Protein inhibitor of cyclic adenosine 3':5'-monophosphate phosphodiesterase in retina.

A protein acting as inhibitor of cyclic 3':5'-nucleotide phosphodiesterase (EC 3.1.4.1.) activity was found in the ox retina tissue. An inhibitor from one tissue (ox retina) effectively cross-inhibited a phosphodiesterase from another tissue (rat brain), indicating a lack of tissue specificity. Kinetic analysis showed that inhibition was independent of the time of preliminary incubation of the inhibitor with enzyme but dependent on its concentration in the reaction mixture. An inhibitor decreased the V of the enzyme and had no effect on its Km for cyclic adenosine-3':5'-monophosphate. The inhibitory effect was more pronounced with cyclic adenosine-3':5'-monophosphate than with cyclic guanosine-3':5'-monophosphate used as substrates of the reaction. The extractable form of the phosphodiesterase of the retina rod outer segments was much more sensitive to the inhibitory action than the membrane-bound one. The binding of labeled cyclic adenosine-3':5'-monophosphate to the inhibitory protein was shown not to occur. The inhibitor was sensitive to trypsin treatment, indicating that it was a proten attempt was mode to purify the inhibitory factor. Gel filtration indicated that the inhibitor had a molecular weight of 38 000.

The effects of melatonin and L-DOPA on the diurnal rhythms of free amino acids content in the rat retina.

The effects of melatonin and dopamine precursor L-3,4-dihydroxyphenylalanine (L-DOPA) intraperitoneal administration on the rhythms of free amino acids content in the retina of rats were studied. The authors found that the levels of those amino acids, which are protein constituents but not neurotransmitters in the rat retina, change diurnally with maximum at 3-6 h after light onset. Diurnal changes of Ala, Arg, Asn, Ile, Met, Ser, Trp, and Val content persisted in the retina of rats maintained at constant darkness. This fact confirms the true circadian nature of these rhythms. Constant lighting abolished diurnal changes of the content of all amino acids with the exception of Trp. Daytime but not nighttime administration of melatonin decreased the levels of Ala, Asn, Gln, Ile, Met, and Ser down to nocturnal values. Diurnal changes of amino acids content vanished in melatonin-injected rats. The effect of melatonin administration disappeared when the protein synthesis was inhibited by cycloheximide. The effect of intraperitoneal administration of L-DOPA on the levels of free amino acids was opposite the effect of melatonin administration. L-DOPA increased nocturnal levels of Gly, Thr, Trp, and Val but had no effect on the daytime amino acids content. As in the case of melatonin administration, significant diurnal changes of amino acid levels disappeared in L-DOPA-injected rats. The authors hypothesize that melatonin and dopamine can serve as zeitgebers-antagonists of amino acids content rhythms in the rat retina.

Active sites of the cyclic GMP phosphodiesterase gamma-subunit of retinal rod outer segments.

Monoclonal antibodies were prepared to the gamma-subunit of the cGMP phosphodiesterase. One of them gamma p-1, suppresses the activation of phosphodiesterase through the alpha-subunit of transducin. The gamma-subunit fragment 24-45 rich in Arg and Lys residues is involved in gamma p-1 binding and is essential for the gamma-subunit interaction with transducin. Carboxypeptidase Y cleaves off seven amino acid residues from the C-terminus of the gamma-subunit resulting in phosphodiesterase activation. Thus, the C-terminal fragment of gamma-subunit participates in phosphodiesterase inhibition.

Immunochemical study of the cyclic nucleotide system of retinal photoreceptor membranes: antibodies raised to phosphodiesterase, its protein inhibitor and GTP-binding proteins.

Monospecific precipitating antibodies raised to phosphodiesterase, its protein inhibitor and GTP-binding proteins of bovine retina photoreceptor membranes were obtained. The characterization of the antibodies was carried out by immunochemical methods and according to their functional properties as determined by their effect on enzyme activities. The antibodies were used to study the distribution of immunolike proteins in different animal retinas and to purify the inhibitor protein by the method of immunoaffinity chromatography.

[Modification of the retina photoreceptor membranes and temperature stability of rhodopsin]. / Modifikatsiia fotoretseptornykh membran setchatki i temperaturnaia stabil'nost' rodopsina.

