to regulatory sites of the transport protein. By this mechanism, subtle changes in the concentration of cyclic GMP could promote large changes in the flux of Ca2+ ions across the disc membrane.

Animals↗