ADP-Ribosylation of Membrane Proteins in Cholinergic Nerve Terminals

Abstract: Lysed Torpedo synaptosomes or washed synaptosomal membranes were incubated with [³²P]NAD⁺ and subjected to electrophoresis on SDS-polyacrylamide gels. More than eight membrane proteins were ADP-ribosylated. The most intensely labeled proteins were those of M_r= 62,000 and 82,000. Radiolabeling was more intense in synaptosomes than in other subcel-lular fractions. Cholera toxin caused ribosylation of additional synaptosomal proteins with M_r= 42,000 and (in some preparations) 49,000. Neither endogenous nor cholera toxin-catalyzed ADP-ribosylation required added guanyl nu-cleotides. Cholera toxin increased the adenylate cyclase activity of synaptosomal membranes, suggesting that the cholera toxin substrates are regulatory components of adenylate cyclase in these synaptosomes.     

Influence of Calcium on Dopamine Synthesis and Tyrosine Hydroxylase Activity in Rat Striatum

Abstract: We have investigated three aspects of the relationship between calcium and tyrosine hydroxylase activity in rat striatum. In the first series of experiments, we examined the hypothesis that the rise in dopamine synthesis during increased impulse flow results from a calcium-induced activation of tyrosine hydroxylase. Calcium (12.5–200 μ M ) had no effect when added to crude enzyme or enzyme partially purified by gel filtration. Moreover, incubation of synaptosomes with excess calcium (up to 3.5 m M ) had little or no effect on dopamine synthesis. Incubation with the depolarizing alkaloid veratridine (75 μ M ) did increase dopamine synthesis, but did not alter the activity of tyrosine hydroxylase subsequently prepared from the synaptosomes, despite the presumed rise in intracellular calcium. In the second series we examined the hypothesis that increased dopamine synthesis after axotomy results from activation of tyrosine hydroxylase owing to a decrease in intracellular calcium. Addition of the calcium chelator EGTA (100 μ M ) to crude or partially purified enzyme was without effect, whereas incubation of synaptosomes with EGTA (500 μM ) decreased cell-free enzyme activity. In the third experimental series we examined the relationship between calcium and activation of tyrosine hydroxylase by dibutyryl cyclic AMP. EGTA failed to alter the increase in the activity of tyrosine hydroxylase prepared from synaptosomes incubated with dibutyryl cyclic AMP. However, it blocked the increase in synaptosomal dopamine synthesis and dopamine content normally produced by the cyclic AMP analogue. Thus, tyrosine hydroxylase does not appear to be activated by either increases or decreases in calcium availability. However, calcium may be important for the maintenance of basal tyrosine hydroxylase activity, and may play an indirect role in the expression of tyrosine hydroxylase activation produced by other means.     

Measurement of Methionine-Enkephalin [Arg6,Phe7] in Rat Brain by Specific Radioimmunoassay Directed at Methionine Sulphoxide Enkephalin[Arg6,Phe7]

Abstract: A specific and sensitive radioimmunoassay procedure for Metenkephalin[Arg⁶,Phe⁷] which allows its measurement in regions of the rat brain is described. The antiserum was raised against the methionine sulphoxide derivative of the peptide, and all samples and standards were oxidized with hydrogen peroxide prior to use in the assay with chloramine T-oxidized ¹²⁵I-labelled Met(O)-enkephalin[Arg⁶,Phe⁷]. The only significant cross-reactivity was 30% with the reduced heptapeptide Met-enkephalin[Arg⁶,Phe⁷]. The assay showed less than 0.15% cross-reactivity with fragments of the heptapeptide and with leucine-enkephalin-containing peptides. Acid acetone extraction of rat striatum followed by Sephadex G-50 chromatography and reverse-phase high pressure liquid chromatography showed that essentially all immunoreactivity co-chromatographed with Met-enkephalin[Arg⁶,Phe⁷]. This confirmed the specificity of the assay and showed that the striatum does not contain a high concentration of larger molecular weight forms with the heptapeptide at the COOH terminus. Distribution of the heptapeptide followed that of methionine enkephalin, with highest concentrations in the globus pallidus, intermediate levels in caudate-putamen and hypothalamus, and low levels in cortex and cerebellum.     

