Solubilization and characterization of kainate receptors from goldfish brain

The binding of [3H]kainate to goldfish brain membrane fragments was investigated. Scatchard analysis revealed a single class of binding sites in Tris-HCl buffer with a Kd of 352 nM and a Bmax of 3.1 pmol/mg wet weight. In Ringer's saline, [3H]kainate bound with a Bmax of 1.8 pmol/mg wet weight and a Kd of 214 nM. Binding in Ringer's saline, but not Tris-HCl buffer, displayed positive cooperativity with a Hill coefficient of 1.15. The [3H]kainate binding sites were solubilized in Ringer's saline using the nonionic detergent n-octyl-beta-D-glucopyranoside. Approximately 30-50% of the total number of membrane-bound binding sites were recovered on solubilization. The Kd of [3H]kainate for solubilized binding sites was approximately 200 nM. The rank order of potency for glutamatergic ligands at inhibiting [3H]kainate binding was identical and the competitive ligands had similar Ki values in both membranes and solubilized extracts. In membrane preparations, [3H]kainate displayed a two component off-rate with koff values of 0.97 min-1 and 0.07 min-1; in solubilized extracts, however, only a single off-rate (koff = 0.52 min-1) was observed. The hydrodynamic properties of n-octyl-beta-D-glucopyranoside solubilized [3H]kainate binding sites was investigated by sucrose density centrifugation. A single well defined peak was detected which yielded a sedimentation coefficient of 8.3 S. The results presented in this report suggest that goldfish brain may provide an ideal system in which to study kainate receptor biochemistry.     

Protein metabolism in goldfish brain

Abstract— Protein metabolism of goldfish brain was studied in vivo by means of intraperitoneal or intracranial injections of [³H]leucine and compared with concomitant studies in the mouse. Heterogeneity of turnover values was observed. Long turnover times were seen relative to other organs examined. The free amino acid pools of goldfish brain were determined, and the fate of tritium from labelled leucine was followed at various times after injection. Following ‘chasing’ with large amounts of unlabelled leucine or protein inhibitors shortly after isotope injection, further incorporation was arrested, but examination of the labelled protein over a period of 2 weeks indicated a slow decay, similar to that seen without ‘chasing’. Possible use of ‘pulse-chase’ experiments in vivo in animals is discussed in relation to behavioural studies.     

Studies on gangliosides of goldfish brain

Abstract— Purified gangliosides from goldfish brain have been separated on thin layer chromatography. Incorporation of radioactivity into gangliosides from intracranially- injected [³H]galactose was not affected by concentrations of puromycin (170 μg intra- cranially) known to exert behavioural effects. Puromycin (500 μg intracranially) inhibited the incorporation of radiolabelled galactose into infant rat brain gangliosides, a result which confirms a previous report in the literature.     

Characterization of the nicotinic acetylcholine receptor isolated from goldfish brain.

We have studied the binding of alpha-bungarotoxin to a particulate fraction of goldfish brain enriched in synaptosomes. The binding is specific and saturable and exhibits the pharmacological properties of a nicotinic cholinergic receptor. Equilibrium binding measurements yield a single dissociation constant (KD) of 0.92 nM. Kinetic analysis revealed one association rate constant and two dissociation rate constants. Dissociation constants calculated from kinetic measurements were 1.9 nM and 12.5 pM. The toxin . receptor complex is readily solubilized in nonionic detergent. The isoelectric point of the toxin . receptor complex was found to be 5.00 +/- 0.01. Sedimentation velocity analysis in sucrose/H2O and sucrose/D2O gradients in conjunction with Sepharose 4B chromatography and diffusion experiments yielded a sedimentation constant of 11.45, a partial specific volume of 0.79 cm3/g for the toxin . receptor . detergent complex, and a molecular weight of approximately 340,000 for the toxin . receptor complex.     

Diversity of gene expression in goldfish brain

The sequence complexity of nuclear and polysomal RNA from goldfish brain and kidney was measured by RNA-driven hybridization reactions with single-copy [3H]DNA. At saturation, brain nuclear and polysomal RNA were complementary to 23.2 and 6.7% of the DNA probe, respectively. In contrast to these findings, nuclear and polysomal RNA from kidney hybridized to 16.1 and 3.1% of the single-copy DNA, values that were significantly lower than that obtained in the CNS. Taken together, the results focus attention on the striking diversity of gene expression in goldfish brain and extend to lower vertebrates the observation that nervous tissue expresses significantly more genetic information than other somatic tissues or organs.     

