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Biomedical subjects

N Miki

Publications and source records attributed to N Miki.

At least 217 records · Page 12Linked to original sources
PubMed1976

Interaction of Vibrio cholerae toxin with sarcoma 180 cell membranes.

Three discrete phases are discernible in the activation, by Vibrio cholerae toxin, of adenylate cyclase in fragments of sarcoma 180 cell membranes. In the first, or preparatory, phase the toxin must be exposed to dithiothreitol or nicotinamide adenine dinucleotide (NAD) in the absence of the membranes. In the second phase, the prepared toxin is dissociated to yield a macromolecular cyclase-activating factor (MCAF) in the presence of the membranes. In the third phase, membrane basal adenylate cyclase is activated by MCAF in the presence of NAD. The integrity of the catecholamine or beta-receptor associated with sarcoma adenylate cyclase is irrelevant in the activation of cyclase by MCAF. This activation proceeds undiminished even if the beta-receptor is desensitized or blocked by propranolol.

Adenosine Triphosphate↗
PubMed1975

Purification and properties of the light-activated cyclic nucleotide phosphodiesterase of rod outer segments.

Frog (Rana catesbiana) rod outer segment disc membranes contain a cyclic nucleotide phosphodiesterase (EC 3.1.4.17) which is activated by light in the presence of ATP. This enzyme is firmly bound to the disc membrane, but can be eluted from the membrane with 10 mM Tris-HCl buffer, pH 7.4 and 2 mM EDTA. The eluted phosphodiesterase has reduced activity, but can be activated approximately 10-fold by polycations such as protamine and polylysine. The eluted phosphodiesterase can no longer be activated by light in the presence of ATP, that is, activation by light apparently depends on the native orientation of phosphodiesterase in relationship to other disc membrane components. The eluted phosphodiesterase was purified to homogeneity as judged by analytical polyacrylamide gel electrophoresis and polyacrylamide gel isoelectric focusing. The over-all purification from intact retina was approximately 925-fold. The purification of phosphodiesterase from the isolated rod outer segment preparation was about 185-fold with a 28% yield. Phosphodiesterase accounts for approximately 0.5% of the disc membrane protein. The eluted phosphodiesterase (inactive form) has a sedimentation coefficient of 12.4 S corresponding to an approximate molecular weight of 240,000. Sodium dodecyl sulfate polyacrylamide gel electrophoresis separates the purified phosphodiesterase into two subunits of 120,000 and 110,000 daltons. With cyclic 3':5'-GMP (cGMP) as substrate the Km for the purified phosphodiesterase is 70 muM. Protamine increases the Vmax without changing the Km for cGMP. The isoelectric point (pI) of the native dimer is 5.7. Limited exposure of the eluted phosphodiesterase (inactive form) to trypsin produces a somewhat greater activation than is obtained with 0.5 mg/ml of protamine. The trypsin-activated phosphodiesterase has a sedimentation coefficient of 7.8 S corresponding to an approximate molecular weight of 170,000. The 110,000-dalton subunit is much less sensitive to trypsin hydrolysis and the 120,000-dalton subunit is rapidly replaced by smaller fragments. On the basis of the molecular weight of the purified phosphodiesterase (240,000) and the concentrations of phosphodiesterase and rhodopsin in the rod outer segment, it is estimated that the molar ratio ophosphodiesterase to rhodopsin in the rod outer segment is approximately 1:900. Since all of the disc phosphodiesterase molecules are activated when 0.1% of the rhodopsins are bleached, we conclude that in the presence of ATP 1 molecule of bleached rhodopsin can activate 1 molecule of phosphodiesterase.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1975

A link between rhodopsin and disc membrane cyclic nucleotide phosphodiesterase. Action spectrum and sensitivity to illumination.