Active sulfate absorption in rabbit ileum: Dependence on sodium and chloride and effects of agents that alter chloride transport

Summary Unidirectional fluxes of³⁵SO₄ across and into rabbit ileal epithelium were measured under short-circuit conditions, mostly at a medium SO₄ concentration of 2.4mm. Unidirectional mucosa (m)-to-serosa (s) ands-to-m fluxes (J _ms,J _sm) were 0.456 and 0.067 moles hr^–1 cm^–2, respectively.J _ms was 2.7 times higher in distal ileum than in mid-jejunum. Ouabain abolished net SO₄ transport (J _net) by reducingJ _ms. Epinephrine, a stimulus of Cl absorption, had no effect on SO₄ fluxes. Theophylline, a stimulus of Cl secretion, reducedJ _ms without affectingJ _sm, causing a 33% reduction inJ _net. Other secretory stimuli (8-Br-cAMP, heat-stable enterotoxin, Ca-ionophore A23187) had similar effects. Replacement of all Cl with gluconate markedly reducedJ _net through both a decrease inJ _ms and an increase inJ _sm. The anion-exchange inhibitor, 4-acetoamido-4-isothiocyano-2,2-sulfonic acid stilbene (SITS), when added to the serosal side, reducedJ _ms by 94%, nearly abolishingJ _net. SITS also decreasedJ _sm by 75%. Mucosal SITS (50 m) was ineffective. 4,4-diisothiocyano-2,2-sulfonic acid stilbene (DIDS) had effects similar to SITS but was less potent. Measurements of initial rates of epithelial uptake from the luminal side (J _me) revealed the following: (1)J _me is a saturable function of medium concentration with aV _max of 0.94 moles hr^–1 cm^–2 and aK _1/2 of 1.3mm; (2) replacing all Na with choline abolishedJ _me; (3) replacing all Cl with gluconate increasedJ _me by 40%; (4) serosal SITS had no effect onJ _me; and (5) stimuli of Cl secretion had no effect onJ _me or increased it slightly. Determination of cell SO₄ with³⁵SO₄ indicated that, at steady-state, the average mucosal concentration is 1.1 mmoles per liter cell water, less than half the medium concentration. Cell SO₄ was increased to 3.0mm by adding SITS to the serosal side. Despite net transport rates greater than 1.4 Eq hr^–1 cm^–2, neither addition of SO₄ to the SO₄-free medium nor addition of SITS to SO₄-containing medium altered short-circuit current. The results suggest that (1) ileal SO₄ absorption consists of Na-coupled influx (symport) across the brush border and Cl-coupled efflux (antiport) across the basolateral membrane; (2) the overall process is electrically neutral; (3) the medium-to-cell Cl concentration difference may provide part of the driving force for net SO₄ absorption; and (4) since agents affecting Cl fluxes (both absorptive and secretory) have little effect on SO₄ fluxes, the mechanisms for their transcellular transports are under separate regulation.     

Crystallization of alfalfa mosaic virus coat protein as a T = 1 aggregate

Reassembled alfalfa mosaic virus coat protein was partially digested with trypsin to remove the first 26 amino acids (Bol et al., 1974). These particles are empty icosahedral protein shells built with 60 alfalfa mosaic virus protein subunits. This aggregate has been crystallized in two different crystal forms, one of which diffracts X-rays to at least 3.4 Å resolution. The type I crystals (space group $\text{P6}{}_3\text{, a = 200}\text{A}\text{?}\text{, c = 314}\text{A}\text{?}$) contain two particles per cell separated by 195 Å with each sitting on a 3-fold axis. The type II crystals contain three particles per cell in space group P3₁or P3₂ ($\text{a = 201}\text{A}\text{?}\text{, c = 485}\text{A}\text{?}$). Other T = 1 viral particles have very similar diameters.     

