TWO PROTEINS PURIFIED FROM LOBSTER NERVE EXTRACT : ISOLATION AND PHYSICOCHEMICAL CHARACTERIZATION

1. A protein component, fraction B, of lobster nerve extracts has been isolated and purified by differential ultracentrifugation and precipitation with zinc acetate. 2. Physicochemical data obtained from this protein and from fraction C are summarized. 3. Fraction B is present in lobster nerve extracts in higher concentration (relative to fraction A) than in blood. 4. A second component, fraction C, of sedimentation constant S₂₀^o = 13.2 has been isolated from lobster nerve extracts.     

Studies of nerve proteins; isolation and physicochemical characterization of a protein from lobster nerve

1. A procedure is described by which several protein constituents may be obtained from extracts of lobster claw nerves. One of these fractions, designated fraction A, representing 10 per cent of the total non-dialyzable material of the extracts, has been obtained in relatively (85 per cent) pure form. 2. This component has been characterized with respect to its physicochemical properties, particle shape, dimensions, and absorption spectrum.     

Resolution of the methane mono-oxygenase of Methylococcus capsulatus (Bath) into three components. Purification and properties of component C, a flavoprotein.

1. Ion-exchange chromatography resolves the methane mono-oxygenase from soluble extracts of Methylococcus capsulatus (Bath) into three fractions. 2. Fractions A and B are comparatively stable at 0 degrees C, whereas fraction C is very unstable unless kept in the presence of sodium thioglycollate (1-10 mM) or dithiothreitol (5-10mM). 3. The active component from fraction C was purified some 80-fold. 4. Purified component C has mol. wt. 42000. Its solutions are yellow with absorption maxima at 270 and 465 nm and a shoulder at 395 nm. The 465 nm peak is abolished by reduction with NADH or sodium dithionite, or by photoreduction in the presence of EDTA. A new spectral species, probably a neutral flavin semiquinone, is observed on partial reduction of component C. 5. No copper was detected in samples of purified component C, but the protein contains 1.3-1.5 atoms of iron/molecule. 6. On boiling, component C releases a yellow-green fluorescent material that has been identified as FAD from its absorption and fluorescence spectra and by t.l.c. 7. Component C contains 1 mol of FAD/mol of protein.     

Surface Antigens of Smooth Brucellae

Surface antigens of smooth brucellae were extracted by ether-water, phenol-water, trichloroacetic acid, and saline and examined by immunoelectrophoresis and gel diffusion with antisera from infected and immunized rabbits. Ether-water extracts of Brucella melitensis contained a lipopolysaccharide protein component, which was specific for the surface of smooth brucellae and was correlated with the M agglutinogen of Wilson and Miles, a polysaccharide protein component devoid of lipid which was not restricted to the surface of smooth brucellae and was not correlated with the smooth agglutinogen (component 1), and several protein components which were associated with internal antigens of rough and smooth brucellae. Immunoelectrophoretic analysis of ether-water extracts of B. abortus revealed only two components, a lipopolysaccharide protein component, which was correlated with the A agglutinogen, and component 1. Component 1 from B. melitensis and B. abortus showed identity in gel diffusion tests, whereas component M from B. melitensis and component A from B. abortus showed partial identity with unabsorbed antisera and no cross-reactions with monospecific sera. Attempts to prepare monospecific sera directly by immunization of rabbits with cell walls or ether-water extracts were unsuccessful. Absorption of antisera with heavy fraction of ether-water extracts did not always result in monospecific sera. It was concluded (as has been described before) that the A and M antigens are present on a single antigenic complex, in different proportions depending upon the species and biotype, and that this component is a lipopolysaccharide protein complex of high molecular weight that diffuses poorly through agar gel. Components 1, A, and M were also demonstrated in trichloroacetic acid and phenol-water extracts. With all extracts, B. melitensis antigen showed greater diffusibility in agar than B. abortus antigens. After mild acid hydrolysis, B. abortus ether-water extract was able to diffuse more readily.     