Frog (Rana pipiens) rod outer segment disc membranes contain guanosine 3',5'-cyclic monophosphate phosphodiesterase (EC 3.1.4.1.c) which, in the presence of ATP, is stimulated 5- to 20-fold by illumination. The effectiveness of monochromatic light of different wavelengths in activating phosphodiesterase was examined. The action spectrum has a maximum of 500 nm, and the entire spectrum from 350 to 800 nm closely matches the absorption spectrum of rhodopsin, which is apparently the pigment which mediates the effects of light on phosphodiesterase activity. trans-Retinal alone does not mimic light. Half-maximal activation of the phosphodiesterase occurs with a light exposure which bleaches 1/2000 of the rhodopsins. Half-maximal activation can also be achieved by mixing 1 part of illuminated disc membranes in which the rhodopsin is bleached with 99 parts of unilluminated membranes. Regeneration of bleached rhodopsin by addition of 11-cis-retinal is illuminated disc membranes reverses the ability of these membranes to activate phosphodiesterase in unilluminated membranes. If the rhodopsin regenerated by 11-cis-retinal is illuminated again, it regains the ability to activate phosphodiesterase. These studies show that the levels of cyclic nucleotides in vetebrate rod outer segments are regulated by minute amounts of light and clearly indicate that rhodopsin is the photopigment whose state of illumination is closely linked to the enzymatic activity of disc membrane phosphodiesterase.

Adenosine Triphosphate↗
PubMed1975

Cholera toxin activation of adenylate cyclase in cancer cell membrane fragments.

Activation of adenylate [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] by cholera toxin (84,000 daltons, 5.5 S) is demonstrated in plasma membrane fragments of mouse ascites cancer cells. The activation of adenylate cyclase is mediated by a macromolecular cyclase activating factor (MCAF), which has a sedimentation constant of 2.7 S and a molecular weight of about 26,000. MCAF is derived from, and may be identical to the "A fragment" of cholera toxin. Generation of MCAF depends on prior interaction of cholera toxin with either dithiothreitol, NADH, NAD, or a low-molecular-weight component (less than 700 daltons) present in cytoplasm. Subsequent exposure of this pretreated cholera toxin to cell membranes from a variety of mouse ascites cancer cells is followed rapidly by the appearance of MCAF, which no longer requires dithiothreitol, NADH, or NAD for the activation of adenylate cyclase. Activation of adenylate cyclase by MCAF in ascites cancer cell membrane fragments is not reversed by repeated washing of these membrane fragments. Adenylate cyclase in normal cell membrane fragments fails to respond either to cholera toxin or MCAF in the presence of dithiothreitol. In striking contrast, the adenylate cyclase in membrane fragments from five ascites cancer cells responds to either MCAF or native cholera toxin preincubated with dithiothreitol, NADH, or NAD.

Adenylyl Cyclases↗
PubMed1973

Regulation of cyclic nucleotide concentrations in photoreceptors: an ATP-dependent stimulation of cyclic nucleotide phosphodiesterase by light.

Regulation of cyclic nucleotide concentrations in rod outer segments (Rana pipiens) has been further examined. The present studies show that illumination markedly diminishes the concentration of cyclic nucleotides in suspensions of photoreceptor membranes, but the locus of regulation is cyclic nucleotide phosphodiesterase (EC 3.1.4.c) (light-stimulated) and not adenylate cyclase. There is a marked disproportionality between bleaching of rhodopsin and stimulation of phosphodiesterase. Bleaching only 0.6% of the rhodopsin produces half the stimulation produced by bleaching 100% of the rhodopsin. The process of activation of phosphodiesterase by light is in two steps, a light-dependent step followed by an ATP-dependent step. Illumination (in the absence of ATP) produces a trypsin-resistant, heat-labile, macromolecular stimulator. In the presence of 0.75 mM ATP (GTP or ITP) this stimulator produces a greater than 5-fold increases in the V(max) of photoreceptor phosphodiesterase without changing the K(m). At physiological substrate concentrations (10(-7) M) the rate of hydrolysis of cyclic GMP is 23 times greater than that of cyclic AMP. The light-produced stimulator appears unique to the photoreceptor membranes and does not activate phosphodiesterase in other tissues.

Adenosine Triphosphate↗