Crystallization,crystal structure analysis and molecular model of the third domain of Japanese quail ovomucoid,a Kazal type inhibitor

The third domain of Japanese quail ovomucoid, a Kazal type inhibitor, has been crystallized and its crystal structure determined at 2.5 Å resolution using multiple isomorphous replacement techniques. The asymmetric unit contains four molecules. In the crystal the molecules are arranged in two slightly different octamers with approximate D4 symmetry. The molecules are held together mainly by interactions of the N-terminal residues, which form a novel secondary structural element, a β-channel.The molecule is globular with approximate dimensions 35 Å × 27 Å × 19 Å. The secondary structural elements are a double-stranded anti-parallel β-sheet of residues Pro22 to Gly32 and an α-helix from Asn33 to Ser44. The reactive site Lys18-Asp19 is located in an exposed loop. It is close to Asn33 at the N terminus of the helical segment. The polypeptide chain folding of ovomucoid bears some resemblance to other inhibitors in the existence of an anti-parallel double strand following the reactive site loop.     

Comparison of content with synthesis,uptake and breakdown of GABA in substantia nigra of unoperated,hemitransected and kainate-treated rats

The synthesis, high affinity uptake and degradation of GABA were compared with GABA content measured simultaneously in the nigras of unoperated, hemitransected and kainate-treated rats. In intact animals nigral GABA concentration was linearly related to glutamate decarboxylase activity, but not to GABA aminotransferase (GABA-T) or GABA uptake. One week after hemitransection all three parameters showed a linear reduction with GABA content, suggesting striatonigral GABA neurones may account for as much as 90% of the GABA and GAD, 31% of the GABA-T and 58% of GABA uptake sites in whole nigra. Nigral kainate treatment unexpectedly reduced all four parameters in parallel by as much as 50%, consistent with the formation of an unselective lesion.     

Improved high-affinity binding of 3H-apomorphine with a subcellular membrane fraction of mammalian brain tissue

The binding of low concentrations of [³H](?)apomorphine to preparations of calf and rat forebrain tissue was evaluated. Fractionation of crude homogenates to prepare a membrane fraction (P₄) of striatal or caudate homogenates increased the proportion of saturable to total binding from 33% to over 80%, and increased the apparent density of binding sites from 94 to 681 fmol/mg protein. Binding in calf caudate P₄ tissue was protein-dependent and optimal at pH = 7.0 to 7.5, and T = 20 to 25°C; at higher temperatures tissue binding sites appeared to degrade. The half-time of association and dissociation at 22°C were, respectively, 14.0 and 18.5 min; equilibration was complete in 60 min. Kinetic characteristics of high-affinity binding obtained from association and dissociation constants and from saturation isotherms were similar (K_d = 2.1 to 3.4 nM). The pharmacology of competition for ³H-APO suggests selectivity for dopamine-agonist interactions. These results indicate that the P₄ membrane preparation may be useful for the evaluation of dopamine-agonist binding sites or “receptors.”     

Location of antibiotic resistance determinants, copy control, and replication functions on the double-selective streptococcal cloning vector pGB301

Summary The physical map of the streptococcal cloning vector pGB301 (9.8 kb) has been extended by localizing the cleavage sites of restriction endonucleases AvaII, AvaI, BclI, BstEII, and PvuII. The latter four enzymes cleaved pGB301 at unique sites. Insertion of chromosomal DNA from Staphylococcus aureus strain 3Ar^–m^– into the single BstEII site of pGB301 led to inactivation of the plasmid's chloramphenicol resistance determinant. Twelve deletion derivatives of pGB301 were isolated either by in vitro manipulation of pGB301 or as spontaneous deletion mutants following transformation of Challis by mixtures of recombinant plasmids. The overlapping deletions which spanned a continuous sequence of 7.7 kb ranged in size from 0.3 kb to 4.1 kb and allowed to localize the chloramphenicol and MLS-resistance determinants, the copy control function, and the replication region on the physical map of pGB301. Plasmid pGB301 together with its deletion mutants constitutes a valuable tool for further molecular cloning experiments in streptococci.