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B E Knox

Publications and source records attributed to B E Knox.

At least 37 records · Page 2Linked to original sources
PubMed1998

Enhancement of opsin activity by all-trans-retinal.

The rod cell photoreceptor apoprotein, opsin, activates the G-protein, transducin, although at a much reduced level than light-activated rhodopsin. The ability of all-trans-retinal to enhance opsin apoprotein activity was investigated using a guanyl nucleotide exchange assay on transducin. All-trans-retinal enhanced opsin activity in a concentration-dependent manner. At high concentrations of all-trans-retinal, the activity of the all-trans-retinal-opsin complex was comparable to that from an equimolar amount of metarhodopsin(II). However, in contrast to metarhodopsin(II), the active all-trans-retinalopsin complex did not require a stable Schiff base linkage between opsin and all-trans-retinal. The lack of a stable Schiff base and differences in activity at high pH imply that opsin and all-trans-retinal form a complex that is distinct from metarhodopsin(II). The ability of all-trans-retinal to stimulate the transduction cascade may be a source of post-bleach noise in photoreceptors.

Animals↗
PubMed1998

Cloning and expression of a Xenopus short wavelength cone pigment.

The short wavelength visual pigment from Xenopus responsible for vision in the blue/violet portion of the spectrum was characterized by sequence spectroscopic analysis. The amino acid sequence was deduced by sequencing clones isolated by reverse transcription PCR, from retinal cDNA and genomic libraries. The gene contains 5 exons spanning 8.4 kb of genomic DNA and produces an mRNA of 2.4 kb in length. The deduced amino acid sequence predicts a protein of 347 amino acids with 76-78% identity to other short wavelength opsins. The mRNA encoding the Xenopus violet pigment was detected using in situ hybridization in cones, comprising a few percent of the total photoreceptors in the adult retina. The Xenopus violet opsin cDNA, modified to contain an epitope from the carboxyl terminus of bovine rhodopsin, was expressed in COS1 cells by transient transfection and analysed by UV-visible absorption spectroscopy. The protein expressed in COS1 cells migrated at 34 kD and was glycosylated at a single site in the amino terminus, exhibiting a diffuse pattern on SDS PAGE similar to bovine rhodopsin expressed in COS1 cells. Following incubation with 11-cis retinal, a light-sensitive pigment was formed with the lambdamax=425+/-2 nm. A Schiff base linkage between retinal and the violet opsin was demonstrated by acid denaturation. Xenopus violet opsin was sensitive to hydroxylamine in the dark, reacting with a half-time of 5 min at room temperature. This is the first group S pigment for amphibians. The pigment was expressed and purified from COS1 cells in a form that has permitted for the first time determination of the extinction coefficient, reactivity to hydroxylamine and presence of a Schiff base.

Amino Acid Sequence↗
PubMed1997

Modulation of opsin apoprotein activity by retinal. Dark activity of rhodopsin formed at low temperature.

The bovine opsin apoprotein activates transducin, although at a much reduced level than light-activated rhodopsin (Surya, A., Foster, K., and Knox, B. (1995) J. Biol. Chem. 270, 5024-5031). The ability of retinal to modulate opsin apoprotein activity was investigated using a guanyl nucleotide exchange assay on transducin. 11-cis-Retinal reacted with opsin at 22 degrees C to (a) reform pigment having maximal absorbance at 500 nm and (b) reduce opsin activity by >80%. Pigment formation also occurred at 0 degrees C with a t1/2 of 260 min. However, unlike rhodopsin formed at 22 degrees C (R22), the rhodopsin formed at 0 degrees C (R0) activated transducin with the same half-saturating concentration as opsin in an exhaustive binding assay. Thus, the formation of a protonated Schiff base associated with 500 nm absorbance does not by itself lead to the inactivation of opsin. The R0 conformation was partially inactivated by incubation at 22 degrees C (t1/2 = 61 +/- 9 min), suggesting that it may be an intermediate conformation in the regeneration of rhodopsin.

Animals↗
PubMed1997

Activation of transducin by a Xenopus short wavelength visual pigment.

