Chemical modification by diethylpyrocarbonate of an essential histidine residue in 3-ketovalidoxylamine A C-N lyase

3-Ketovalidoxylamine A C-N lyase of Flavobacterium saccharophilum is a monomeric protein with a molecular weight of 36,000. Amino acid analysis revealed that the enzyme contains 5 histidine residues and no cysteine residue. The enzyme was inactivated by diethylpyrocarbonate (DEP) following pseudo-first order kinetics. Upon treatment of the inactivated enzyme with hydroxylamine, the enzyme activity was completely restored. The difference absorption spectrum of the modified versus native enzyme exhibited a prominent peak around 240 nm, but there was no absorbance change above 270 nm. The pH-dependence of inactivation suggested the involvement of an amino acid residue having a pKa of 6.8. These results indicate that the inactivation is due to the modification of histidine residues. Substrates of the lyase, p-nitrophenyl-3-ketovalidamine, p-nitrophenyl-alpha-D-3-ketoglucoside, and methyl-alpha-D-3-ketoglucoside, protected the enzyme against the inactivation, suggesting that the modification occurred at or near the active site. Although several histidine residues were modified by DEP, a plot of log (reciprocal of the half-time of inactivation) versus log (concentration of DEP) suggested that one histidine residue has an essential role in catalysis.     

Immunohistochemical studies of the serotonergic supraependymal plexus in the mammalian ventricular system, with special reference to the characteristic reticular ramification

Distributional and morphological features, especially characteristics of the ramification of serotonin-containing supraependymal fibers (SEF), were studied in the ventricular systems of mammals (mouse, rat, guinea pig, rabbit, cat, dog, monkey) by means of a modified peroxidase antiperoxidase technique, using antiserotonin antiserum prepared in our laboratory. SEF were present in all ventricular systems, except on the third ventricle floor and in the choroid plexus. The density of SEF was higher in the smaller species. In the rat, light- and scanning electron microscopical SEF were almost completely abolished 1 week after intraventricular administration of 5,6-dihydroxytryptamine. Ramification of SEF was complicated; the SEF formed a true network with frequent anastomosing. In the ventricular system of rats rendered hydrocephalic by kaolin administration, the mode of axonal branching in the supraependymal plexus could best be analyzed by the scanning electron microscope because the meshes of the plexus were spread out.     

Characteristics of a Urate-Degrading Diamine Oxidase-Peroxidase Enzyme System in Soybean Radicles

The cadaverine content of soybean radicles showed a maximumpeak 3–4 days after planting. The variation coincidedwith radicle uricase activity during seed germination. The uricase activity could not be fractionate when the bufferpH for the extraction was at 6.0. The addition of 1 M KCl orNaCl to the buffer allowed the extraction of the uricase activity,but an addition of 1 M MgCl₂ or BaCl₂ inhibited this enzyme'sactivity. The urate-degrading enzyme system was purified 248-fold permilligram of protein from soybean radicles. The respective K_mvalues of the diamine oxidase activity for cadaverine and ofthe urate-degrading activity for hydrogen peroxide and uratewere 1.25, 2.93 and 50.3 µM. Analysis by gel electrophoresisof the partially purified enzyme fraction revealed that theurate-degrading enzyme system consisted of a peroxidase thatdegrades urate with hydrogen peroxide and a diamine oxidasethat releases hydrogen peroxide. These data are evidence that a urate-degrading diamine oxidaseand peroxidase system exists in soybean radicles and that thereaction rate of urate-degradation is controlled by the concentrationof cadaverine. (Received November 28, 1984; Accepted April 8, 1985)     

Differentiation of Photorespiratory Activity between Mesophyll and Bundle Sheath Cells of C4 Plants II. Peroxisomes of Panicum miliaceum L.

