Leonard G. Davis

Identification of an endogenous peptide-ligand for the benzodiazepine receptor.

Abstract An inhibitor of 3H-diazepam binding with characteristics that distinguish it from the other endogenous ligands reported for the benzodiazepine receptor was obtained from bovine brain. After isolation by gel filtration and ion exchange chromatography, the inhibitory factor was found to be a weakly charged molecule of approximately 3000 daltons. Although heat stable, the activity of the factor can be destroyed by treatment with papain. This factor, presumably peptide in nature, inhibited 3H-diazepam binding competitively and in a concentration dependent fashion.

Opioid Peptides and Protein Phosphorylation

The identification and isolation of naturally occur ring neuropeptides with morDhine-like activity was first reported by Hughes (1975) and co-workers (HUGHES, et al, 1975). These compounds were named methionine-enkephalin and leucine-enkephalin and have the amino-acid sequences of Tyr-Gly-Gly-Phe-Met and Tyr-Gly-Gly-Phe-Leu, respectively. Subsequently, a 31 amino-acid peptide with similar pharmacological and biochemical properties was described (COX, et al, 1975; BRADBURv, et al, 1976; GUILLEMAN, et al, 1976). Sequential amino-acid analysis of this larger peptide, named s-endorphin, demonstrated a similarity to the C-terminal portion (#61-91) in the sequence of s-lipotropin (LI and CHUNG, 1976: see Figure 1). Using the mouse vas deferens, the guinea pig ileum, or the opiate receptor assays, it was shown that these neuropeptides act in vitro similar to alkaloid opiates like morphine (HUGHES, 1975; HUGHES, et al, 1975; LI and CHUNG, 1976; SIMANTOV and SNYDER, 1976; GRAF, et al, 1976). These endogenous opioidlike neuropeptides have been shown to interact differentially with the opiate receptors of brain (LORD, et al, 1976).

Distribution of endogenously phosphorylated proteins in subcellular fractions of rat cerebral cortex

The cerebral cortex from adult rats was separated into several subcellular fractions by using established methods of differential and sucrose density gradient centrifugation. Aliquots from each fraction were incubated with γ-32P-ATP, in the presence and absence of adenosine 3′,5′-monophosphate (cyclic AMP), and its protein constituents were separated by means of SDS-slab gel electrophoresis. Fractions containing nuclei, synaptosomes, myelin, microsomes, and soluble proteins each showed a characteristic pattern of protein staining and of endogenously phosphorylated proteins detected by autoradiography of the gels. Cyclic AMP-stimulated phosphorylation of proteins with MW 78K and 84K can serve as markers for membranes of synaptic origin, while cyclic AMP-independent phosphorylation of low-molecular-weight proteins (15K–20K) is characteristic of myelin. The finding of different phosphoproteins in various subcellular fractions may be related to the diversity of cellular functions known to be regulated by phosphorylative activity.

Endorphin-regulated protein phosphorylation in brain membranes

Abstract This study was initiated to determine whether opioid peptides exert direct effects on the phosphorylation of specific proteins in membranes from rat neostriatum. It was found that low concentrations of β-endorphin (0.1–10nM) inhibit the phosphorylation of specific proteins designated F and H (M.W. 47,000 and 10–20,000 respectively). In addition, β-endorphin produced an overall stimulation of phosphate incorporation into other membrane proteins, the phosphorylation of which is dependent on calcium ions. The stimulatory effects were blocked by naloxone, but the inhibitory effects were not. The regulation of membrane protein-phosphorylation by endorphins may constitute a biochemical mechanism mediating for some of the physiological affects of these peptides on neuronal function.

Selective changes in the phosphorylation of endogenous proteins in subcellular fractions from cyclic amp-induced differentiated neuroblastoma cells

The endogenous phosphorylation of specific proteins was studied in subcellular fractions from proliferating and cAMP-induced differentiated neuroblastoma cells. Fractions containing nuclear, membrane-bound, and cytosolic proteins were incubated with [γ-32P]ATP, in the presence and absence of added cyclic nucleotides. Phosphate incorporation into specific proteins was determined by slab-gel electrophoresis of sodium dodecyl sulfate-solubilized reaction products. Cytosol fractions from differentiated cells demonstrated a twofold increase in cAMP-dependent phosphorylation of a specific protein with apparent mol wt of 59,000 daltons and a comparable decrease in cAMP-independent phosphorylation of another protein (97,000). The nuclear fraction of differentiated cells showed an increase in the cAMP-independent phosphorylation of two nonhistone proteins (110,000 and 102,000). Membrane fractions from differentiated cells exhibited a differential decrease in endogenous phosphorylation of specific proteins. Selective alterations in the phosphorylation of specific proteins in various subcellular components may be important biochemical events associated with the increased levels of differentiated functions in neuroblastoma cells in culture.

