Effects of Dietary Amino Acids on Brain Amino Acids and Transmitter Amines in Rats with a Portacaval Shunt

The etiologic relationship between disturbances in metabolism of amino acids and amines and hepatic coma was investigated by examining the effects of diets containing various mixtures of amino acids on brain amine metabolism in rats with a portacaval shunt, using a method for simultaneous analysis of amino acids and amines. Rats with a portacaval shunt were fed on four different amino acid compositions with increased amounts of various amino acids suspected to be etiologically related to hepatic coma, such as methionine, phenylalanine, tyrosine, and tryptophan. The animals were killed 4 weeks after operation. During the experimental period, these animals did not become comatose, but exhibited various behavioral abnormalities. Marked increase in the plasma and brain levels of the augmented amino acids, especially methionine and tyrosine, were observed in rats with a portacaval shunt. Brain noradrenaline, dopamine, and serotonin levels were significantly decreased when the brain tyrosine level was increased. These results indicate that in rats with a portacaval shunt the dietary levels of amino acids greatly influence the brain levels of both amino acids and transmitter amines.     

Immobilized mitochondrial electron transport particle for NADH determination

Nonphosphorylating electron transport particles (ETP) prepared from beef heart mitochondrion were immobilized in agar gel. The immobilized ETP showed an oxidase activity to both NADH and succinate. The immobilized ETP was reusable. An electrochemical device for the determination of either NADH or succinate was assembled consisting of the membrane-bound ETP and an oxygen probe. The response to succinate was specifically inhibited by the addition of malonate.     

Kinetic study of denaturation and subsequent reduction of disulfide bonds of lysozyme by the rapid ultrasonic absorption measurement

A New technique for the rapid measurement of ultrasonic absorption with a sampling interval of 5 msec has been developed and applied to the kinetic study of denaturation and subsequant redution of hen egg-white lysozyme. The lysozyme is denatured by guanidine hydrochloride (GuHCl) orLiBr, and afetr denaturation by GuHCl, its disulfide bonds are reduced by dithiothreitol (DTT). The ultrasonic adsorption coefficient at 9 MHz increases with denaturation but decreases with reduction. The rate constant of denaturation by GuHCl obtained from the rime variance of ultasonic agrees well with that from uv absorption and optical rotation. The time variance if absorption after GuHCl and Dtt have been simultaneously added exhibits two rate constants. Analysis of the constants as functions of regeant concentrations indicates that the intermediates state between native and reduced states is not necessarily the completely denatured state but depends on the concentartions of GuHCl and DTT.     

Pumpkin (Cucurbita sp.) seed globulin I. Purification, characterization, and subunit structure

A heat stable globulin present in the cotyledons of pumpkinseeds was prepared as crystals which were soluble in a dilutesaline solution below pH 4.5 or in a solution with a high ionicstrength at neutral pHs. The protein was nearly homogeneousby ultracentrifuge analysis, and had a molecular weight of about112,000 daltons. Sodium dodecyl sulfate-polyacrylamide gel electrophoresisseparated the globulin into two subunits, and ß,corresponding to molecular weights of about 63,000 and 56,000daltons, respectively. By reduction of disulfide bonds, thetwo subunits were each separated into two polypeptide chainswith molecular weights of around 36,000 and 22,000 daltons,judged by gel electrophoresis. The amino acid composition ofwhole globulin indicated high contents of arginine, glutamicacid and aspartic acid. The total number of half-cystine residuewas nine and only one residue was shown to be free. The subunitstructure of the globulin is discussed. The protein has beenshown to have oxaloacetate decarboxylase activity, and thisfact was confirmed. However, the activity decreased markedlyat pH 4.5 in a fairly short period. It did not require Mn⁺⁺,and the K_m for oxaloacetate was determined to be 4.1 mM. (Received April 9, 1976; )     

Kinetics of proton transfer reactions of lysozyme with p-nitrophenol near neutral pH—a study of dynamic properties of Glu 35 and His 15

Kinetics of proton transfer between lysozyme and a pH indicator p-nitrophenol (p-Np) were measured by the temperature-jump method in a pH range of 6.0–7.0. Two well-defined relaxation processes were observed. The fast process (τ ? 15 μsec) was also observed for a lysozyme derivative succinylated at the terminal α-amino group of Lys 1. Therefore, the fast process was found to be attributable to the proton transfer reaction of His 15 with p-Np. The slow process (τ ? 50 μsec) was found to be characteristic of the proton transfer reaction of Glu 35, because it disappeared completely in solution containing a lysozyme derivative having an ester crosslink between the carboxyl group of Glu 35 and indol C-2 of Trp 108. The rate constants for proton transfer from Glu 35 and His 15 to p-Np were found to be 9 × 10⁶/sec/M (±65%, 23°C) and 3 × 10⁸/sec/M (±20%, 25°C), respectively. These data indicate that the proton of the carboxyl group of Glu 35 is kinetically stabilized in lysozyme.     