Biosynthesis of monosulfogangliotriaosylceramide and GM2 by N-acetylgalactosaminyltransferase from rat brain

Some properties of the enzyme activity that catalyzes the transfer of N-acetylgalactosamine from UDP-N-acetylgalactosamine to exogenous lactosylceramide-II3-sulfate (SM3) and N-acetylneuraminosyllactosylceramide (GM3) were studied using the enzyme preparation solubilized from the 100,000 X g pellet of 6-day-old rat brain. The products from SM3 and GM3 were identified as gangliotriaosylceramide-II3-sulfate (SM2) and N-acetylneuraminosylgangliotriaosylceramide (GM2), respectively, by TLC-autoradiography. Optimal conditions for both activities were similar: pH (Hepes-NaOH), 7.0-7.5; detergent (heptylthioglucoside), 0.64% and Mn2+, 5-10 mM. The concentrations of the detergent optimal for both enzyme activities were also examined at various concentrations of the acceptors. The lower the amounts of acceptors, the less the amounts of detergent that were required, and vice versa, for the maximum activities. The acceptor-saturation curve for SM2 synthesis was triphasic, exhibiting a sigmoidal region at lower concentrations, a hyperbolic region and finally a descending region. For GM2 synthesis, the curve was biphasic without the descending region. The donor-saturation curves were classical hyperbolic ones for both syntheses. The Km values calculated for SM3 and GM3 were 0.37 and 0.19 mM, respectively, when the data corresponding to the hyperbolic regions were used for the double-reciprocal plots. The Km values for UDP-N-acetylgalactosamine in the SM2- and GM2-synthesis were 82 and 26 microM, respectively. SM3 and GM3 were the best acceptors for this enzyme preparation. From the results of the acceptor competition study, it was suggested that the two synthetic reactions are catalyzed by a single enzyme.     

Rapidly-labelled, acidic phospholipids of the goldfish brain

Homogenates and particulate fractions of goldfish brain incorporated radioactivity from γ-[³²P]ATP selectively into acidic phospholipids during brief periods of incubation. Phosphatidate and lysophosphatidate became strongly labelled and activity was also found in phosphatidyl inositol phosphate and in phosphatidyl inositol diphosphate. When tetraphenylborate (a K⁺-complexing agent) was added, a selective stimulation of incorporation of ³²P into phosphatidate occurred. The addition of perchlorate (also known to bind K⁺) did not produce a similar stimulation, nor did the addition of K⁺ block the stimulation by tetraphenylborate. The stimulation of the labelling of phospholipids by tetraphenylborate appeared to be the result of multiple actions. Besides the evidence that it acted by stimulating the phosphoinositide phosphodiesterase of brain, data were obtained suggesting that it stimulated diglyceride kinase and blocked endogenous destruction of ATP as well. The stimulation by tetraphenylborate was blocked by addition of atropine but not of arecoline.     

Ependymins are expressed in the meninx of goldfish brain

Summary Ependymins are dimeric glycoproteins found in the extracellular fluid of goldfish brain. They were originally observed because of their enhanced turnover rates after learning. In this paper we present the first investigation concerning the expression of these secretory proteins in goldfish brain via in situ hybridization with synthetic oligonucleotides and cRNA probes. It is shown that ependymin-mRNAs are predominantly expressed in the meninx surrounding the brain and in an invaginated part of the meninx called the cavum cranii. These results have been confirmed by immunhistochemical analysis. This indicates that, in fish, the meninx synthesizes a major protein constituent of the cerebrospinal fluid; furthermore, this suggests that the functional sites of ependymins are removed from the place of their synthesis. Distribution between different compartments may be achieved via the open communication system of the perivascular spaces.     

