Comparison of high-affinity binding of [3H]GABA to subcellular particles of rat brain and liver

The binding of [³H]GABA and retention of [¹⁴C]sucrose have been studied in freshly prepared synaptosomal-mitochondrial (P₂) fractions of rat cerebral cortex and liver using bicarbonate-buffered medium (containing 147 mEq/liter of N⁺), and in frozen/thawed crude membrane fractions of rat whole brain and liver using Na⁺-free Tris HCl medium. GABA-sensitive sites (GSS) and bicucul-line-methiodide-(BMI-) sensitive sites (BMI-SS) were defined as those amounts of [³H]GABA that were sensitive to the displacement by 10^–3 M unlabeled GABA or BMI. In the presence of added Na⁺, two high-affinity GABA-binding processes were detected in the P₂fraction of cerebral cortex. The lower-affinity process (likely related mainly to uptake sites) hadK _B10^–5 M,B _max for GSS3 nmol/mg protein, andB _max for BMI-SS0.5 nmol/mg protein, whereas the higher-affinity process (likely related to synaptic GABA receptors) hadK _B10^–7 M,B _max for GSS43 pmol/mg protein, andB _max for BMI-SS2 pmol/mg protein. Only the higher-affinity process was detected in the liver P₂ fraction and it hadK _B3.7×10^–8 M,B _max for GSS0.48 pmol/mg protein, andB _max for BMI-SS0.1 pmol/mg protein (i.e., about 1/100 and 1/20 the receptiveB _max values of cerebral cortex). This binding process of the liver P₂ fraction could represent sites involved in mitochondrial GABA transport. In Na⁺-free Tris HCl medium, high-affinity [³H]GABA binding appeared to exist in frozen/thawed membrane preparations of both brain and liver when data were expressed on a protein basis. However, this binding to liver membranes was not displaceable by 10^–3 M unlabeled GABA, and when these data were expressed on a weight basis and corrected for [³H]GABA present in trapped supernatant fluid of the pellets, no [³H]GABA binding was detected in the liver preparation.     

Properties of ecto-(inoganic) pyrophosphatase of nervous system cells in culture. Activation upon partial release of sialic acid from the cell surface.

Clonal line NN hamster astroblasts and clonal line N18 neuroblasts were treated with phospholipase C-free, protease-free, and hemolysin-free Clostridium perfringens sialidase, at a low level (5 X 10(-3) units/ml) so as to maintain cell intactness and to avoid spurious protein effects. A rapid, regular release of sialic acid was achieved. An approximately 9-fold increase in ecto-pyrophosphatase activity could be brought about by action of C. perfringens sialidase for 10 min. Since the sialidase preparations were employed at a level which gave a very low concentration of extraneous protein, and the preparations were free of demonstrable phospholipase C and protease activities, these effects appear to relate specifically to removal of cell surface sialic acid. Neutral p-nitrophenyl phosphatase was activated under the same conditions, but activity remained low compared with pyrophosphatase. Progress curves for activation of the two enzymes were dissimilar. Ecto-pyrophosphatase of NN and N18 cells had an absolute requirement for Mg2+ both before and after removal of cell surface sialic acid. In the presence of near optimum Mg2+ (5 mM), other divalent cations were inhibitory at a low level (10(-1)mM). The effect of Mg2+ concentration, as well as inorganic pyrophosphate concentration, upon ecto-pyrophosphatase activity was shown to obey Michaelis-Menten kinetics for the control activity and for the sialidase-enhanced activity of both cell types. Km for Mg2+ and for pyrophosphate remained constant upon ecto-pyrophosphatase enhancement by sialic acid removal; increase in enzymatic activity was accounted for entirely by an increase in Vmax.     

Brain UDPgalactose: ceramide galactosyltransferase Purification of a catalytically active protein obtained after proteolytic digestion.

A procedure for the purification of UDPgalactose--2-hydroxyacylsphingosine galactosyltransferase (EC 2.4.1.45) including detergent extraction, ion-excharge chromatography and proteolytic digestion was developed. The active fraction obtained by this procedure had about 100 times higher specific activity than microsomes. Enzymic activity resisted destruction by pronase treatment at 4 degrees C. Agarose gel chromatography indicated the presence of an enzyme-phospholipid-detergent complex with a molecular weight between 400 000 and 500 000. Intact phospholipids seemed to be required for full enzymic activity as evidenced by the drastic loss of activity upon treatment with phospholipase A or C.     

SUPEROXIDE DISMUTASE OF MAMMALIAN NERVOUS SYSTEM

Superoxide dismutase was assayed in portions of the nervous system (a) by inhibition of tetrazolium reduction by oxygen radicals, generated enzymically, and (b) by inhibition of tetrazolium reduction by oxygen radicals generated by oxidation of NADH in presence of phenazine methosulphate. Superoxide dismutase activity was found in beef brain, retina and adrenal medulla, as well as in brain, retina and lungs of adult and of newborn rats. Preliminary experiments with rats exposed to hyperbaric oxygen showed no alteration of enzyme activities in tissues of newborn and adult animals. The possible role of superoxide dismutase in the nervous system is discussed.     

Infectivity of phage P2 DNA in presence of helper phage

Summary Phenol extracted deoxyribonucleic acid of temperate bacteriophage P2 infects E. coli strains C and K 12 with about equal efficiency. Infection occurs only if the bacteria exposed to P2 DNA are simultaneously infected with a related helper phage. Deoxyribonuclease completely destroys the infectivity of the DNA extract. The kinetics of the development of competence and the dependence of the number of infectious units on the multiplicity of infection of helper phage are compared with those of the DNA system. The molecular weight of P2 DNA was determined by sedimentation in a sucrose density gradient to be 2.20±0.2x10⁷.     

Antibody response of young animals to bacteriophages of different immunological behaviour: Φ X 174 and T2

The antibody response of piglets to the two immunologically unrelated phages Φ X 174 and T2 was studied. Antibodies against both antigens were observed already on the third day after immunization. Some differences were noted in the immunological behaviour of the phages: They have different eliminating curves from the circulation of the immunized animal and different requirements for the thermolabile serum component of the complement or cofactor for the neutralizing reaction. This factor is necessary for neutralizing phage T2, but not for phage Φ X 174.     

Synthesis of virus deoxyribonucleic acid during abortive infection of simian cells by human adenoviruses

Rapp, Fred (Baylor University College of Medicine, Houston, Tex.), Lawrence A. Feldman, and Manley Mandel. Synthesis of virus deoxyribonucleic acid during abortive infection of simian cells by human adenoviruses. J. Bacteriol. 92:931-936. 1966.-Inoculation of green monkey kidney cells (GMK) with adenovirus types 2 or 12, under conditions where neither infectious virus was synthesized, resulted in an increase in the uptake of H(3)-thymidine into deoxyribonucleic acid (DNA). Extraction of the DNA from infected cells, followed by identification by isopycnic analysis in CsCl gradients, revealed the presence of virus DNA. Cells infected with adenovirus type 2 yielded DNA giving bands with peak densities of 1.699 g/ml [GMK DNA with 40 moles% guanine + cytosine (GC)] and 1.714 g/ml (adenovirus type 2 DNA with 55 moles% GC). Cells infected with adenovirus type 12 also yielded the GMK DNA and a band at 1.706 g/ml (adenovirus type 12 DNA with 47 moles% GC). The rate of synthesis of adenovirus type 2 DNA in KB cells (productive cycle) and in GMK cells infected only with adenovirus (nonproductive cycle) or with adenovirus and simian virus 40 (adeno-productive cycle) was not significantly different.