Enzymes of the cyclic GMP metabolism in bovine retina (original) (raw)
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Localization of cGMP-dependent protein kinase isoforms in mouse eye
2000
PURPOSE To examine the expression of the major isoforms of cyclic guanosine monophosphate (cGMP)-dependent protein kinase (cGK) in mouse eye. METHODS Immunohistochemical localization of cGMP in mouse eye cryosections was performed using an anti-cGMP antibody, followed by visualization with indirect fluorescence microscopy. The presence of types Ialpha, Ibeta, and II cGK mRNAs in mouse eye extracts was determined initially by RNase protection analysis. Further localization of cGK I and II mRNAs on cryosections was accomplished by in situ hybridization using digoxigenin-labeled cRNA probes and an alkaline phosphatase-conjugated anti-digoxigenin antibody. Finally, cGK I protein was localized to subcellular areas within the retina using an anti-cGK I-specific primary antibody. RESULTS In initial immunohistochemical experiments cGMP was present in numerous regions and layers within the eye and retina. Subsequent RNase protection studies demonstrated that cGK Ialpha, Ibeta, and II mRNAs w...
Dynamics of Cyclic GMP Synthesis in Retinal Rods
Neuron, 2002
, 2 1989). As a result, the intracellular [Ca 2ϩ ] falls. This drop and Denis A. Baylor 1 in [Ca 2ϩ ] causes coordinated changes that antagonize 1 Department of Neurobiology the response to light. The Ca 2ϩ signal is thought to de-Stanford University Medical Center crease rhodopsin activity (Kawamura and Murakami, Stanford, California 94305 1991), increase the sensitivity of the channel for cGMP 2 The Mary D. Allen Laboratory for Vision Research (Hsu and Molday, 1993), and increase the activity of GC Doheny Eye Institute and (Lolley and Racz, 1982). To date, the specific quantita-Departments of Ophthalmology and Cell tive contribution of each Ca 2ϩ-dependent process to rod and Neurobiology function has only been inferred (see Koutalos et al., Keck School of Medicine of the 1995b; Nikonov et al., 2000). We therefore sought to University of Southern California directly determine how rapidly and powerfully Ca 2ϩ feed-Los Angeles, California 90033 back to GC acts in intact rods. 3 Center for Neuroscience and Figure 1 is a diagram of the negative feedback loop Department of Psychiatry that couples the rate of cGMP synthesis to the size of the University of California, Davis inward membrane current. A change in the intracellular Davis, California 95616 [cGMP] rapidly changes the inward current, and thus, the intracellular free [Ca 2ϩ ]. The change in [Ca 2ϩ ] is detected by GCAPs, which alter the rate of synthesis of Summary cGMP to oppose the initial change in [cGMP]. The steady-state gain of the feedback loop, g L , is not known. In retinal rods, Ca 2؉ exerts negative feedback control This parameter is defined as the ratio of the relative on cGMP synthesis by guanylate cyclase (GC). This change in the rate of synthesis of cGMP to the relative feedback loop was disrupted in mouse rods lacking change in the [cGMP], both changes being small. The guanylate cyclase activating proteins GCAP1 and loop gain is perhaps the single most important property GCAP2 (GCAPs Ϫ/Ϫ). Comparison of the behavior of of the loop, for it determines how powerfully small wild-type and GCAPs Ϫ/Ϫ rods allowed us to investigate changes in [cGMP] are nulled in the steady state. Since the role of the feedback loop in normal rod function. the cooperativity of channel activation (n ch) by cGMP is We have found that regulation of GC is apparently the 3 (Haynes et al., 1986; Zimmerman and Baylor, 1986; only Ca 2؉ feedback loop operating during the single Ruiz et al., 1999), a small relative change in [cGMP] will photon response. Analysis of the rods' light responses produce a 3-fold larger change in the relative Ca 2ϩ influx and cellular dark noise suggests that GC normally rein the steady state (see Experimental Procedures). Likesponds to light-driven changes in [Ca 2؉ ] rapidly and wise, if the activation of GCs by GCAPs is linear for highly cooperatively. Rapid feedback to GC speeds small changes in [Ca 2ϩ ], the relative change in the rate the rod's temporal responsiveness and improves its of synthesis will be n GC-fold greater than the relative signal-to-noise ratio by minimizing fluctuations in change in the [Ca 2ϩ ], where n GC is the cooperativity of cGMP. Ca 2ϩ regulation of GCs. Therefore, the ratio of the relative change in the rate of synthesis to the relative change
Cyclic GMP-phosphodiesterase of rd retina: Biosynthesis and content
Experimental Eye Research, 1988
Deficient cGMP-phosphodiesterase (cGMP-PDE) activity results in elevated levels of cGMP in the rd retina before any pathological signs are observed. Since the enzyme is present in rd retina, although it is barely activated by light, we determined whether its synthesis starts at the same time as that of cGMP-PDE in normal retina, or if either its synthesis is halted or degradation of the enzyme increases before the degeneration of the visual cells. We found that synthesis of cGMP-PDE in rd retina is comparable with that in normal retina while the photoreceptors are viable but that cGMP-PDE content is lower than that of normal retina before the visual cells begin to degenerate. Our results suggest that cGMP-PDE is more susceptible to degradation in rd than in normal photoreceptors or, alternatively, that proteolytic enzyme(s) involved in the degradation of cGMP-PDE are in higher concentration or more active in the defective than in the normal retina.