The polypeptide and the phospholipid components of axon plasma membranes.

The axon plasma membrane fraction isolated from garfish olfactory nerve was analyzed for its polypeptide composition by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. There were present over 20 well-resolved polypeptide components in this membrane, and eleven of them, with an apparent molecular weight range of 22,000-130,000, accounted for most of the membrane proteins. None of the major polypeptide species present in the membrane appeared to be glycoprotein. Based on electrophoretic mobility on sodium dodecyl sulfate-polyacrylamide gel, eight of the major polypeptides found in garfish nerve membrane appeared to be also present in the axon plasma membrane isolated from lobster walking leg nerve. Both garfish and lobster nerve membranes contained high concentration of lipids (66-76%) which were essentially cholesterol and phospholipids. The classes of phospholipids present were phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, phosphatidylinositol and sphingomyelin. Lobster nerve membrane also contained about 3% phosphatidic acid. Assays for acetylcholinesterase in axon plasma membrane fractions isolated from different nerve sources showed a wide variation, ranging from a specific activity of 2.4 for garfish nerve to 312.5 for lobster nerve membrane.     

Isolation and some physico-chemical properties of a new neurospecific protein 10-40-4 from human brain

A method for isolation of a neurospecific protein 10-40-4 from human brain has been elaborated. This procedure includes immunoaffinity chromatography of a Sepharose 4B-IgG fraction of rabbit antisera against the protein fraction containing the antigen. The isolated protein cannot be detected in protein extracts of various organs and human blood serum by immunochemical methods. This indicates that the protein is specific for nervous tissue. The values of molecular weight (74 000) and pI (4.7) of the isolated protein suggest that the protein does not contain the carbohydrate component and reveals limited tissue specificity. The properties of protein 10-40-4 differ from those of the well-known neurospecific proteins, such as S-100, enolase 14-3-2 and glial fibrillar acid protein GFA.     

The lobster nerve sodium channel: solubilization and purification of the tetrodotoxin receptor protein

Solubilization and purification of the tetrodotoxin (TTX) binding protein of the lobster walking-leg nerve Na+ channel were carried out utilizing [3H]tetrodotoxin [( 3H]tetrodotoxin) as a marker. The nerve membrane was solubilized with Lubrol-PX and the Na+ channel protein was purified with diethylaminoethyl Bio-Gel A, Bio-Gel hydroxylapatite powder and two Sepharose 6B columns. Care was taken to keep the temperature of the Na+ channel preparation as close to 1 degrees C as possible and to use solutions (pH 7.5) that contain Na channel protectors, i.e., egg phosphatidylcholine/Lubrol-PX mixture, TTX, EDTA, EGTA, phenylmethylsulfonyl fluoride, pepstatin A, iodoacetamide, antipain, phosphoramidon, soybean trypsin inhibitor, leupeptin and bacitracin. From an initial specific binding of 20.1 pmol of [3H]TTX/mg protein for the solubilized membrane, the binding increased to 1241 pmol/mg protein for the most active fraction of the last Sepharose 6B column. The [3H]TTX specific binding of the Sepharose 6B fractions correlated with a large peptide of Mr 260,000 (240-280K), although other peptides were also present in lesser amounts.     

The Xenopus YB3 protein binds the B box element of the class III promoter

We have isolated a Xenopus cDNA encoding the YB3 protein which binds specifically to the B box promoter element of class III genes. Northern analysis shows YB3 is expressed in a variety of adult tissues. Fractionation of oocyte S150 extracts demonstrates YB3 is present in phosphocellulose fraction IIIC, as well as in the fraction isolated by B box DNA affinity chromatography. Silver staining indicates that YB3, or a protein of the same mobility in SDS gels, is the most abundant component in either fraction.     

