Calmodulin in bovine rod outer segments.

Kohnken, Russell E.; Chafouleas, James G.; Eadie, D M; Means, Anthony R.; McConnell, David G.

doi:10.1016/s0021-9258(18)43305-4

Cited by 70 publications

(6 citation statements)

References 40 publications

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“…4, the Kd for the binding of the modulator to the target protein would have to be greater than ~5 #M. Such a binding interaction between the hypothetical modulator and its target protein is atypically weak. The Kd'S between calcium binding proteins and their target proteins typically range from 10-9-10 -8 M (Picton et al, 1980;Adelstein and Klee, 1981;Kohnken et al, 1981;Vallet et al, 1981), that is, 2-3 orders of magnitude lower than that which would be consistent with these data. Although the model is cast in terms of a target protein with a concentration equal to that of Rh kinase, we point out that the conclusion applies equally well to all the cGMP cascade components that compositely determine the dim-flash or linear PDE kinetics of Fig.…”

Section: Discussionsupporting

confidence: 54%

Calcium dependence of the activation and inactivation kinetics of the light-activated phosphodiesterase of retinal rods.

Barkdoll

Pugh

Sitaramayya

1989

The Journal of General Physiology

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The Ca ~+ dependence of the kinetics and light sensitivity of light-activated phosphodiesterase was studied with a pH assay in toad and bovine rod disk membranes (RDM), and in a reconstituted system containing GTP-binding protein, phosphodiesterase and rhodopsin kinase. Three statistics, peak hydrolytic velocity, turnoff time, and time to peak velocity, were measured. ATP decreased phosphodiesterase light sensitivity nearly 10-fold and accelerated the dim-flash kinetics of cGMP hydrolysis when compared to those with GTP alone. Ca ~+ reversed all of the effects of ATP, Ca ~+ increased peak velocity, turnoff time, and time to peak velocity, to the values obtained with GTP alone. The Ca ~+ dependence of peak velocity and turnoff time can be characterized as hyperbolic saturation functions with a K0.5 for Ca 2+ of 1.0-1.5 mM in toad RDM. In bovine RDM the Ca 2+ dependence of peak velocity and turnoff time has a K0. 5 of 0.1 mM Ca ~+. The Ca ~+ dependence in the reconstituted system is similar to that in bovine RDM for peak velocity (K0.s = 0.1 mM Ca 2+) but differs for turnoff time (K0. s = 2.5 mM Ca2+). We tested the hypothesis that a soluble modulator, normally required to confer submicromolar Ca 2+ sensitivity, was too dilute in our assay by comparing data obtained at one RDM concentration with those obtained at 10-fold higher RDM, and therefore a constituent protein, concentration. We observe no difference and present a formal analysis of these data that excludes the hypothesis that the soluble modulator binds its target protein with Kd < 5/~M. The lack of submicromolar Ca ~+ dependence of any of the steps in the cGMP cascade that underlie cGMP phosphodiesterase activation and inactivation in vitro argues against Ca 2+ regulation of these steps having a significant role in the light adaptation of the intact rod.

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Section: Discussionsupporting

confidence: 54%

Calcium dependence of the activation and inactivation kinetics of the light-activated phosphodiesterase of retinal rods.

Barkdoll

Pugh

Sitaramayya

1989

The Journal of General Physiology

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“…The data presented here demonstrate the presence of CaMand Ca2+-dependent CaM binding proteins in highly purified, intact amphibian ROS. In bovine ROS, CaM is present at the level of about 450 ng of CaM/mg of protein, and the molar ratio of rhodopsin to CaM is 700:1 (Kohnken et al, 1981). In our study which utilized intact frog ROS, CaM was present at a level of about 420 ng/mg of ROS protein, and the molar ratio of rhodopsin to CaM was about 800:1.…”

Section: Discussionmentioning

confidence: 48%

“…A series of recent studies show that the regulation of biological processes by Ca2+ and CaM often depends upon the CaM binding proteins (Kakiuchi & Sobue, 1982; Manalen & Klee, 1984). In bovine ROS, the presence of CaM has also been described (Kohnken et al, 1981). The importance of CaM and its binding proteins for Ca2+-dependent cellular, regulation, and the accumulated evidence suggesting that photoreceptor Ca2+ levels are dynamically regulated (Korenbrot, 1985;Fain & Schroder, 1985), prompted our interest in CaM and its binding proteins in frog ROS. In this study, we demonstrate the presence and 1 Abbreviations: ROS, rod outer segment(s); CaM, calmodulin; DTSSP, S.S'-dithiobisCsulfosuccinimidyl propionate); RIA, radioimmunoassay; EGTA, ethylene glycol bis(0-aminoethyl ether)-7V,./V,./V',./V'tetraacetic acid; SDS, sodium dodecyl sulfate; Tris-HCl, trisfhydroxymethyl)aminomethane hydrochloride.…”

mentioning

confidence: 99%

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Calmodulin and calmodulin binding proteins in amphibian rod outer segments