Phototransduction in cones differs significantly from that in rods in sensitivity, kinetics, and recovery following exposure to light. The contribution that the visual pigment makes in determining the cone response was investigated biochemically by expressing a Xenopus violet cone opsin (VCOP) cDNA in COS1 cells and assaying the light-dependent activation of transducin. Light-exposed VCOP stimulated [35S]guanosine 5'-(gamma-thio)triphosphate nucleotide exchange on bovine rod transducin in a time-dependent manner with a half-time for activation of 0.75 min, similar to that of bovine rhodopsin. In exhaustive binding assays, VCOP and rhodopsin activity showed similar concentration dependence with half-maximal activation occurring at 0.02 mol of pigment/mol of transducin. Although VCOP was able to activate as many as 12 transducins per photoisomerization, rhodopsin catalyzed significantly more. When assays were performed with lambda > 420 nm illumination, VCOP exhibited rapid regeneration and high affinity for the photoregenerated 11-cis-retinal. Recycling of the chromophore and reactivation of the pigment resulted in multiple activations of transducin, whereas a maximum of 1 transducin per VCOP was activated under brief illumination. The decay of the active species formed following photobleaching was complete in <5 min, approximately 10-fold faster than that of rhodopsin. In vitro, VCOP activated rod transducin with kinetics and affinity similar to those of rhodopsin, but the active conformation decayed more rapidly and the apoprotein regenerated more efficiently with VCOP than with rhodopsin. These properties of the violet pigment may account for much of the difference in response kinetics between rods and cones.

Alcohol Dehydrogenase↗
PubMed1996

Characterization of the Xenopus rhodopsin gene.

The abundant Xenopus rhodopsin gene and cDNA have been cloned and characterized. The gene is composed of five exons spanning 3.5 kilobase pairs of genomic DNA and codes for a protein 82% identical to the bovine rhodopsin. The cDNA was expressed in COS1 cells and regenerated with 11-cis-retinal, forming a light-sensitive pigment with maximal absorbance at 500 nm. Both Southern blots and polymerase chain reaction amplification of intron 1 revealed multiple products, indicating more than one allele for the rhodopsin gene. Comparisons with other vertebrate rhodopsin 5 upstream sequences showed significant nucleotide homologies in the 200 nucleotides proximal to the transcription initiation site. This homology included the TATA box region, Ret 1/PCE1 core sequence (CCAATTA), and surrounding nucleotides. To functionally characterize the rhodopsin promoter, transient embryo transfections were used to assay transcriptional control elements in the 5 upstream region using a luciferase reporter. DNA sequences encompassing -5500 to +41 were able to direct luciferase expression in embryo heads. Reporter gene expression was also observed in embryos microinjected with reporter plasmids during early blastomere stages. These results locate transcriptional control elements upstream of the Xenopus rhodopsin gene and show the feasibility of embryo transfections for promoter analysis of rod-specific genes.

Amino Acid Sequence↗
PubMed1995

Transducin activation by the bovine opsin apoprotein.

The interaction of the bovine opsin apoprotein with transducin in rod outer segment membranes was investigated using a guanyl nucleotide exchange assay. In exhaustive binding experiments, opsin activates transducin, with half-maximal exchange activity occurring at 0.8 mol of opsin/mol of transducin. The opsin activity was light-insensitive, hydroxylamine-resistant, unaffected by stoichiometric concentrations of retinaloxime, and more heat-labile than rhodopsin. The t1/2 of transducin activation in the presence of excess opsin was 8.5 min, compared with 0.7 min for metarhodopsin (II). The second-order rate constants were determined to be 0.012 pmol of guanosine 5'-(gamma-thio)triphosphate (GTP gamma S) bound per min/nM opsin and 0.35 pmol of GTP gamma S bound per min/nM metarhodopsin (II). Opsin was able to activate more than one transducin, although there appeared to be a turnover-dependent inactivation of the apoprotein. Opsin showed a broad pH range (5.8-7.4) for optimal activity, with no activity in buffers of pH > 9, whereas metarhodopsin (II) exhibited activity at pH > 9. Regulation of opsin activity by stoichiometric amounts of retinal was observed, with inhibition by 11-cis-retinal and stimulation by all-trans-retinal. A model for opsin activity is proposed.