The effect of modification of photoreceptor membranes of the bovine retina on the termodynamical parameters that characterize heat denaturation of rodopsin was studied. The highest increase of the rate constant and the corresponding maximal drop of the free energy change of heat denaturation of the pigment were obtained by using 7 M urea or 25% Triton X-100 in the presence of 5.10(-4) M EDTA. After chipping off one third of the protein from the rodopsin molecule by papain treatment a significant decrease of the slope of the Arrenius curve and a maximal decrease of entropy change compared to the parameters known for heat denaturation of the pigment in native photoreceptor membranes were found. Modification of the lipid components of the photoreceptor membranes (treatment with Triton X-100 and phospholipase C) reduced the thermostability of rodopsin. Maximal changes were obtained at Triton X-100 concentrations 0.1--1%, further concentration increas (1--25%) did not lead to significant changes. Phospholipase C treatment resulted in a decrease of free energy change and an increase of entropy change without affecting entalpy changes, accompaning the heat denaturation of rodopsin. Bivalent cations (Ca2+, Mg2+) increased the termostability of rodopsin both in photoreceptor membranes and in solutions to 25% Triton X-100.

[Interaction of leucine with the plasma membranes of catfish taste buds]. / Vzaimodeistvie leitsina s plazmaticheskimi membranami vkusovykh lukovits karlikovogo somika.

The binding of L-[3H]leucine by the plasma membranes from the catfish Ictalurus nebulosus taste organ was studied. The two types of specific binding centers for amino acid with high (KD = 2,5 X 10(-10) M) and low (KD = 1,04 X 10(-9) M) affinities were found. Concentrations of high and low affinity centers were 11,85 nmol/mg and 26 nmol/mg respectively. The structural rearrangement of the surface layer of the chemoreceptor membrane was revealed by spin label technique using 5-doxylstearic acid at leucine concentrations 10(-4)-10(-9) M.

[Enzymes of the outer segments of the retinal rods: The problem of localization and coupling with rhodopsin]. / Fermenty naruzhnykh segmentov palochek setchatki: Problema lokalizatsii i sopriazhennosti s rodopsinom.

The problem of the presence of enzymes in retina rod outer segments is summarized using both literature and the author's own data. Difficulties in the solving of this problem are analyzed. It has been shown that some enzymes involved in the primary mechanisms of the photoreceptor process are localized in the inner segment of the photoreceptor cell at a distance from the photoreceptor membranes. A conclusion is drawn that the enzymes necessary for rhodopsin (or its retinal-part) conversions as well as those ensuring synthesis and decay of cyclic nucleotides are concentrated in the outer segments. These data served as a ground for a conclusion about a strictly "biochemial" specialization of particular parts of the photoreceptor cell in which tightly linked chemical transformations proceed in different compartments. Besides, some unusual properties of the plasma membrane of the photoreceptor cell are noticed, in particular, the absence in part of this membrane of some typical marking enzymes. The interrelations between rhodopsin and enzymes as well as a possible participation of Ca-ions in this process are examined. It is supposed that one part of the rhodopsin molecule may act as a Ca2+ binding protein.

[Phosphodiesterase of cyclic nucleotides]. / Fosfodiesteraza tsiklicheskikh nukleotidov.

Problems are considered on form multiplicity, purification and molecular weight of cyclic nucleotides phosphodiesterase. A supposition is made that the molecular weight of the catalytic subunit of the enzyme for most studied objects is about 60000. The catalytic subunit may form di- and trimers and be associated with regulatory proteins of different type. The problem of phosphodiesterase regulation is analyzed on the basis of potentialities of the equilibrium shift between the protein subunits of the enzyme; the role of cyclic nucleotides as well as of triphosphonucleotides are shown to influence the regulation of the enzymic activity. In some cases the mechanism of changes in the activity of phosphodiesterase bound with the receptor is shown to be similar to that for adenylate cyclase. In particular, the role of GTP and one of the protein subunits of phosphodiesterase in this process is stated.