Production of a Doubly Chiral Compound, (4R,6R)-4-Hydroxy-2,2,6-Trimethylcyclohexanone, by Two-Step Enzymatic Asymmetric Reduction

A practical enzymatic synthesis of a doubly chiral key compound, (4R,6R)-4-hydroxy-2,2,6-trimethylcyclohexanone, starting from the readily available 2,6,6-trimethyl-2-cyclohexen-1,4-dione is described. Chirality is first introduced at the C-6 position by a stereoselective enzymatic hydrogenation of the double bond using old yellow enzyme 2 of Saccharomyces cerevisiae, expressed in Escherichia coli, as a biocatalyst. Thereafter, the carbonyl group at the C-4 position is reduced selectively and stereospecifically by levodione reductase of Corynebacterium aquaticum M-13, expressed in E. coli, to the corresponding alcohol. Commercially available glucose dehydrogenase was also used for cofactor regeneration in both steps. Using this two-step enzymatic asymmetric reduction system, 9.5 mg of (4R,6R)-4-hydroxy-2,2,6-trimethylcyclohexanone/ml was produced almost stoichiometrically, with 94% enantiomeric excess in the presence of glucose, NAD⁺, and glucose dehydrogenase. To our knowledge, this is the first report of the application of S. cerevisiae old yellow enzyme for the production of a useful compound.     

Direct interaction of post-synaptic density-95/Dlg/ZO-1 domain-containing synaptic molecule Shank3 with GluR1 alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor

A class of scaffolding protein containing the post-synaptic density-95/Dlg/ZO-1 (PDZ) domain is thought to be involved in synaptic trafficking of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors during development. To clarify the molecular mechanism of AMPA receptor trafficking, we performed a yeast two-hybrid screening system using the cytoplasmic tail of the GluR1 subunit of AMPA receptor as a bait and identified a synaptic molecule, Shank3/ProSAP2, as a GluR1 subunit-interacting molecule. Shank3 is a PDZ domain-containing multidomain protein and is predominantly expressed in developing neurons. Using the glutathione S-transferase pull-down assay and immunoprecipitation technique we demonstrated that the GluR1 subunit directly binds to the PDZ domain of Shank3 via its carboxyl terminal PDZ-binding motif. We raised anti-Shank3 antibody to investigate the expression of Shank3 in cortical neurons. The pattern of Shank3 immunoreactivity was strikingly punctate, mainly observed in the spines, and closely matched the pattern of post-synaptic density-95 immunoreactivity, indicating that Shank3 is colocalized with post-synaptic density-95 in the same spines. When Shank3 and the GluR1 subunit were overexpressed in primary cortical neurons, they were also colocalized in the spines. Taken together with the biochemical interaction of Shank3 with the GluR1 subunit, these results suggest that Shank3 is an important molecule that interacts with GluR1 AMPA receptor at synaptic sites of developing neurons.     

Chemiosmotic coupling of ion transport in the yeast vacuole: Its role in acidification inside organelles

Acidification inside the vacuo-lysosome systems is ubiquitous in eukaryotic organisms and essential for organelle functions. The acidification of these organelles is accomplished by proton-translocating ATPase belonging to the V-type H⁺-ATPase superfamily. However, in terms of chemiosmotic energy transduction, electrogenic proton pumping alone is not sufficient to establish and maintain those compartments inside acidic. Current studies have shown that thein situ acidification depends upon the activity of V-ATPase and vacuolar anion conductance; the latter is required for shunting a membrane potential (interior positive) generated by the positively charged proton translocation. Yeast vacuoles possess two distinct Cl^– transport systems both participating in the acidification inside the vacuole, a large acidic compartment with digestive and storage functions. These two transport systems have distinct characteristics for their kinetics of Cl^– uptake or sensitivity to a stilbene derivative. One shows linear dependence on a Cl^– concentration and is inhibited by 4,4-diisothiocyano-2,2-stilbenedisulfonic acid (DIDS). The other shows saturable kinetics with an apparentK _m for Cl^– of approximately 20 mM. Molecular mechanisms of the chemiosmotic coupling in the vacuolar ion transport and acidification inside are discussed in detail.     

Structure and thermal behavior of a cellulose I-ethylenediamine complex

We prepared highly crystalline samples of a cellulose I-ethylenediamine (EDA) complex by immersing oriented films of algal (Cladophora) cellulose microcrystals in EDA at room temperature for a few days. The unit-cell parameters were determined to be a = 0.455, b = 1.133, and c = 1.037 nm (fiber repeat) and gamma = 94.02 degrees. The space group was P2(1). On the basis of unit cell, density, and thermogravimetry analyses, the asymmetric unit is composed of one anhydrous glucose residue and one EDA molecule. The chemical and thermal stabilities of the cellulose I-EDA complex were also investigated by the use of X-ray diffraction. When the cellulose I-EDA complex was immersed in methanol or water at room temperature, cellulose III I or I beta was obtained, respectively. However, immersion in a nonpolar solvent such as toluene did not affect the crystal structure of the complex. The cellulose I-EDA complex was stable up to a temperature of approximately 130 degrees C, whereas the boiling point of EDA is 117 degrees C. This thermal stability of the complex is probably caused by intermolecular hydrogen bonds between EDA molecules and cellulose. When heated above 150 degrees C, the cellulose I-EDA complex decomposed into cellulose I beta.