Animals↗
PubMed1995

Rhodopsin from the fish, Astyanax: role of tyrosine 261 in the red shift.

PURPOSE: To isolate and characterize the rhodopsin cDNA from the fish, Astyanax fasciatus, and to determine the effect of tyrosine 261 on its spectral tuning. METHODS: The rhodopsin cDNA was cloned using reverse transcription-polymerase chain reaction amplification and then sequenced. A mutant, Y261F, was generated by site-directed mutagenesis. Both wild type and mutant were transiently expressed in COS-1 cells, regenerated with 11-cis retinal, and purified by immunoaffinity chromatography. Ultraviolet-visible spectrophotometry was used to determine wavelength of maximum absorption. RESULTS: A fasciatus rhodopsin cDNA exhibits 80% amino acid identity with bovine rhodopsin. In contrast to all known rhodopsins, this rhodopsin contains a tyrosine instead of a phenylalanine at amino acid position 261. Indeed, this particular amino acid replacement has been implicated in the long wavelength absorption of the red cone pigment. Site-directed mutagenesis was used to change the Astyanax amino acid 261 to phenylalanine (Y261F). Expression of the Y261F mutant in COS-1 cells showed an absorbance maximum of 496 nm, compared to 504 nm for the wild type pigment. CONCLUSIONS: A naturally occurring fish rhodopsin is red shifted about 8 nm due to one critical amino acid substitution.

Amino Acid Sequence↗
PubMed1993

Light-induced currents in Xenopus oocytes expressing bovine rhodopsin.

1. We have investigated the functioning of bovine rod opsin, which is efficiently synthesized from RNA made by in vitro transcription, following injection into Xenopus oocytes. We found that oocytes expressing the gene for opsin exhibit light-dependent ionic currents only after pigment generation by incubation with 11-cis-retinal. These currents are similar to the endogenous muscarinic acetylcholine (ACh) response of oocytes, but their amplitude is substantially smaller. 2. In order to optimize the conditions for obtaining light-induced currents in RNA-injected oocytes, the native ACh response was examined under several conditions. It was found that elevated external calcium markedly enhances the muscarinic response and that these currents have a non-linear dependence on membrane voltage, increasing substantially with depolarization. 3. Using the optimal conditions for evoking the largest ACh responses, (28 mM [Ca2+]o, 0 mV, omission of serum and Hepes from the media), the light-evoked currents obtained in RNA-injected oocytes were remarkably enhanced, and responses to multiple light stimuli could be obtained. 4. The light response appeared to desensitize, even after long periods of recovery and pigment regeneration. By contrast, the ACh responses continued to appear normal. These results suggest that desensitization of photoresponses expressed in Xenopus oocytes involve changes at early stages of the pathway, resulting in a reduced ability of rhodopsin to couple to the endogenous signalling system.

Acetylcholine↗
PubMed1992

Light-stable rhodopsin. I. A rhodopsin analog reconstituted with a nonisomerizable 11-cis retinal derivative.

With the aim of preparing a light-stable rhodopsin-like pigment, an analog, II, of 11-cis retinal was synthesized in which isomerization of the C11-C12 cis-double bond is blocked by a cyclohexene ring built around the C10 to C13-methyl. The analog II formed a rhodopsin-like pigment (rhodopsin-II) with opsin expressed in COS-1 cells and with opsin from rod outer segments. The rate of rhodopsin-II formation from II and opsin was approximately 10 times slower than that of rhodopsin from 11-cis retinal and opsin. After solubilization in dodecyl maltoside and immunoaffinity purification, rhodopsin-II displayed an absorbance ratio (A280nm/A512nm) of 1.6, virtually identical with that of rhodopsin. Acid denaturation of rhodopsin-II formed a chromophore with lambda max, 452 nm, characteristic of protonated retinyl Schiff base. The ground state properties of rhodopsin-II were similar to those of rhodopsin in extinction coefficient (41,200 M-1 cm-1) and opsin-shift (2600 cm-1). Rhodopsin-II was stable to hydroxylamine in the dark, while light-dependent bleaching by hydroxylamine was slowed by approximately 2 orders of magnitude relative to rhodopsin. Illumination of rhodopsin-II for 10 s caused approximately 3 nm blue-shift and 3% loss of visible absorbance. Prolonged illumination caused a maximal blue-shift up to approximately 20 nm and approximately 40% loss of visible absorbance. An apparent photochemical steady state was reached after 12 min of illumination. Subsequent acid denaturation indicated that the retinyl Schiff base linkage was intact. A red-shift (approximately 12 nm) in lambda max and a 45% recovery of visible absorbance was observed after returning the 12-min illuminated pigment to darkness. Rhodopsin-II showed marginal light-dependent transducin activation and phosphorylation by rhodopsin kinase.