[Influence of effectors of hormone-sensitive adenylate cyclase on the activation system of photostimulated cyclic nucleotide phosphodiesterase from outer rod segments]. / Vliianie éffektorov gormonchuvstvitel'noi adenilattsiklazy na sistemu aktivatsii fotostimuliruemoi fosfodiesterazy tsiklicheskikh nukleotidov v naruzhnykh segmentakh palochek setchatki.

The effect of preincubation of preparations of the outer segments of optic rods with the nonhydrolyzed analog GTP-guanilyl-5'-imidodiphosphate (Gpp(NH)p) and NaF, the combined effect of these agents as well as the action of (NH4)2SO4 (10-800 mM), MgSO4 (2-50 mM) and induction of peroxide oxidation of lipids are studied as applied to the catalytic activity of phosphodiesterase of cyclic nucleotides. Gpp(NH)p and NaF are shown to be tightly bound to GTP-binding proteins (G-proteins) of outer segments of optic rods, additional activation of phosphodiesterase in the presence of Gpp(NH)p being observed after preincubation with NaF and subsequent washing of the membrane. A problem on different binding sites of the ion F and Gpp(NH)p on G-proteins is discussed. It is found that (NH4)2SO4 does not affect the basal activity of phosphodiesterase but inhibits the activating effect of Gpp(NH)p and NaF on the enzyme. Induction of peroxide oxidation of lipids prevented by the addition of ionol (antioxidant) in a dose of 5.10(-4) M has the same effect. Changes in the concentration of Mg2+ in the medium influence insignificantly the basal activity of phosphodiesterase but are necessary for manifestation of the activating effect of Gpp(NH)p and NaF.

[Localization and causes of lipid peroxidation disorders in the rat cerebral cortex in the early stages of hereditary retinal degeneration]. / Lokalizatsiia i prichiny narushenii perekisnogo okisleniia lipidov v kore mozga krys na rannikh stadiiakh nasledstvennoi degeneratsii setchatki.

It is established that previously observed increased rate of the induced lipid peroxidation in brain tissue of rats with hereditary retinal degeneration as compared with normal rats is due to the change of the rate of this process in the microsome cortex brain fraction and was not observed in the mitochondrial-synaptosomal and nuclear fractions. The content of nonheme iron ions in microsome cortex brain fraction of the Campbell rats is decreased by 35% and of the Fe ion was in the reduced form as compared with the Wistar rats. The ratio of Fe2+/Fe3+ in this fraction of the Campbell rats will be 5.21; Wistar rats--0.51. The increase of the reduced form of the Fe ion may be a result of the increased rate of the glucose-6-phosphate dehydrogenase activity in cortex brain tissue of the Campbell rats. We accept change of the content and the forms of the Fe ions in the microsome cortex brain fraction as a cause of the increased rate of induced lipid peroxidation in brain of the Campbell rats. All the observed phenomena are manifested at the early stage of life and indicated that different metabolic disorders can be observed in the Campbell rats not only in the retina and eye pigment epithelium but also in the brain tissue.

[Effect of estradiol on 3',5'-AMP-phosphodiesterase activity in rat uterus. Participation of hormone receptors, the role of guanyl nucleotides]. / Vliianie éstradiola na 3',5'-AMP-fosfodiésterazu tkani matki krys. Uchastie gormonal'nogo retseptora, rol' guanilovykh nukleotidov.

Estradiol (10(-7)-2 . 10(-5)M) is shown to inhibit the activity of phosphodiesterase of the soluble fraction of the mature rat womb tissue and to have no effect on the activity of adenylate cyclase of this tissue membrane fraction and on the phosphodiesterase of the tissue of the brain, heart and outer segments of the rod-cell. When blocking the estradiol cytosol receptors by clomiphene there is no inhibitory effect of the hormone on the activity of phosphodiesterase of the womb tissue preparations. Specific binding of estradiol by the cytosol receptors increases in the presence of guanylic nucleotides (10(-5)-10(-4)M); ATP does not affect this process. In the presence of guanyl-5-ilimidodiphosphate (Gpp(NH)p), a nonhydrolyzed analog of GTP, clomiphene does not block receptor binding of estradiol. ATP and GTP (10(-6)-10(-5)M), contrary to Gpp (NH)p, inhibit the phosphodiesterase activity. Independent of their effect on the enzymic activity, all the studied nucleotides partially or completely eliminate the inhibitory effect of estradiol on phosphodiesterase.

