Biomedical subjects

H Ohguro

Publications and source records attributed to H Ohguro.

51 records · Page 3Linked to original sources

PubMed1993

Identification of a novel gamma-subunit from bovine brain GTP binding regulatory proteins (Gi/o).

Heterogeneity of the gamma-subunit of G proteins has been demonstrated by cDNA cloning and by partial sequence analyses. We have isolated two intact beta gamma-subunit isoforms from bovine brain Gi/o mixture, in which only gamma subunits are distinct (Sohma, H., et al. (1992) Biochem. Biophys. Res. Commun. 184, 175-182). In this study, we isolated the gamma-subunit isoforms, gamma-I and gamma-II, and examined their amino acid sequences. Both gamma-I and gamma-II had blocked N-terminal amino acid residues, and the terminal amino acids of both were able to be truncated by an acylamino-acid-releasing enzyme. Gamma-I seemed to be identical with the gamma-subunit reported elsewhere, while the gamma-II appeared to be a novel protein. Antibodies to synthetic peptides based on the part of the amino acid sequences of gamma-I and gamma-II reacted specifically to gamma-I and gamma-II, respectively.

Amino Acid Sequence↗

PubMed1993

[Light-induced dephosphorylation of phosphoproteins in rod outer segments in bovine and frog retina].

Several lines of evidence have suggested that protein phosphorylation and dephosphorylation may play an important role in the regulation of metabolism and signal transduction processes. In our present study, bovine and frog retinas were incubated in Krebs' solution containing [32P] H3PO4 for the labelling of all phosphoproteins. Then, photoreceptor outer segments were isolated from each retina, and further incubated under dark or light conditions. In such conditions, several phosphoproteins were dark- or light-dependently dephosphorylated. Interestingly, the light-dependent dephosphorylated 39 kDa protein as well as the 35-36 kDa protein was commonly observed in both bovine and frog retinas. The 35-36 kDa protein is considered to be the same as the 33 kDa protein that has previously been shown to be phosphorylated light-dependently, whereas 39 kDa protein is thought to be a novel protein that undergoes light-dependent dephosphorylation in retinal photoreceptor outer segments. Thus, these proteins were thought to have significant roles in the visual transduction processes.

Animals↗

PubMed1992

Two gamma-subunits, gamma-I and gamma-II, complex with the same beta-subunits in bovine brain G-proteins (Gi/o).

When a mixture of bovine brain G-proteins (Gi/o) was loaded onto an octyl sepharose column in the presence of AlF4-, alpha-subunits of molecular weights 39 kDa and the 41 kDa were eluted separately, followed by the appearance of two distinct peaks containing beta gamma-subunits (beta gamma-I, beta gamma-II). Both beta gamma-I and beta gamma-II possessed identical beta-subunits but different gamma-subunits. The molecular weights of the two gamma-subunits determined by SDS-polyacrylamide gel electrophoresis both in the presence and absence of urea were 4.5 kDa (gamma-I) and 5.0 kDa (gamma-II). Tests indicated that the two isolated gamma-subunits are intact and have not undergone proteolysis. The amino acid composition of gamma-I appeared to be distinct from that of gamma-II. Therefore, this method is a simple procedure for isolating beta gamma-I and beta gamma-II.

Amino Acids↗

PubMed1992

Fisher's syndrome without total ophthalmoplegia.

A 16-year-old boy with acute ophthalmoplegic polyneuritis (Fisher's syndrome) exhibited bilateral internal ophthalmoplegia, but only lateral rectus and superior oblique eye movements were affected. Gaze-evoked nystagmus was also present on left lateral gaze. Pupillary reflex recovered 7 weeks after onset, but neither cholinergic supersensitivity nor light-near dissociation were present during the recovery process. This is the first such case to be reported in which the site of the lesion responsible for the pupillomotor impairment was the Edinger-Westphal nucleus.

Adolescent↗

PubMed1992

Lack of association of Ca(2+)-calmodulin with the beta gamma-subunits of the photo-receptor G protein (transducin).