Biochemical Journal, 1985
Light-activated hydrolysis of cyclic GMP is achieved through the photoexcitation of rhodopsin, a process which then triggers the replacement of GDP for GTP by a retinal guanosine 5′-triphosphatase referred to as ‘transducin’. The transducin-GTP complex then switches on the phosphodiesterase [Fung, Hurley & Stryer (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 152-156]. The bovine transducin consists of an alpha-subunit (39000 Mr), which is a GTP-binding component, together with a beta-(37000 Mr) and a gamma-subunit (10000 Mr). We have purified retinal transducin from cow, pig, chick and frog. The enzyme specific activities and sodium dodecyl sulphate/polyacrylamide-gel-electrophoretic profiles indicate that this enzyme is similar in all species except the frog. Whereas the bovine, pig and chick transducins consist of major 37000- and 39000-Mr components, that of the frog consists of a single 75000-Mr component. Labelling of the GTP-binding components with the photoaffinity label 8-azidogu...
Guanylate cyclase in rod outer segments of the toad retina
FEBS Letters, 1986
Guanylate cyclase activity was measured in disrupted rod outer segments of the toad retina. The experiments showed that cGMP is synthesized from GTP at a rate of 3 f 1 nmol/min per mg protein. In darkness this value is largely independent of the CaZ+ concentration, while it is enhanced by flashes of light of increasing intensity upon lowering Ca from lo-' to 10e8 M. In view of recent observations that shortly after a flash of light calcium activity inside the photoreceptor cell decreases, it seems likely that calcium plays a regulatory role in cGMP metabolism in visual excitation.
Experimental Eye Research, 1982
Truncated rod photoreceptors containing outer segments with inner segment attachments u-err prepared from bovine retinas. In the absence of a high-energy donor, the preparation transformed cyclic GMP to 5'.GMP, guanosine and a small amount of cyclic XMP. In the presence of ATP. the preparation formed GDP and GTP f rom 5'.GMP. and GTP endogenously formed was converted to cyclir GMP. Conditions for optimal activity of the above reactions were defined and a rank ordering of the specific activities of the reactions established. The specific activity of each enzyme was greater in truncated photoreceptors than in homogenates of retina. The efficiency of the metabolic cycle to resynthesize cyclic GMP from 5'.GMP in vivo probably depends upon the energy state of the visual cell and upon the degree of compartmentation of substrates within the inner and outer segment of the photoreceptor.
Journal of Biological …, 2000
Retinal cGMP phosphodiesterase (PDE) is regulated by Pγ, the regulatory subunit of PDE, and GTP/Tα, the GTP-bound α subunit of transducin. In the accompanying paper (Matsuura, I., Bondarenko, VA, Maeda, T., Kachi, S., Yamazaki, M., Usukura, J., Hayashi, F., and ...
Guanylate cyclases and associated activator proteins in retinal disease
2010
Two isoforms of guanylate cyclase, GC1 and GC2 encoded by GUCY2D and GUCY2F, are responsible for the replenishment of cGMP in photoreceptors after exposure to light. Both are required for the normal kinetics of photoreceptor sensitivity and recovery, although disease mutations are restricted to GUCY2D. Recessive mutations in this gene cause the severe early-onset blinding disorder Leber congenital amaurosis whereas dominant mutations result in a later onset less severe cone-rod dystrophy. Cyclase activity is regulated by Ca 2? which binds to the GC-associated proteins, GCAP1 and GCAP2 encoded by GUCA1A and GUCA1B, respectively. No recessive mutations in either of these genes have been reported. Dominant missense mutations are largely confined to the Ca 2?binding EF hands of the proteins. In a similar fashion to the disease mechanism for the dominant GUCY2D mutations, these mutations generally alter the sensitivity of the cyclase to inhibition as Ca 2? levels rise following a light flash.