[Aminobenzoic acid derivatives as specific inhibitors of cyclic nucleotide phosphodiesterase in the rat uterus]. / Proizvodnye aminobenzoinoi kisloty kak spetsificheskie ingibitory fosfodiésterazy tsiklicheskikh nukleotidov matki krys.

It is shown, that p-aminobenzoic acid and its derivatives (p-acetylaminobenzoic acid and p-aminobenzoic acid hydrazide) in the concentration of 10(-6) M are the potent inhibitors (40% below the control specimens) of the phosphodiesterase activity of cyclic nucleotides in the soluble fraction of the adult rat uterus. These drugs exerted no action on the adenylate cyclase activity in membrane fractions. The inhibition is only specific to the uterus enzyme and is not revealed for other tissues. The inhibition is found to be of incompetitive character Ki for p-aminobenzoic acid hidrazide being equal to 3.2 microM.

[Adenyl cyclase and adenosine-3',5'-monophosphate phosphodiesteraze: their nature, properties and regulation]. / Adeniltsiklaza i fosfodiésteraza adenozin-3',5'-monofosfata: priroda, svoistva i reguliatsiia

Modern data on the nature, properties and pathways of regulation enzymes, participated metabolism of cyclic, adenosine-3',5'-monophosphate, are described. Much consideration is being given to the problem of regulation of the phosphodiesterase activity due to the effect of Ca2+-binding proteins.

[Protein inhibitor of cyclic nucleotide phosphodiesterase in the retina in hereditary degeneration]. / Belkovyi ingibitor fosfodiésterazy tsiklicheskikh nukleotidov v setchatki pri ee nasledstvennoi degeneratsii.

Thermostable protein fraction from retina of rats with hereditary retinal dystrophy (Hunter and Campbell strains) did not inhibit cyclic nucleotide phosphodiesterase. At the same time an inhibitory component, found in retina of Wistar rat, rabbit, frog and lamprey, was similar to the component from bovine retina. Quantity of the inhibitory component in normal rat retina decreased considerably within postnatal period (12 days--3 months). Thermostable proteins, isolated from Campbell rat retina, differed from that of Hunters' one by electrophoretic properties while both preparations were dissimilar to the protein of normal rat. Protein bands, containing inhibitory component from dystrophic rat retina, appear to be less distinct as compared to those of normal rat. These proteins, eluated from the bands of Campbell rats, activated phosphodiesterase but the preparations from Hunter rats did not influence on it.

[Rhabdomere adenosine triphosphatase systems of the squid Todarodes sloanei pacificus]. / Adenozintrifosfataznye sistemy rabdomerov kal'mara Todarodes sloanei pacificus

Highly purified fraction of squid photoreceptor membranes exhibits relatively low activity of Na+,K+-ATPase. No correlation was observed between the distribution of rhodopsin and Na+,K+-ATPase activity among photoreceptor membrane subfractions obtained from rhabdomeres. On the basis of these data, it is suggested that Na+,K+-ATPase and rhodopsin are localized in different membrane structures. In most purified fractions of rhabdomeres, the ratio of rhodopsin to total protein amounted up to 45--50%. In Triton X-100 extracts from membrane fractions, significant enrichment in rhodopsin content was observed.

[Decreased content of non-heme iron in the blood of patients with peripheral tapetoretinal abiotrophy]. / Snizhenie soderzhaniia negemovogo zheleza v syvorotke krovi u bol'nykh s perifericheskoi tapetoretinal'noi abiotrofiei.

Measurement of non-heme iron in the sera of patients with peripheral tapetoretinal abiotrophy aged 10-72 years with stages I-IV of the disease showed a statistically significant (26-30%) decrease of this parameter in comparison with healthy controls. Total iron-binding capacity of the serum was unchanged. The results are discussed with regard for previous reports about changed iron homeostasis in animals with hereditary retinal degeneration (Campbell rats) and the role of iron in the development and formation of nervous tissues.