We previously reported that the beta gamma-subunit of transducin (T beta gamma) is composed of two components, T beta gamma-1 and T beta gamma-2 with distinctive gamma-subunits, T gamma-1 and T gamma-2, respectively. T beta gamma-2 enhances GTP binding to the alpha-subunit of transducin (T alpha) in the presence of a photobleaching intermediate of rhodopsin, while T beta gamma-1 is an inactive component with little enhancement ability (Fukada, Y., Ohguro, H., Saito, T., Yoshizawa, T., and Akino, T. (1989) J. Biol. Chem. 264: 5937-5943). To further elucidate the functional differences between T beta gamma-1 and T beta gamma-2, we examined the association of T beta gamma s with Ca(2+)-calmodulin, and the effect of Ca2+ on binding of GTP to T alpha in the presence of either T beta gamma-1 or T beta gamma-2. Ca2+ had no effect on the GTP binding activity of transducin and T beta gamma s could not associate with Ca(2+)-calmodulin, indicating that the relationship of T beta gamma with Ca(2+)-calmodulin of is different from that of the brain G protein.

Animals↗

PubMed1991

Carboxyl methylation and farnesylation of transducin gamma-subunit synergistically enhance its coupling with metarhodopsin II.

A heterotrimeric G-protein in vertebrate photoreceptor cells is called transducin (T alpha beta gamma), whose gamma-subunit is a mixture of two components, T gamma-1 and T gamma-2. T gamma-2 is S-farnesylated and partly carboxyl methylated at the C-terminal cysteine residue, whereas T gamma-1 lacks the modified cysteine residue. To elucidate the physiological significance of the double modifications in T gamma, we established a simple chromatographic procedure to isolate T gamma-1, methylated T gamma-2 and non-methylated T gamma-2 on a reversed phase column. Taking advantage of the high and reproducible yield of T gamma from the column, we analyzed the composition of T gamma subspecies in the T alpha-T beta gamma complex which did not bind with transducin-depleted rod outer segment membranes containing metarhodopsin II. The binding of T alpha-T beta gamma with the membranes was shown to require the S-farnesylated cysteine residue of T gamma, whose methylation further enhanced the binding. This synergistic effect was not evident when T alpha was either absent or converted to the GTP-bound form which is known to dissociate from T beta gamma. Thus we concluded that a formation of the ternary complex, T alpha-T beta gamma-metarhodopsin II, is enhanced by the farnesylation and methylation of T gamma. This suggests that the double modifications provide most efficient signal transduction in photoreceptor cells.

Amino Acid Sequence↗

PubMed1991

Structure and function of gamma-subunit of photoreceptor G-protein (transducin).

1. The gamma-subunit of the vertebrate photoreceptor GTP-binding protein (transducin) is S-farnesylated at the C-terminal cysteine residue, with a part of the residue being methyl-esterified at the alpha-carboxyl group. 2. Functionally, the modified cysteine residue is implicated in efficient coupling of the alpha- and beta gamma-subunits, and indispensible for expressing GTP-binding activity. 3. Similar modifications, isoprenylation and methyl-esterification of the C-terminal cysteine residue have been found in a variety of proteins involved in signal transduction and growth regulation processes. However, it seems likely that the physiological roles of the modifications are different for the various proteins.

Amino Acid Sequence↗

PubMed1990

Farnesylated gamma-subunit of photoreceptor G protein indispensable for GTP-binding.

Transducin, composed of subunits T alpha, T beta and T gamma, is a member of a heterotrimeric G-protein family, and transduces the light signal in visual cells. We have recently found that bovine T beta gamma can be separated into two components. T beta gamma-1 and T beta gamma-2, each of which has its own gamma-subunit, T gamma-1 and T gamma-2, respectively. T beta gamma-2 enhances the binding of GTP to T alpha in the presence of metarhodopsin II by about 30-fold compared with T beta gamma-1. Here we show that a farnesyl moiety is attached to a sulphur atom of the C-terminal cysteine of T gamma-2 (active form), a part of which is additionally methyl-esterified at the alpha-carboxyl group. In T gamma-1 (inactive form), however, such modifications are missing. Thus, the farnesyl moiety attached to the gamma-subunit is indispensable for the GTP-binding activity of transducin. This suggests that a similar modification may occur in the gamma-subunits of other heterotrimeric G proteins involved in biological signal transduction processes.

Amino Acid Sequence↗

PubMed1990

A specific beta gamma-subunit of transducin stimulates ADP-ribosylation of the alpha-subunit by pertussis toxin.