Activation of protein kinase C from B lymphocytes by lipid A

The effect of lipid A, a residue of the lipopolysaccharide molecule, on protein kinase C from B lymphocytes has been studied. Lipid A activates and promotes the translocation of protein kinase C from the soluble to the particulate membrane fraction in a cell-free system reconstituted with purified enzyme and membranes isolated from B lymphocytes. These results demonstrate that the activating effect of lipopolysaccharide on protein kinase C from B cells is due to the lipid moieties of this molecule.     

On the Specificity of 125I-α-Bungarotoxin Binding to Axonal Membranes

Abstract: ¹²⁵I-α-Bungarotoxin (α-BGT) was used to characterize the binding sites for cholinergic ligands in lobster walking leg nerve membranes. The toxin binding component has been visualized histochemically on the external surfaces of intact axons and isolated axonal membrane fragments. Binding of α-BGT to nerve membrane preparations was demonstrated to be saturable and highly reversible ( K _D^app± 1.7 ± 0.32 × 10^-7 M; B _max± 249 ± 46 pmol/mg protein) at pH 7.8, 10 mM-Tris buffer. Binding showed a marked sensitivity to ionic strength that was attributable to the competitive effects of inorganic cations (particularly Ca²⁺ and Mg²⁺) in the medium. ¹²⁵I-α-BGT binding could be inhibited by cholinergic drugs (atropine ≅ d -tubocurarine > nicotine > carbamylcholine ≅ choline) and local anesthetics (procaine > tetracaine = lidocaine), but was unaffected by other neuroactive compounds tested (e.g., tetrodotoxin, 4-aminopyridine, quinuclidinyl benzilate, octopamine, bicuculline, haloperidol, ouabain). The pharmacological sensitivity of toxin binding resembles that of nicotine binding to axonal membranes, but differs significantly from nicotinic cholinergic receptors described in neuromuscular junctions, fish electric organs, sympathetic ganglia, and the CNS. The possible physiological relevance of the axonal cholinergic binding component and its relationship to α-BGT binding sites in other tissues are discussed.     

Isolation and characterization of thioredoxin f from the filamentous cyanobacterium, Anabaena sp. 7119

Two thioredoxin fractions had previously been reported to occur in Anabaena 7119 by Buchanan and co-workers (Yee, B. C., dela Torre, A., Crawford, N. A., Lara, C., Carlson, D. E., and Buchanan, B. B. (1981) Arch. Microbiol. 130, 14-18). These proteins were detected by their ability to activate spinach fructose-1,6-bisphosphatase (Fru-P2-ase). The partially purified proteins resembled similar thioredoxins found in spinach chloroplasts and were designated thioredoxin f (Tf) for the fraction most effective in activating spinach Fru-P2-ase and thioredoxin m (Tm) for the fraction most effective in activating spinach NADPH-malate dehydrogenase. Using the assay system of Yee and co-workers, we were able to separate and purify to homogeneity two thioredoxin fractions from Anabaena extracts. Tm corresponded to the thioredoxin fraction we had isolated and studied previously (Gleason, F. K., and Holmgren, A. (1981) J. Biol. Chem. 256, 8301-8309). The other fraction, Tf, was characterized further. Unlike the thioredoxins found in higher plants, the cyanobacterial thioredoxins do not appear to be related. Anabaena thioredoxin f has a Mr = 25,500 as compared to the more usual Mr = 12,000 for Tm. From a comparison of the amino acid composition, Tf is not obviously a dimer or otherwise related to Tm. Tf has one active center cystine disulfide. Anabaena Tf activates spinach Fru-P2-ase very efficiently but has very little activity with spinach malate dehydrogenase. Anabaena Tf, unlike Tm, does not reduce the homologous ribonucleotide reductase. Anabaena Tf also does not activate a partially purified preparation of Anabaena Fru-P2-ase. We conclude that the cyanobacterial Tf is a unique protein with no structural or functional properties in common with other thioredoxins.     

