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Biomedical subjects

R Roskoski

Publications and source records attributed to R Roskoski.

At least 73 records · Page 4Linked to original sources
PubMed1980

Adenosine cyclic 3',5'-monophosphate dependent protein kinase: active site directed inhibition by Cibacron Blue F3GA.

Cibacron Blue F3GA, the polycyclic blue chromophore of Blue Dextran 2000, inhibits the catalytic subunit of bovine brain protein kinase. The rate of inactivation exhibits a hyperbolic dependence on the dye concentration. This suggests that an enzyme-dye complex forms prior to inactivation. Protein and peptide substrates or MgATP protects the enzyme against dye inactivation. Kinetic measurements show that the dissociation constant is approximately 100 micro M and the maximal rate of inactivation is 0.13 min-1 at 22 degree C. Inactivation is temperature and time dependent. Exhaustive dialysis, gel filtration, or the addition of substrate fails to reactivate inhibited enzyme. The failure to reverse the inhibition suggests that the dye forms a covalent complex with the enzyme. Denaturation by sodium dodecyl sulfate also fails to dissociate the dye from enzyme. The hyperbolic kinetics, moreover, suggest that the dye functions as an active site directed reagent. The holoenzyme is resistant to Cibacron Blue inactivation. Addition of cAMP converts the enzyme to a form susceptible to inhibition. In agreement with our previous studies, these results also suggest that the regulatory subunit shields, either physically or functionally, the active site of the catalytic subunit.

Animals↗
PubMed1979

Net uptake of aspartate by a high-affinity rat cortical synaptosomal transport system.

Reuptake of neuroactive amino acids by high affinity transport systems (Km approximately 10 micrometer) is thought to terminate the action of these substances. Since homoexchange can complicate the interpretation of uptake experiments, it is necessary to demonstrate net inward transport of neuroactive amino acids before uptake can be considered as a likely mechanism for transmitter inactivation. When rat cortical synaptosomes are incubated with 10 micrometer [14C]L-aspartate, net (chemical) and apparent (radioactive) uptake into the synaptosomal fractions are equivalent. Although there is net aspartate uptake into the synaptosome fraction, aspartate exchange can be demonstrated in a variety of conditions. Net uptake exhibits the characteristics of high-affinity transport systems including Na+-and temperature-dependence. Furthermore, KCl (or RbCl)--1 micrometer--are required for net uptake but not radioactive or apparent uptake. LiCl, NH4Cl, CsCl, and choline chloride fail to support net uptake. Ouabain (0.1 micrometer) inhibits net uptake to a greater extent than apparent uptake. Although glutamate inhibits aspartate uptake (and vice versa), the net uptake of the combination is greater than that of each alone. The demonstration of net uptake of aspartate by a high-affinity system is consonant with the idea that this system may play a role in its inactivation in the synaptic region.

Animals↗
PubMed1979

Choline acetyltransferase activity in rat and guinea pig heart following vagotomy.

Choline acetyltransferase (CAT), which serves as a marker for the preganglionic and postganglionic parasympathetic cholinergic neurons in heart, was measured in heart regions in rats and guinea pigs after either a right or a left unilateral cervical vagotomy or a sham operation. One day after a left vagotomy in rat, CAT activity was decreased in the region of the SA and AV nodes. In rats killed on the 2nd, 4th, and 8th day after left vagotomy, the activity of CAT in these regions increased to the control level. CAT activity in the remainder of the heart did not decrease. In guinea pig 1 wk after a left vagotomy, CAT activity was decreased in the anterior interventricular septum but not in other regions. Selective increases in enzyme activity were noted in the right atrial appendage following left vagotomy and in the superior interventricular septum following right vagotomy. These results suggest that compensatory increases in CAT activity occur that may be related to collateral sprouting similar to that observed in the frog heart.

Animals↗
PubMed1978

Choline acetyltransferase activity in rat heart after transplantation.

Choline acetyltransferase (CAT) catalyzes the biosynthesis of acetylcholine according to the chemical equation: Acetyl-CoA + Choline in equilibrium Acetylcholine + CoA. To demonstrate the neuronal relationship of this enzyme, CAT activity was measured in hearts to which the extrinsic innervation was completely interrupted by extirpation of the heart from a donor animal and transplantation in a heterotopic position into the recipient's abdominal cavity. After the animals were killed at 8 days, enzyme activity in the transplanted hearts was measured and compared with that of the recipient. Choline acetyltransferase activity decreased 98% in the right atrium of the transplanted denervated hearts, 94% in the SA node, 87% in the left atrium, 80% in the right ventricle, and about 50% in the interventricular septum and left ventricle. These data show that sectioning the extrinsic parasympathetic innervation to the heart results in variable decreases in CAT activity, a finding which confirms that CAT activity is related to the extrinsic cholinergic innervation. The extrinsic (preganglionic) component of the parasympathetic system is nonuniform and supplies predominantly the specialized regions of the heart with conduction tissue. The residual enzyme activity is related to intrinsic (postganglionic) parasympathetic neurons. The intrinsic component is uniformly distributed to both specialized and contractile regions of the heart.