Effects of decapitation-stress on the phosphorylation of cortical membrane proteins

The endogenous phosphorylation of membrane-bound proteins was studied in preparations from the cerebral cortex of rats sacrificed by immersion in liquid nitrogen or by decapitation. Compared to quick-frozen rats, samples from decapitated animals demonstrated a two-fold increase in 32P-phosphate incorporation into specific protein bands with apparent molecular weights of 56K, and 52K (designated E1 and E2) and a decreased incorporation into a phosphoprotein of 47K (designated F). The phosphorylation of two proteins (78K and 34K) in membranes from decapitated rats was found to be highly stimulated by exogenously added cyclic AMP. On the other hand, the phosphorylation of specific protein bands in preparations from quick frozen rats was minimally affected by addition of cyclic AMP. The results indicate that conditions which lead to increases in cyclic AMP levels in the brain in situ induce specific changes in phosphorylative activity, and these can be detected by assaying isolated membrane fragments in vitro.

The metabolism and structure of phosphoglycoproteins in rat brain

Glycoproteins that contain phosphohexosyl groups were found to be present in the myelin- and synaptosomal-enriched fractions as well as in the microsomes of rat brain. The kinetics of flow of intraperitoneally injected [32P]phosphate suggests that the phosphate is enzymatically added in structures found in the microsomal fraction. The newly synthesized phosphoglycoproteins then appear in the soluble fraction of the synaptosomes and in the cytosol, prior to incorporation into the membranes of the synaptosomes and myelin. Phosphoglycopeptides recovered from the phosphoglycoprotein contain 3 Mannose units per N-acetylglucosamine residue; one of the mannose residues is phosphorylated. [13C]NMR studies indicate that the phosphoglycopeptides contain a chitobiose group and more than four sugar residues. Thus, the phosphomannoglycopeptides from rat brain contain an average of 2 N-acetylglucosamine, 6 mannose, and two phosphate moieties per oligosaccharide chain. Enzymatic treatment with α-mannosidase failed to remove the phosphomannose, although some mannose residues were released. Thus, the phosphorylated mannose is not removed by the glycosidase and terminal nonphosphorylated mannose residues are present in the oligosaccharide. The phosphate residues are removed by treatment with alkaline phosphatase.

Glycoprotein catabolism in brain tissue in the lysosomal enzyme deficiency diseases.

Glyocoproteins of brain tissue contain two major classes of heteropolysaccharide side chain, both of which are attached to the polypeptide chain of the protein moiety by means of an alkali-stable beta-aspartylglycosamine linkage (1). These carbohydrate-rich structures are readily liberated as soluble glycopeptides upon treatment of the denatured glycoproteins with proteolytic enzymes such as papain or pronase. The acidic sialoglycopeptides thus released account for approximately 65% of the glycoprotein-carbo-hydrate in brain tissue. These substances contain N-acetylneuraminic acid, galactose, mannose, N-acetylglucosamine, and fucose; some of these heteropolysaccharide polymers also contain sulfate-ester groups. A hypothetical structure for these glycopeptides is depicted (Fig. 1).

Inhibition of [3H]diazepam binding by an endogenous fraction from rat brain synaptosomes

Borkman, R. F., Dalrymple, A,, Lerman, S. (1977) Photochem. Photobiol. 26: 129-132 Davies, A. K., Navaratnam, S. , Phillips, G. 0. (1976) J. Chem. Soc. Perkin Trans. 2: 25-27 Foote, C . S. (1976) in: Pryor, W. A. (ed) Free Radicals in Biological Systems, Vol 11. Academic Press, New York, chapter 3, pp 69--145 Guilbault, G. G. (1973) Practical Fluorescence. Dekker, New York, pp 23, 599 Kurzel, R., Wolbarsht, M., Yamanachi, B., Staton, G., Borkman, R. F. (1973) Nature (London) 241: 132133 Magnus, I. A. (1976) Dermatological Photobiology. Blackwell, Oxford, pp 213-215 McCormick, J. P., Thornason, T. (1978) J. Am. Chem. SOC. 100: 312-313 Merkel, P. B., Kearns, D . R. (1972) Ibid. 94: 10291030 Moore, D. E., Tamat, S. R . (1980) J. Pharm. Pharmacol.

Hemodialyses and Schizophrenia Effects of Hemodialyses on Schizophrenic Symptoms and Dialysate Endorphin Levels

The use of hemodialyses in schizophrenia can be traced back to 1960 when a group of researchers in Switzerland noted improvement in three of five acute schizophrenic patients following hemodialyses (Thoelen et al., 1960; Feer et al.,1960). In 1977, Wagemaker and Cade in a preliminary study reported remission in chronic schizophrenic patients following weekly hemodialyses (Wagemaker and Cade, 1977). The encouraging results of these uncontrolled studies have generated much speculation among some psychiatrists, and the effect of hemodialyses on schizophrenia has become a controversial issue. The notion that schizophrenic patients may benefit from hemodialyses is intriguing and has prompted several groups of researchers to embark on controlled studies to assess the effects of hemodialyses on schizophrenic patients.