Nagao¹,

Yamazaki²,

Bitensky³

1987

Biochemistry

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The calmodulin (CaM) content of fully intact frog rod outer segments (ROS) has been measured. The molar ratio between rhodopsin and total CaM in ROS is 800:1. This is in good agreement with the data reported for bovine ROS CaM [Kohnken, R. E., Chafouleas, J. G., Eadie, D. M., Means, A. R., & McConnell, D.G. (1981) J. Biol. Chem. 256, 12517-12522]. In the absence of Ca2+, the ROS membrane fraction contains only 4% of total ROS CaM. In contrast, in the presence of Ca2+, 15% of total ROS CaM is found in the membrane fraction. For half-maximal binding of CaM to CaM-depleted ROS membranes, 3 X 10(-7) M Ca2+ is required. This CaM binding is inhibited by trifluoperazine. CaM binding proteins in the ROS membrane fraction are identified by using two different methods: the overlay method and the use of 3,3'-dithiobis(sulfosuccinimidyl propionate) (DTSSP), a bifunctional cross-linking reagent. Ca2+-dependent CaM binding proteins with apparent molecular weights of 240,000, 140,000, 53,000, and 47,000 are detected in the ROS membrane fraction by the overlay method. Anomalous, Ca2+-independent CaM binding to rhodopsin is also detected with this method, and this CaM binding is inhibited by the presence of Ca2+. With the bifunctional cross-linking reagent, DTSSP, three discrete proteins with molecular weights of 240,000, 53,000, and 47,000 are detected in the native ROS membrane fraction. CaM binding to rhodopsin is not detected with this method. Moreover, while the Mr 140,000 band is not detected with DTSSP, a smeared band with a molecular weight between 78,000 and 93,000 is identified (with DTSSP) in the ROS membrane fraction.(ABSTRACT TRUNCATED AT 250 WORDS)

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“…The molecular identity of the endogenous modulator is under investigation. Calmodulin exists at high concentration in the rod outer segment (Kohnken et al, 1981;Bauer, 1996). Hsu and Molday (1993) first reported that calmodulin causes a Ca ϩϩ -dependent shift in K 1/2 in rod channels of thoroughly washed bovine outer segment membrane vesicles (see also Hsu and Molday, 1994;Bauer, 1996).…”

Section: Discussionmentioning

confidence: 99%

Calcium Modulation of Ligand Affinity in the Cyclic GMP–gated Ion Channels of Cone Photoreceptors

Hackos

Korenbrot

1997

The Journal of General Physiology

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To investigate modulation of the activation of cGMP-gated ion channels in cone photoreceptors, we measured currents in membrane patches detached from the outer segments of single cones isolated from striped bass retina. The sensitivity of these channels to activation by cGMP depends on the history of exposure to divalent cations of the membrane's cytoplasmic surface. In patches maintained in 20 μM Ca++ and 100 μM Mg++ after excision, the current amplitude dependence on cGMP is well described by a Hill equation with average values of K 1/2, the concentration necessary to activate half the maximal current, of 86 μM and a cooperativity index, n, of 2.57. Exposing the patch to a solution free of divalent cations irreversibly increases the cGMP sensitivity; the average value of K 1/2 shifts to 58.8 μM and n shifts to 1.8. Changes in cGMP sensitivity do not affect other functional parameters of the ion channels, such as the interaction and permeation of mono- and divalent cations. Modulation of cGMP activation depends on the action of an endogenous factor that progressively dissociates from the channel as Ca++ concentration is lowered below 1 μM. The activity of the endogenous modulator is not well mimicked by exogenously added calmodulin, although this protein competes with the endogenous modulator for a common binding site. Thus, the modulation of cGMP affinity in cones depends on the activity of an unidentified molecule that may not be calmodulin.

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Calmodulin in bovine rod outer segments.

Cited by 70 publications

References 40 publications

Calcium dependence of the activation and inactivation kinetics of the light-activated phosphodiesterase of retinal rods.

Calcium dependence of the activation and inactivation kinetics of the light-activated phosphodiesterase of retinal rods.

Calmodulin and calmodulin binding proteins in amphibian rod outer segments

Calcium Modulation of Ligand Affinity in the Cyclic GMP–gated Ion Channels of Cone Photoreceptors

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