Animals↗
PubMed1978

Regional choline acetyltransferase activity in the guinea pig heart.

Choline acetyltransferase is the enzyme that catalyzes the biosynthesis of acetylcholine, the neurotransmitter of the pre- and postganglionic parasympathetic system. To assess the extent of parasympathetic innervation, enzyme activity was measured in specialized and contractile regions throughout the guinea pig heart. Enzyme activity in the right atrial appendage was 137 nmol g(-1) hr(-1). Activity was greatest in the region of the sinoatrial node (187 nmol g(-1) hr(-1). Also, enzyme activity was high in the regions of the atrioventricular node (153 nmol g(-1) hr(-1), the proximal conduction bundles (133 nmol g(-1) hr(-1), and the base of the anterior papillary muscle of the right ventricle (179 nmol g(-1) hr(-1), which contains the moderator band and Purkinje fibers. In contrast, the enzyme activity in the inferior interventricular septum and the free walls of the right and left ventricles, which are more predominantly contractile tissue, was 67 +/- 6,108 +/- 14, and 56 +/- 11 nmol g(-1) hr(-1), respectively. This activity is significantly lower than in the right atrial appendage. These results suggest that the density of parasympathetic innervation is similar in all the components of the conduction system, from the sinoatrial node to Purkinje tissues. Furthermore, the parasympathetic innervation of regions specialized for conduction is up to four times more dense than that of contractile regions.

Animals↗
PubMed1977

Net uptake of gamma-aminobutyric acid by a high affinity synaptosomal transport system.

Reuptake of gamma-aminobutyric acid (GABA) by a high affinity transport system in nerve endings in the central nervous system is thought to terminate the action of this postulated neurotransmitter. This hypothesis has been challenged since the demonstration of exchange between synaptosomal and exogenous GABA (G. Levi and M. Raiteri, Nature 250: 735, 1974). In our studies, rat cortical synaptosomes were incubated (25 degrees C) in various media containing 10 muM 14C-GABA. After the synaptosomes were removed by centrifugation, 14C and total GABA (fluorometric assay) in the resulting supernatant were measured. Uptake of labeled GABA, detected by a decrease in medium radioactivity, is Na+- and K+-dependent. Net GABA uptake, however, does not parallel 14C-GABA translocation. Exchange accounts for 20 to 70% of radiolabeled GABA accumulation depending upon the experimental conditions. On the other hand, GABA-deficient synaptosomes (prepared by treatment with 56 mM KCl and 1 mM CaCl2) show equivalent net and radiolabeled GABA uptake in Ringer's solution containing 1 to 4 mM KCl and 60 to 150 mM NaCl (average 4.6 nmol of GABA accumulated per mg of synaptosomal protein). Net and 14C-GABA uptake by GABA-deficient synaptosomes are identical at various pH values (6.0-8.5), synaptosomal protein concentrations (0.4-3.5 mg/ml) and temperatures (5-37 degrees C). Although GABA homoexchange may contribute significantly to radiolabel accumulation by synaptosomes containing higher GABA levels (9.5-9.9 nmol/mg), homoexchange is limited in GABA-depleted synaptosomes. Our results are consistent with the proposal that presynaptic GABA capture by a high affinity system in vivo may terminate the action of this neuroactive amino acid.

Aminobutyrates↗
PubMed1975

Human brain and placental choline acetyltransferase: purification and properties.

Choline acetyltransferase (EC 2.3.1.6) catalyzes the biosynthesis of acetylcholine according to the following chemical equation: acetyl-CoA + choline in equilibrium to acetylcholine + CoA. In addition to nervous tissue, primate placenta is the only other animal source which contains appreciable acetylcholine and its biosynthetic enzyme. Human brain caudate nucleus and human placental choline acetyltransferase were purified to electrophoretic homogeneity using ion-exchange and blue dextran-Sepharose affinity chromatography. The molecular weights determined by Sephadex G-150 gel filtration and sodium dodecyl sulfate gel electrophoresis are 67000 plus or minus 3000. N-Ethylmaleimide, p-chloromercuribenzoate, and dithiobis(2-nitrobenzoic acid) inhibit the enzyme. Dithiothreitol reverses the inhibition produced by the latter two reagents. The pKa of the group associated with N-ethylmaleimide inhibition is 8.6 plus or minus 0.3. A chemically competent acetyl-thioenzyme is isolable by Sephadex gel filtration. The enzymes from the brain and placenta are thus far physically and biochemically indistinguishable.

Acetyltransferases↗