Faculty Bibliography

The effects of elevated levels of GABA, glycine, or taurine on the rate of protein synthesis in plasma, brain, liver, and muscle of adult mice were measured in in vivo experiments after a flooding dose of labeled valine. Elevation of these amino acids caused hypothermia; keeping the animals in an incubator maintained physiological body temperature. The increase in GABA or glycine did not affect the rate of protein synthesis in these tissues to a significant degree. The increase in taurine levels caused inhibition of valine incorporation in plasma, liver, and muscle, while brain protein synthesis was unaffected. When glycine was increased in brain, the uptake of labeled free valine in the brain was greater

Brain proteins of BALB/cBy mice were labeled for a period of 8 days by a single intraperitoneal injection of valine. Following this the mice received 10% ethanol and protein breakdown was estimated from the release of label from brain proteins. Ethanol intake resulted in a significant inhibition of cerebral protein breakdown in vivo as measured in whole brain and in subcellular brain fractions (myelin, synaptosomes, mitochondria, microsomes, and nuclei). The intake of 10% ethanol for 4.5 months resulted in minor alterations in amino acid levels; increase in some and decrease in others were observed in plasma and brain, but most of the changes were not significant (P greater than 0.05). The uptake of AIB in brain was decreased 17% by prolonged ethanol intake

The supernatant obtained from mouse brain homogenates contains material that inhibits the saturable binding of [3H]nicotine in mouse cerebral cortex. This inhibitory material was further purified by heat denaturation, ultrafiltration through an Amicon PM-10 membrane filter, and gel chromatography on Sephadex G-10. The material inhibited the binding of [3H]acetylcholine with the same potency as it did that of [3H]nicotine. It also had some affinity for the sites that specifically bind [3H]D-Ala, D-Leu enkephalin, but had much lower affinity for the binding sites for [3H]quinuclidinyl benzilate (QNB), [3H]spiroperidol, [3H]naloxone, or [3H]imipramine. Acid hydrolysis destroyed the activity. These preliminary results suggest the presence in brain of 'nicotinelike' substances, one of which may be the endogenous ligand for the sites that specifically bind [3H]nicotine

In measurements of high affinity transport in tissue slices, the incubation medium is often treated as an 'infinitely large pool'. External substrate concentrations, even at the micromolar level, are assumed to be constant and metabolic interactions between tissue and medium are neglected. In the present report we describe experiments in which glutamic and aspartic acid uptake by mouse brain slices were studied using techniques that could test these assumptions. Cerebral hemispheres were cut into 0.1 mm sections and about 90 mg of tissue incubated in 10 ml of oxygenated medium. After 45 minutes of equilibration, radioactive substrates were added and the concentrations and specific activities of the amino acids and their metabolites in the medium were determined. During the first 10 min following substrate addition, rapid decreases in glutamic and aspartic acid concentrations in the medium were accompanied by large decreases in specific activity caused by the continuous release of these amino acids from the tissue. In addition, extensive conversion of both substrates to glutamine and the preferential accumulation of this metabolite, in the medium, was found. These results demonstrate that metabolism and release occur simultaneously with uptake during transport experiments in vitro and that these processes can take place in specific tissue compartments. It is therefore necessary to measure the tissue and medium concentration levels of amino acids along with their radioactivity in such experiments, since all three processes (transport, metabolism, and compartmentation) are interrelated in the clearance of amino acids from the incubation medium and probably from the extracellular spaces in vivo as well

Significant positive correlation between the ligand binding values for beta-adrenergic receptors and those for muscarinic cholinergic receptors was found in five inbred strains of mice (r = 0.84) and in different vertebrate species (r = 0.99). Comparative analysis of analogous receptor studies done on various brain regions in other laboratories revealed high positive correlation between regional binding values for these two receptors. In conjunction with the work presented here, an overlap in the genetic determination of beta-adrenergic and muscarinic cholinergic receptors is suggested

Enkephalin-containing polypeptides derived from pro-enkephalin A, pro-enkephalin B, or pro-opiomelanocortin were inhibitors of enkephalin degradation by aminoenkephalinases purified from cytosol or membranes. Of the peptides, Argo-Met-enkephalin was the most potent inhibitor for the aminoenkephalinases, with an IC50 of about 0.6 microM, it was more effective than bestatin (IC50 = 0.8-1.0 microM). This inhibition was partly due to substrate competition. Argo-Met-enkephalin was hydrolyzed by aminoenkephalinases to form Arg, Tyr, and Gly-Gly-Phe-Met in a substrate-inhibited manner. The hexapeptide also inhibited the breakdown of Arg- and Tyr-beta-naphthylamide by the membrane aminoenkephalinase. Since Argo-Met-enkephalin did not inhibit leucine aminopeptidase, it was a more selective inhibitor than bestatin of Met-enkephalin breakdown by aminopeptidases. Argo-Met-enkephalin inhibited enkephalin breakdown by synaptosomal plasma membranes but not by brain slices. Our data suggest that in addition to their possible role as opioids, the enkephalin-containing polypeptides may be regulators of enkephalin levels

In our studies on newly synthesized compounds for their potential analgesic effect, we decided for purposes of convenience and economy to investigate non-mammalian sources for the presence of enkephalin degrading enzymes. An aminopeptidase that catalyzes the hydrolysis of the tyrosylglycyl bond of leucine- and methionine enkephalin was purified from the mesophiles Pseudomonas sp ATCC 11299a (Ps) and Chromobacterium violaceum ATCC 12540 (Cv). Each preparation also hydrolyzed to varying extents neutral dipeptides, tripeptides, tetrapeptides and amino acid beta-naphthylamides. The Ps enzyme has a pH optimum of 6.8, Km of 80 microM and a Vmax of 6.7 nmoles/min/mg of protein. The Cv enzyme has a pH optimum of 6.8-7.2, Km of 111 microM and a Vmax of 42 nmoles/min/mg of protein. Both are sulfhydryl enzymes since they are activated by dithiothreitol (DTT) and inactivated by p-chloro- and p-hydroxymercuribenzoate. They are not glycoproteins since they pass unretained through a Con A-Sepharose column. The activity lost by dialysis against EDTA can be restored, wholly or in part, by Co+2, Mg+2, Mn+2 and Ni+2; ions exerting an inhibitory effect were A1+3, Cd+2, Cu+2, Hg+2 and Zn+2. From a range of organic compounds, the greatest inhibition was elicited by the microbial peptides amastatin and bestatin. Several dipeptide analogues of bestatin, synthesized from DL-threo-2-amino-3-hydroxy-3-phenylpropanoic acid (AHPP) as the N-terminal residue in order to define the stereospecific requirements of the alpha, beta-functional groups for maximal activity, were not as active as the parent compound