ISOLATION AND CHARACTERIZATION OF PLASMA MEMBRANE FRACTIONS FROM GARFISH LEPISOSTEUS OSSEUS OLFACTORY NERVE

Garfish Lepisosteus osseus olfactory nerve, because of its large size and the unusually high concentration of axonal membrane, is an excellent source of axonal membrane. A procedure is described for the isolation of two types of plasma membranes from the nerve which are obtained in yields of about 20 mg (fraction I) and 1.5 mg (fraction II) per g of wet nerve. Both membrane fractions consist mostly of rounded membrane vesicles, with a unit membrane thickness of ~7.5 nm. The two membrane fractions are different in their lipid to protein ratios, Na-K ATPase activities, polypeptide patterns on sodium dodecyl sulfate (SDS) gel electrophoresis, and fatty acid compositions. They have similar phospholipid composition. On the basis of the relative concentration of axonal and Schwann cell plasma membranes in the nerve, the Na-K ATPase activities of the two membrane fractions and a comparison of the properties of the membrane fractions to those of squid and lobster nerve membrane preparations, fraction I seems to be the axonal membrane and fraction II the Schwann cell plasma membrane. Fraction I has a low protein to lipid ratio. Its polypeptide pattern on SDS gel appears to be much more complex as compared to that of fraction II membrane.     

Comparative studies on the structure and aggregative properties of the myosin molecule. III. The in vitro aggregative properties of the lobster myosin molecule.

The solubility of rabbit skeletal and lobster abdominal muscle myosin has been studied in monovalent salt solutions as a function of pH (over the range 4.75 to 8.5) and ionic strength (50-500 mM). Rabbit skeletal muscle myosin was found to precipitate over a narrower pH range than the lobster abdominal muscle myosin but at equivalent pH values and ionic strengths the former exhibited greater solubility. Comparison of the solubility of rabbit myosin, per se with that of light meromyosin and lobster myosin with its equivalent proteolytically produced fragment (fraction B1) showed that both rod fragments were more soluble than their parent molecules. Under conditions of low solubility (low ionic strength and pH) the quantitiy of protein in solution remained essentially constant with increasing total protein, thus suggesting that the aggregation phenomenon is of a phase transition type. Examination of the aggregates by electron microscopy revealed that rabbit myosin formed classical, elongate, spindle-shaped filaments similar to those previously observed by others. In contrast lobster myosin only formed short, dumbbell-shaped filaments 0.2-0.3 mum long. Consideration of the pH ranges over which aggregation occurred suggests that protonation of histidine residues may be involved in rabbit myosin filament formation while for lobster myosin, aggregation may involve protonation of epsilon-amino or guanidino groups. The possible relationship between the distribution of these groups along the rod portion of the myosin molecule and the formation of elongate filaments has been explored.     

Cytolytic activity of Naegleria fowleri cell-free extract

The cytotoxic activity of a cell-free extract of Naegleria fowleri amebae on B103 rat nerve cells in culture was investigated. The cell-free extract was prepared by subjecting lysed amebae to centrifugation at 100,000 g for 1 h, precipitation of the supernatant fluid with 30-60% saturated ammonium sulfate, and desalting by group exclusion chromatography utilizing Sephadex G-25. The supernatant fluid recovered from this procedure was termed the soluble fraction. The Naegleria cytotoxic activity present in the soluble fraction was assayed by 51Cr released from labeled B103 cells. The Naegleria soluble fraction, when added to nerve cells, elicited blebs on the B103 target cell surface within 5 min after exposure to the fraction. Later, holes were observed in the B103 cell plasma membrane. These alterations were never observed on untreated B103 cells. Phospholipase A, phospholipase C, and protease activities were associated with the desalted ammonium sulfate-precipitable cytotoxic activity of N. fowleri cell-free lysate. The cytotoxic activity was impaired by ethylenediamine-tetraacetate (EDTA), phospholipase A inhibitor (Rosenthal's reagent), heating at 50 degrees C for 15 min, or incubation at pH 10 for 60 min. Repeated freeze-thawing and inhibitors of proteolytic enzymes had no effect on the cytotoxic activity. Small amounts of ethanol (5% v/v) enhanced cytotoxic activity of the fraction. Phospholipases A and C, as well as other as yet unidentified cytolytic factors may be responsible for producing 51Cr release from target cells by the soluble fraction of N. fowleri extracts.     