Transducin (T alpha and T beta gamma) is a GTP-binding protein involved in the visual transduction process in a rod outer segment. We have previously demonstrated that T beta gamma is a mixture composed of two components, T beta gamma-1 and T beta gamma-2, with distinctive gamma-subunits, T gamma-1 and T gamma-2, respectively (Fukada et al., 1989, J. Biol. Chem., 264, 5937-5943). To investigate the interaction between T alpha and the two components of T beta gamma, the effect of either T beta gamma-1 or T beta gamma-2 on the ADP-ribosylation of T alpha catalyzed by pertussis toxin (IAP) was examined. T beta gamma-2 stimulated the ADP-ribosylation of T alpha by IAP, while T beta gamma-1 displayed almost no enhancement of the ADP-ribosylation. Addition of T beta gamma-1 to the mixture of T alpha and T beta gamma-2 had no effect on the ADP-ribosylation of T alpha. These results indicate that T alpha and T beta gamma-2 form a complex that serves as a substrate of IAP in the ADP-ribosylation reaction, while T beta gamma-1 has a little affinity for T alpha. It was suggested that T gamma-2 is an essential subunit for T beta to interact with T alpha.

Adenosine Diphosphate Ribose↗

PubMed1990

Binding of GTP to transducin is not inhibited by arrestin and phosphorylated rhodopsin.

In the presence of a photobleaching intermediate of unphosphorylated or phosphorylated rhodopsin (Rh*), the binding of GppNHp to transducin was measured with or without arrestin for elucidation of the shut-off mechanism of the visual transduction process in bovine rod outer segments. The ability of Rh* to catalyze the formation of the transducin-GppNHp complex in the absence of arrestin was independent of the degree of phosphorylation of Rh*. Furthermore, the catalyzing ability of the phosphorylated Rh* was not reduced by the addition of arrestin. These observations indicate that the interaction between phosphorylated Rh* and transducin was not inhibited by arrestin. Thus, the hypothesis was not supported that the PDE shut-off process is a simple competition between transducin and arrestin for binding to phosphorylated Rh*.

Animals↗

PubMed1990

Functional heterogeneity of beta gamma-subunit of frog transducin.

1. Transducin subunits (T alpha and T beta gamma) were purified from freshly dissected frog (Rana catesbeiana) retinas. It was found that purified T beta gamma is composed of three components which can be separated from each other by an anion exchange column chromatography under nondenaturing conditions. 2. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses of these three components demonstrated that each contains T beta (mol. wt 35,000) and T gamma (mol. wt approximately 8000). 3. Only one of the three components retained an ability to enhance the binding of GppNHp to T alpha in the presence of a photobleaching intermediate of rhodopsin, while the others showed very low abilities to enhance the binding. 4. These observations, together with the similar findings on bovine T beta gamma, strongly suggest that the functional heterogeneity of T beta gamma is conserved in vertebrate photoreceptor cells.

Animals↗

PubMed1989

Beta gamma-subunit of bovine transducin composed of two components with distinctive gamma-subunits.

During the process of transduction of a photon signal in vertebrate rod outer segments, transducin, a guanine nucleotide binding protein, mediates between a photobleaching intermediate of rhodopsin and a cGMP-phosphodiesterase. We report here that the beta gamma-subunit of bovine transducin (T beta gamma) characterized so far consists of two components (T beta gamma-1 and T beta gamma-2), which can be separated by anion exchange chromatography under nondenaturing conditions. Both components consisted of two polypeptides of Mr 36,000 (T beta) and about 8,000 (T gamma) in sodium dodecyl sulfate polyacrylamide (13%) gel electrophoresis. On a further analysis by 8 M urea/sodium dodecyl sulfate-polyacrylamide gel electrophoresis, T gamma subunits of T beta gamma-1 and T beta gamma-2 showed Mr values of 8,000 (T gamma-1) and 6,000 (T gamma-2), respectively. Amino acid compositions of both T gamma-1 and T gamma-2 roughly corresponded with that of T gamma previously reported and were quite different from that of gamma-subunit of cGMP-phosphodiesterase. Western blot analysis of freshly isolated rod outer segments by an antiserum raised against a mixture of T beta gamma-1 and T beta gamma-2 revealed the presence of both components in the membranes of a starting material. This observation excludes the possibility that one of the components might be produced artificially in the course of the purification. In the presence of a photobleaching intermediate of either unphosphorylated or phosphorylated rhodopsin, the binding of guanosine 5'-(beta, gamma-imido)triphosphate (GppNHp) to the alpha-subunit of transducin (T alpha) was remarkably enhanced with increasing concentrations of purified T beta gamma-2. On the contrary, T beta gamma-1 retained little ability, if any, to enhance the GppNHp binding to T alpha; the ability of T beta gamma-1 was at least 30 times lower than that of T beta gamma-2. Such a low activity of T beta gamma-1 was attributed to inability for coupling of T alpha with a photobleaching intermediate of rhodopsin. These results indicate that T gamma-2 is essential for the GTP binding of transducin. The role of T gamma-1 in vertebrate photoreceptor cells was discussed.

