Analysis of the kinetics of cyclic AMP hydrolysis by the cyclic GMP-stimulated cyclic nucleotide phosphodiesterase

The kinetics of cAMP hydrolysis by the purified calf liver cGMP-stimulated cyclic nucleotide phosphodiesterase were analyzed in the absence or presence of a number of competitive inhibitors of the methylxanthine type according to a two-site competitive model for allosteric enzymes. Methylxanthines were also classified by graphical analysis of classical competition kinetics at saturating cAMP. This treatment yielded Km/KI ratios which estimated the relative effectiveness of the binding of substrate and inhibitors to the "high affinity" (ES complex) state without establishing individual equilibrium-binding constants of cAMP and inhibitors for specific enzyme states. Individual binding constants for substrate and inhibitors were estimated directly by fitting primary data to the rate equation for the two-site competitive model. The equilibrium dissociation constants for cAMP to the "high" (KS) and "low affinity" (AKS) states were 2.4 +/- 0.8 and 410 +/- 140 microM, respectively. Dissociation constants for various inhibitors to the high (BKI) and low affinity (KI) states were also estimated. The ratio KS/BKI, which directly compared the equilibrium-binding constants of substrate and inhibitors to the high affinity state (ES complex), was in excellent agreement with Km/KI ratios derived from graphical analysis. Whereas a number of the methylxanthine analogues were more effective or as effective as cAMP in binding to the low affinity or "ligand-free" state, only isobutylmethylxanthine was effective as cAMP in binding to the high affinity state (1-methyl-3-isopropylxanthine, and 1,3-dipropylxanthine were somewhat less effective). These findings suggested that allosteric transitions might alter the topography of specific hydrophobic domains at cyclic nucleotide-binding sites and that structural determinants were more stringent for binding to the high affinity state than to the low affinity state.     

Circular Arrangement of Cortical F-actin around the Subapical Region of a Tip-Growing Fern Protonemal Cell

F-actin organization in the tip-growing cells of fern protonematawas investigated by rhodamine-phalloidin staining in two species:Adiantum capillus-veneris and Pteris vittata. Circular arrangementof cortical F-actin was found around the subapical region ofprotonemal cells in both species. In rhizoids, such structureswere absent and the axial filaments appeared to fan out fromthe tip. (Received May 22, 1989; Accepted September 6, 1989)     

Conformational change of bovine serum albumin by heat treatment

The thermal denaturation of bovine serum albumin (BSA) was studied at pH 2.8 and 7.0 in the range of 2–65°C. The relative proportions of -helix, -structure, and disordered structure in the protein conformation were determined as a function of temperature, by the curve-fitting method of circular dichroism spectra. With the rise of temperature at pH 7.0, the proportion of -helix decreased above 30°C and those of -structure and disordered structure increased in the same temperature range. The structural change was reversible in the temperature range below 45°C. However, the structural change was partially reversible upon cooling to room temperature subsequent to heating at 65°C. On the other hand, the structural change of BSA at pH 2.3 was completely reversible in the temperature range of 2–65°C, probably because the interactions between domains and between subdomains might disappear due to the acid expansion. The secondary structure of disulfide bridges-cleaved BSA remained unchanged during the heat treatment up to 65°C at pH 2.8 and 7.0.     

Cell type-specific expression of the genes for the protein kinase C family: down regulation of mRNAs for PKC alpha and nPKC epsilon upon in vitro differentiation of a mouse neuroblastoma cell line neuro 2a

By the use of cloned cDNAs for protein kinase C isozymes alpha, beta I, beta II, gamma, and those for novel protein kinase C, epsilon and zeta, the expression of the corresponding mRNA species was examined in various mouse tissues, human lymphoid cell lines, and mouse cell lines of neuronal origin. In adult brain, mRNAs for all the isozymes of PKC family are expressed. However, the expression of these mRNA species in brain is low at birth. A similar pattern of expression was also observed for beta I/beta II mRNAs in spleen. These expression patterns are in clear contrast to that for beta I/beta II mRNAs in thymus where the mRNAs are expressed at birth and the levels of expression decrease with age. Human lymphoid cell lines express large amounts of PKC beta mRNAs in addition to PKC alpha. Further, nPKC epsilon mRNA is expressed in some of these cell lines. On the other hand, all the mouse cell lines of neuronal origin tested express nPKC epsilon and zeta in addition to PKC alpha. In a mouse neuroblast cell line, Neuro 2a, down modulation of mRNAs for both PKC alpha and nPKC epsilon was observed in association with in vitro differentiation.     