Biosynthesis of phosphatidylserine in rat brain microsomes

1. Rat brian microsomes incorporated L-serine into phosphatidylserine in the presence of 2mM ATP. This reaction was stimulated 2-fold by the addition of phosphatidic acid (0.2 mM) and 5-fold by the addition of nickel (0.5 mM). 2. This phosphatidylserine synthesis was inhibited completely by p-hydroxymercuribenzoate (0.1 mM) and N-ethylmaleimide (1 mM), whereas the Ca2+-dependent phosphatidylserine synthesis was unaffected by these sulfhydryl reagents. 3. The specific activity of the ATP-Ni2+-dependent phosphatidylserine was increased more than 2-fold during active myelination, whereas the Ca2+-dependent system remained unchanged. 4. Preliminary data indicate that pyrophosphatidic acid (p,p'-bis(1,2-diacyl-sn-glycero-3-)pyrophosphate) is the immediate precursor of phosphatidylserine synthesis.     

Biosynthesis in vitro of disialosylneolactotetraosylceramide by a solubilized sialyltransferase from embryonic chicken brain

A sialyltransferase involved in the biosynthesis in vitro of LD1c (NeuAc alpha 2-8NeuGc alpha 2-3Gal beta 1-4Glc-NAc beta 1-3Gal beta 1-4Glc-Cer) has been characterized from 9 to 11-day-old embryonic chicken brains. The CMP-[14C]NeuAc:LM1(alpha 2-8)sialyltransferase (SAT-2) sedimented (75%) at the junction of 0.75 and 1.2 M on a discontinuous sucrose density gradient when still membrane bound. In addition to the biosynthesis of LD1c, the detergent-solubilized (0.4% Nonidet P-40) preparation also catalyzes the transfer of sialic acid to O-8 of sialic acid in GM3 to form GD3 (NeuAc alpha 2-8NeuAc alpha 2 - 3Gal beta 1 - 4Glc - Cer). Substrate inhibition studies indicated that these two reactions are probably catalyzed by the same enzyme, SAT-2. The kinetic parameters of SAT-2 activity were determined. The Km values were 70 and 63 microM with CMP-[14C]NeuAc and LM1, respectively, when the detergent-solubilized supernatant fraction was used as enzyme source. The (alpha 2-8)-linkage between the terminal and penultimate sialic acids was determined using nonradioactive CMP-NeuAc and [Ac-14C]LM1 as substrates (Higashi, H., and Basu, S. (1982) Anal. Biochem. 120, 159-164) for the enzyme, followed by identification of the permethylated [14C]sialic acid of the product by radioautography. At 0.5 mM N-ethylmaleimide, the SAT-2 activity was inhibited 50% whereas SAT-1 and SAT-3 activities (Basu, M., Basu, S., Stoffyn, A., and Stoffyn, P. (1982) J. Biol. Chem. 257, 12765-12769) remained uninhibited.     

Variable homeoviscous responses of different brain membranes of thermally-acclimated goldfish

The effects of thermal acclimation of goldfish upon the bulk fluidity of synaptic, mitochondrial and myelin membrane fractions of brain was determined using steady-state and differential polarised phase fluorimetry. Membrane fluidity decreased in the order, mitochondria>synaptic membranes>myelin. In each case membranes from cold-acclimated goldfish were more fluid than the corresponding membranes of warm-acclimated goldfish, though the adjustment of fluidity in each case was insufficient to compensate for the direct effects of the temperature difference. The extent of fluidity compensation was greatest in the mitochondrial fraction and least in the myelin fraction, indicating heterogeneous responses in different membrane-types. Steady-state and dynamic fluorimetric techniques provided identical estimates of the homeoviscous responses, indicating that despite its short-comings, the steady-state technique provided as good a measure of adaptive responses as the more complex and sophisticated technique.     

Stress induced biosynthesis of a 31 kd-glycoprotein in goldfish brain

The de novo protein synthesis in goldfish brain has been studied under defined stress conditions and after training in an active swimming task. One- and two-dimensional gel electrophoresis of the brain proteins previously labelled in vivo with 35S-methionine indicated that the synthesis of one distinct protein increases after stress but not after successful training. This protein is a glycoprotein with an apparent molecular weight of 31 kd and an isoelectric point of pH 5. It is discussed that the protein may be the ACTH precursor "pro-opiomelanocortin".