Peroxisomes were isolated by sucrose density gradient centrifugationfrom mesophyll and bundle sheath protoplasts of a C₄ plant,Panicum miliaceum L. The equilibrium density in the gradientwas 1.25 for bundle sheath peroxisomes and 1.23 for mesophyllperoxisomes, the former density being similar to that of peroxisomesof wheat mesophyll protoplasts. Photorespiratory and other microbody enzymes were assayed forthe peroxisomes of P. miliaceum to detect possible differentiationat an enzyme level. The specific activities of photorespiratoryenzymes, except for hydroxypyruvate reductase, in bundle sheathperoxisomes were 40–60% of those in wheat peroxisomes,when compared on a protein basis, and only 20–30% in mesophyllperoxisomes. However, peroxisomes from both cell types containedsignificant levels of all the enzymes involved in the photorespiratoryglycolate pathway, when compared with castor bean glyoxysomes.The activity of hydroxypyruvate reductase in the peroxisomesof P. miliaceum was comparable to or higher than that in wheatperoxisomes. Two ß-oxidation enzymes and urate oxidasewere detected in the peroxisomes in a similar level to thatin wheat peroxisomes. These results suggest that the peroxisomes of mesophyll andbundle sheath cells of P. miliaceum are essentially similarto those of C₃ plants, and that they cannot be differentiatedexcept for a difference in equilibrium density in a sucrosegradient. (Received December 24, 1984; Accepted April 9, 1985)     

Properties of porcine platelet myosin. II. Shape change of the molecule

Porcine platelet myosin molecules were examined by electron microscopy for changes in their shape. At high ionic strength, the molecules were morphologically indistinguishable from skeletal muscle myosin, except for a slight difference in the bent regions of their tails. At physiological ionic strength, however, the following important difference was observed between the two myosins. Unlike skeletal muscle myosin, the filaments of nonphosphorylated platelet myosin could be disassembled by stoichiometric ATP into a monomeric form with sharply bent or folded tail, and reassembled after ATP hydrolysis. Similar disassembly changes could be induced by various nucleotide triphosphates (CTP, GTP, ITP, and UTP) and to a lesser extent by ADP, AMP, and AMPPNP. These results suggest that ATP binds to the hydrolytic sites in platelet myosin molecule and induces the molecular shape change.     

Studies on cation-induced membrane vesicle aggregation of porcine intestinal brush borders

Protein alterations during the development of the mouse brain were studied by two-dimensional gel electrophoresis. A protein spot with a molecular weight (MW) of 68,000 and pI value of 5.6 was found in the brain of the 11th day of gestation. Between the 12th and the 14th day of gestation, spots with the same MW and lower pI values appeared progressively. Neuraminidase digestion converted the pI of these acidic spots to 5.6. Thus, increased sialylation appeared to occur during this period. This class of molecules became hardly detectable on the 15th day, and disappeared completely after the 16th day. Analogous spots were present in the heart, liver, and stomach of the embryos, although the increased sialylation was not observed in the liver. No adult organs so far examined showed these spots. On the other hand, two polypeptides (MW 55,000, pI 4.7, and 53,000, pI 4.6) appeared in the brain on the 13th day of gestation and persisted throughout the fetal period. After birth, they became hardly detectable. Furthermore, a spot (MW 48,000, pI 4.8) became newly detectable in the brain 4-5 weeks after the birth.     

Do protozoa conceal a high potency of nucleoside triphosphate hydrolysis present in Toxoplasma gondii?

ATP hydrolytic activity in whole cell homogenates of some protozoa was assayed in the presence or absence of dithiothreitol. The activities in all protozoan cell homogenates, except Toxoplasma gondii, ranged from 0.6 to 32 mumol/mg protein/hr, irrespective of the presence or absence of dithiothreitol. A remarkably higher activity, 11,690 mumol/mg protein/hr, was observed for T. gondii in the presence of dithiothreitol. These results indicate that the higher ATP hydrolytic potency observed for T. gondii is not universal to protozoa, rather it is unique to T. gondii.