Temperature gradient DNA-probe column chromatography: as tools for detection and purification of particular DNAs and RNAs

We have developed the temperature-gradient DNA-probe column chromatography as the new method for detecting and purifying particular DNAs or RNAs accurately. The method has high discrimination resolution, and can detect a single-base change in sample base sequences. The method also has high performances in purification of particular DNAs or RNAs, which has been demonstrated by purification of bovine mitochondria serine t-RNA. None of other method can succeed in complete purification of this t-RNA molecule. The present method can be applied not only to molecular biology as basic tools for purifying and picking-up particular sequences but also to the medical field as diagnostic tools.     

A theoretical study of the dielectric constant of protein

The dielectric properties of a protein molecule were investigated by calculating a 'local dielectric constant' with the aid of normal mode analysis. This local dielectric constant was calculated from the electronic polarization of atoms and the orientational polarization of local dipoles. The former was obtained from atomic polarizations of the whole atoms in a protein molecule. The latter was determined from the fluctuation-dissipation theorem. The degree of dipole fluctuation was calculated from the positional fluctuation of each atom obtained by the normal mode analysis. Assuming a minimum volume for a continuum model, the resulting local dielectric constants ranged from 1 to 20 inside the protein.     

Copper absorption in young men fed adequate and low zinc diets

Copper absorption was measured at two levels of dietary zinc in six healthy young men who were confined to a metabolic unit for a 75 d study of zinc utilization. A diet of conventional foods was fed, providing either 16.5 or 5.5 mg zinc and 1.3 mg copper daily. Copper absorption was determined by feeding⁶⁵Cu, a stable isotope of copper, once during the 16.5 mg Zn diet and near the beginning and end of the 5.5 mg Zn diet. Apparent copper absorption averaged 48.1% when the 16.5 mg Zn diet was fed. This was significantly higher than the averages of 37.2 and 38.5% when the 5.5 mg Zn diet was fed. Absorption also differed significantly among subjects. Fecal copper did not differ between diets or among subjects. All subjects were in positive copper balance at both levels of dietary zinc. These results suggest that a dietary zinc intake slightly above the Recommended Dietary Allowance of 15 mg/d does not increase fecal copper loss and does not interfere with copper absorption.     

Analysis of human plasma as an exposure level monitor for carcinogenic tryptophan pyrolysis products

A high-performance liquid chromatography method for detecting 3-amino-1,4- dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) and 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) in human plasma was developed. Plasma samples of 10 normal subjects were examined. Trp-P-1 and Trp-P-2, carcinogenic tryptophan pyrolysis products, were detected in all specimens, and the concentrations of Trp-P-1 and Trp-P-2 in plasma were 68.31 +/- 24.03 fmoles/ml (mean +/- S.D., n = 10) and 18.79 +/- 4.99 fmoles/ml, respectively. Our results suggest that plasma levels of carcinogenic tryptophan pyrolysis products may be useful indicators for estimating the exposure levels of the dietary carcinogens.     

Chloroplast proliferation based on their distribution and arrangement inAdiantum protonemata growing under red light

Chloroplast proliferation was investigated inAdiantum protonemata growing under continuous red light. Cell division is absent when cells are grown under red light. The chloroplast number increases as the cell length increases, therefore the chloroplasts divide in the absence of cell division. Chloroplasts in the basal part of the filamentous protonemal cell migrate gradually toward the cell apex, but there is no large net migration from the tip to the base or vice versa, indicating that chloroplast division takes place in the apical part of the protonemata. Chloroplast number in the apical 100 μm was maintained at about 200 during cell growth at least over eight days. The chloroplasts were either dumbbell- or ellipsoid-shaped. Dumbbell-shaped chloroplasts are abundant everywhere in a protonema, ranging from 30 to 50% of the total chloroplasts. The dumbbell-shaped chloroplasts attached to or very close to the plasma membrane seem to be the ones that are dividing but the dumbbell-shaped ones in the other regions do not divide. These data support the hypothesis that a signal from the plasma membrane induces the dumbbell-shaped chloroplasts to divide.