Adenosine Triphosphate↗

PubMedDate not supplied

Mechanism of rhodopsin phosphorylation.

A key reaction in the inactivation of rhodopsin is its phosphorylation by rhodopsin kinase. In recent years, extensive studies related to rhodopsin kinase function and enzymatic properties were carried out. Rhodopsin kinase is a Ser/Thr protein kinase and a member of the G protein-coupled receptor kinases sub-family (GRKs) which consists of six recently identified members. Photolyzed rhodopsin is phosphorylated by rhodopsin kinase sequentially, with the first phosphate transferred preferentially to Ser-338, and subsequent phosphates transferred to Ser-343 and Thr-336. The binding of arrestin to the receptor, and reduction of the photolyzed chromophore all-trans-retinal to all-trans-retinol limits physiologically significant phosphorylation at no more than three sites (H. Ohguro, R.S. Johnson, L.H. Ericsson, K.A. Walsh and K. Palczewski, Biochemistry, 33 (1994) 1023). A similar phosphorylation reaction is implicated in most, if not all, G protein-coupled receptors during their desensitization.

Amino Acid Sequence↗

PubMedDate not supplied

Two cases of primary open angle glaucoma with serum autoantibody against retinal ganglion cells.

PURPOSE: To present two cases of primary open angle glaucoma (POAG) with serum autoantibody against retinal 25 kDa or 50 kDa antigen. METHODS: Ocular examinations were performed on a 60-year-old man (case 1) and a 74-year-old woman (case 2). We also performed serological and immunological examinations including Western blot analysis and immunocytochemical analysis. RESULTS: In Western blot analysis, they were found to have serum autoantibody against retinal 25 kDa or 50 kDa antigen, and immunocytochemical analysis revealed that retinal ganglion cell layers were specifically labeled by each patient's IgG. CONCLUSION: We report two interesting cases of POAG with serum autoantibody against retinal 25 kDa or 50 kDa antigen.

Aged↗

PubMedDate not supplied

Clinicopathologic and immunogenetic analysis of mucosa-associated lymphoid tissue lymphomas arising in conjunctiva.

PURPOSE: To identify mucosa-associated lymphoid tissue (MALT) type lymphoma in conjunctival infiltrates. METHODS: Clinical, histopathologic, immunophenotypic, and immunogenotypic studies were performed on 14 patients with conjunctival lymphoid infiltrates. Surgical biopsy specimens were subjected to histopathologic, immunohistochemical, and gene rearrangement analysis. RESULTS: Thirteen of the 14 patients (92.9%) met the diagnostic criteria for MALT lymphoma, and the remaining patient showed morphologic features of diffuse, small lymphocytic lymphoma. Genotypic analysis confirmed immunoglobulin heavy chain gene rearrangement in all of the 12 patients on whom the analysis was performed. Two patients with bilateral lesions exhibited identical immunoglobulin rearrangement patterns in each pair of lesions. All patients were alive at the last follow-up (mean: 39.9 months). Nine of the 14 patients were alive without disease, 4 had localized recurrences, and 1 had a residual tumor. CONCLUSIONS: These findings indicate that conjunctival lymphoid infiltrates usually have the features of MALT lymphoma with genotypic B lymphocytic monoclonality and a favorable prognosis.

Adult↗