Electrophoresis, Polyacrylamide Gel↗
PubMed1990

Optic nerve decompression via the lateral facial approach.

Two cases of visual loss after lateral orbital wall fracture are presented: one with retrobulbar hematoma and evidence of optic nerve compression who failed to respond to lateral canthotomy and high-dose corticosteroid administration, and the second with immediate, total blindness associated with fracture of the bony optic canal. In both, extradural decompression of the orbit and optic nerve was achieved through the lateral facial approach with partial return of visual acuity and without surgical complications. The role of orbital and optic nerve decompression in the management of patients with blindness following orbital trauma is controversial. Orbital decompression may be of value for cases of post-traumatic visual loss unresponsive to medical management. If optic nerve injury is suspected as the cause, the additional step of decompression of the optic nerve is a logical but unproven procedure. The indications for optic nerve decompression are not established and should be considered only within the context of the specific needs of the individual patient.

Adult↗
PubMed1988

Expression of a bovine rhodopsin gene in Xenopus oocytes: demonstration of light-dependent ionic currents.

Xenopus oocytes express a gene encoding bovine rhodopsin as well as its SP6 RNA polymerase-derived transcripts and total retinal mRNA. The opsin produced is in unglycosylated (30 kDa) and two glycosylated (35 kDa and 41 kDa) forms. Incubation of the cells expressing the above proteins with 11-cis-retinal generates rhodopsin, which was purified by immunoaffinity chromatography. The purified protein shows the expected UV/visible absorption spectrum and characteristic light-dependent activation of the rod outer segment GTP binding protein. Oocytes expressing rhodopsin exhibit light-dependent ionic currents that are detected by voltage-clamp techniques.

Animals↗
PubMed1987

Thermal inactivation of electron-transport functions and F0F1-ATPase activities.

Bovine heart submitochondrial particles in suspension were heated at a designated temperature for 3 min, then cooled for biochemical assays at 30 degrees C. By enzyme activity measurements and polarographic assay of oxygen consumption, it is shown that the thermal denaturation of the respiratory chain takes place in at least four stages and each stage is irreversible. The first stage occurs at 51.0 +/- 1.0 degrees C, with the inactivation of NADH-linked respiration, ATP-driven reverse electron transport, F0F1 catalyzed ATP/Pi exchange, NADH and succinate-driven ATP synthesis. The second stage occurs at 56.0 +/- 1.0 degrees C, with the inactivation of succinate-linked proton pumping and respiration. The third stage occurs at 59.0 +/- 1.0 degrees C, with the inactivation of electron transfer from cytochrome c to cytochrome oxidase and ATP-dependent proton pumping. The ATP hydrolysis activity of F0F1 persists to 61.0 +/- 1.0 degrees C. An additional transition, detectable by differential scanning calorimetry, occurring around 70.0 +/- 2.0 degrees C, is probably associated with thermal denaturation of cytochrome c and other stable membrane proteins. In the presence of either mitochondrial matrix fluid or 2 mM mercaptoethanol, all five stages give rise to endothermic effects, with the absorption of approx. 25 J/g protein. Under aerobic conditions, however, the first four transitions become strongly exothermic, and release a total of approx. 105 J/g protein. Solubilized and reconstituted F0F1 vesicles also exhibit different inactivation temperatures for the ATP/Pi exchange, proton pumping and ATP hydrolysis activities. The first two activities are abolished at 49.0 +/- 1.0 degrees C, but the latter at 58.0 +/- 2.0 degrees C. Differential scanning calorimetry also detects biphasic transitions of F0F1, with similar temperatures of denaturation (49.0 and 54.0 degrees C). From these and other results presented in this communication, the following is concluded. (1) A selective inactivation, by the temperature treatment, of various functions of the electron-transport chain and of the F0F1 complex can be done. (2) The ATP synthesis activity of the F0F1 complex involves either a catalytic or a regulation subunit(s) which is not essential for ATP hydrolysis and the proton translocation. This subunit is 10 degrees C less stable than the hydrolytic site. Micromolar ADP stabilizes it from thermal denaturation by 4-5 degrees C, although ADP up to millimolar concentration does not protect the hydrolytic site and the proton-translocation site.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine Diphosphate↗
PubMed1986

A mechanism for exact sensory adaptation based on receptor modification.

We provide a theoretical explanation for the observation that in many sensory systems a step increase in stimulus triggers a response that goes through a maximum and then returns to the basal level. Considered here is a receptor molecule that in the absence of ligand can be found in either of two states R and D. Two more states, RL and DL, are formed upon the addition of ligand L. It is assumed that the receptor triggers activity in a sensory system, and that the activity is proportional to a weighted combination of the fractions of molecules that are in each of the four states. It is shown that judicious choice of the weights can provide both an adequate response and exact adaptation to step increases in stimuli. The interconversion between states may operate without energy expenditure or through covalent modification. In both cases, adaptation is associated with receptor modification that acts as a counterweight to changed external conditions. Application to cAMP secretion in Dictyostelium discoideum and to chemotaxis in bacteria is discussed.

Adaptation, Physiological↗
PubMed1986

A molecular mechanism for sensory adaptation based on ligand-induced receptor modification.

Physiological responses mediated by cell-surface receptors frequently adapt or "desensitize" (i.e., terminate despite persistent occupancy of receptors by ligand). Binding of ligands to the external domains of a wide variety of surface receptors induces covalent modification of their cytoplasmic domains. A mechanism is presented in which the variety of receptor states generated by ligand binding and covalent modification act together to regulate physiological responsiveness. The development of the model is guided by observations of adaptation for chemotaxis in Escherichia coli and adenylate cyclase activation in Dictyostelium. The general features of the marked response and eventual exact adaptation predicted by the model match those observed in the experimental systems.

Adaptation, Physiological↗
PubMed1984

Voltage-driven ATP synthesis by beef heart mitochondrial F0F1-ATPase.

The F0F1-ATPase of the inner mitochondrial membrane catalyzes the conversion of a proton electrochemical energy into the chemical bond energy of ATP (Boyer, P.D., Chance, B., Ernster, L., Mitchell, P., Racker, E., and Slater, E.C. (1977) Annu. Rev. Biochem. 46, 955-1026). To assess the role of the membrane potential (delta psi) in this process and to study the effect of very short pulses on ATP synthesis, we employed a high voltage pulsation method (Kinosita, K., and Tsong, T.Y. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 1923-1927) to induce a delta psi of controlled magnitude and duration in a suspension of submitochondrial particles and F0F1-ATPase vesicles. Cyanide-treated submitochondrial particles were exposed to electric pulses of 10-30 kV/cm of magnitude (generating a peak delta psi of 150-450 mV) and 1-100 microseconds duration. Net [32P]ATP synthesis from [32P]Pi and ADP was observed with maximal values of 410 pmol/mg X pulse for a 30 kV/cm-100-microseconds pulse. This corresponds to a yield of 10-12 mol of ATP per mol of F0F1 complex per pulse. As many as 4 nmol/mg were produced after pulsing the same sample 8 times. By varying the ionic strength of the suspending medium, and consequently the pulse width, it is clearly shown that the synthesis was electrically driven and did not correlate with Joule heating of the sample. Titrations using specific inhibitors and ionophores were performed. The voltage-induced ATP synthesis was 50% inhibited by 0.11 microgram/mg of oligomycin and 2.4 nmol/mg of N,N'-dicyclohexylcarbodiimide. Ionophores and uncouplers had varying degrees of inhibition. The dependence of ATP synthesis on pulse width was nonlinear, exhibiting a threshold at 10 microseconds and a biphasic behavior above this value. Isolated F0F1-ATPase reconstituted into asolectin vesicles also synthesized ATP when pulsed with electric fields. A 35 kV/cm pulse induced the synthesis of 115 pmol of ATP per mg of protein, which corresponds to approximately 0.34 mol of ATP per mol of F0F1-ATPase. This synthesis was also sensitive to oligomycin and dicyclohexylcarbodiimide. The possibility of turnover of the ATPase in microseconds is considered.

Adenosine Triphosphate↗
PubMed1983

The structure of the Ca2+ ATPase as revealed by electron microscopy and image processing of ordered arrays.

Two-dimensional ordered arrays of the membrane-bound Ca2+ ATPase, were formed over a wide range of conditions (i.e., pH, ionic strength, temperature) in the presence of vanadate, and studied by electron microscopy and image processing. These ordered tubular and spherical membrane vesicles of Ca2+ ATPase could also be formed with approximately one bound ATP and between one and two nonchelatable Ca2+ bound. The tubular arrays ranged between 1 and 10 microns in length and had an average flattened diameter of 90 nm, as observed in negatively stained preparations. The basic building blocks of these ordered arrays appear to be linear ribbons of Ca2+ ATPase dimers. Fourier analysis of electron micrographs of these flattened tubes revealed a near-rectangular lattice (lattice angle 73.3 +/- 4.6 degrees with average lattice constants of a = 6.2 +/- 0.25 nm, and b = 11.5 +/- 0.30 nm). The double-stranded ribbons (i.e., parallel to a) are inclined by 56 +/- 3.7 degrees relative to the tube axis in a right-handed sense, as determined from freeze-dried metal-shadowed specimens. Computer averaging of negatively stained arrays reveals a crystallographic dimer of stain-excluding matter. The dimensions of each monomer within this dimer are consistent with established structural parameters, leading us to believe a form of the Ca2+ ATPase, capable of binding at least one ATP and of binding Ca2+ ions, may exist as a dimer in the sarcoplasmic reticulum.

Animals↗
PubMed1981

Synthesis of adenosine triphosphate in respiration-inhibited submitochondrial particles induced by microsecond electric pulses.

Phosphorylation of ADP to ATP was induced in nonrespiring submitochondrial particles (SMP) from rat liver by the application of electric pulses with field strengths of 10-35 kV/cm and a decay time of 60 mus. In all cases respiration was inhibited completely by using cyanide or rotenone. Newly formed ATP was measured by two independent methods, (i) the luciferase/luciferin bioluminescence assay and (ii) synthesis of [(32)P]ATP from ADP and (32)P(i). Both methods gave consistent and essentially identical results. Above 10 kV/cm the amount of ATP synthesized increased with increasing field strength, and at 30 kV/cm, approximately 40 pmol of ATP was synthesized per mg of SMP protein per pulse. ATP synthesis was shown to be related to the field-induced transmembrane potential, not to Joule heating of the suspension. Synthesis was abolished by the uncouplers carbonyl cyanide p-trifluoromethoxyphenylhydrazone, carbonyl cyanide m-chlorophenylhydrazone, and 2,4-dinitrophenol. The ionophores valinomycin and A23187 reduced the level of synthesis by 75% and 50%, respectively. ATP synthesis was also blocked by inhibitors of the F(0)F(1) ATPase complex, oligomycin, N,N'-dicyclohexyl carbodiimide, venturicidin, and aurovertin. The activities of the adenine nucleotide translocator and adenylate kinase, as well as release of bound nucleotides, could be excluded as sources of the new ATP. The data indicate that the minimal applied field at which ATP synthesis could be detected is approximately 8 kV/cm, corresponding to a maximal induced membrane potential of 60 mV in SMP. The maximal synthesis occurred at around 30 kV/cm, or an induced transmembrane potential of 200 mV. The duration of the applied pulse was also found to be critical, with 8 mus being the minimal triggering time for the synthesis. The induction of ATP synthesis in nonrespiring SMP by an externally applied electrical field is a direct demonstration of the transformation, by the mitochondrial inner membrane, of electrical energy into the chemical bond energy of ATP.

Adenosine Triphosphate↗