Raman spectroscopy of nerve fibers. A study of membrane lipids under steady state conditions.

The molecular structures of different nerve fibers kept in good physiological conditions were studied by laser Raman spectroscopy. For myelinated nerves like the rat sciatic nerve, the Raman spectrum is dominated by bands due to the lipid component of the myelin sheath. The temperature dependence of these bands does not reveal any thermotropic phase transition between 0 and 40 degrees C. There is, however, with temperature, a linear increase in the intermolecular disorder that is accompanied by an increase in the number of gauche bonds of the phospholipid acyl chains. For unmyelinated nerves such as the lobster leg nerve, the C-H stretching region of the Raman spectrum is covered by bands arising from the protein component of the axoplasm. However, for the garfish olfactory nerve that has a high density of excitable membranes, phospholipid bands are observed and can be used as intrinsic structural probes of the excitable membranes. The relative intensity of these bands is also temperature dependent.     

Incorporation of radioactive glucosamine into macromolecules at nerve endings

Mice were injected intracerebrally with [¹⁴C]glucosamine, and incorporation into macromolecules in various subcellular fractions of brain was studied at a number of times after administration of the precursor. The [¹⁴C]glucosamine was rapidly incorporated into macromolecules of all the subcellular fractions of brain including both the soluble and particulate fractions of isolated nerve endings. Incorporation into macromolecules in the soluble fraction of nerve endings was quite extensive 3 hr after administration of the precursor and the specific acitvity of this fraction fell thereafter. In contrast there was only slight incorporation of [¹⁴C] leucine into the soluble protein from isolated nerve endings in the first few hours after administration, whereas the other subcellular fractions were maximally labelled at that time. The data suggests that, unlike protein which is largely transported to nerve endings in the axoplasm, there is extensive incorporation of carbohydrate into macromolecules in nerve endings. Whereas the protein component of a glycoprotein or mucopolysaccharide may be transported to the nerve ending from the perikaryon, the structure and function of this protein may be modified at the nerve ending by further incorporation of glucosamine, sialic acid and possibly other carbohydrates. The carbohydrate-containing macromolecules could influence nerve ending function immediately after these final synthetic reactions since these reactions occur at the nerve ending and not in the perikaryon.     

Nonfunctional Tricarboxylic Acid Cycle and the Mechanism of Glutamate Biosynthesis in Acetobacter suboxydans

Acetobacter suboxydans does not contain an active tricarboxylic acid cycle, yet two pathways have been suggested for glutamate synthesis from acetate catalyzed by cell extracts: a partial tricarboxylic acid cycle following an initial condensation of oxalacetate and acetyl coenzyme A. and the citramalate-mesaconate pathway following an initial condensation of pyruvate and acetyl coenzyme A. To determine which pathway functions in growing cells, acetate-1-(14)C was added to a culture growing in minimal medium. After growth had ceased, cells were recovered and fractionated. Radioactive glutamate was isolated from the cellular protein fraction, and the position of the radioactive label was determined. Decarboxylation of the C5 carbon removed 100% of the radioactivity found in the purified glutamate fraction. These experiments establish that growing cells synthesize glutamate via a partial tricarboxylic acid cycle. Aspartate isolated from these hydrolysates was not radioactive, thus providing further evidence for the lack of a complete tricarboxylic acid cycle. When cell extracts were analyzed, activity of all tricarboxylic acid cycle enzymes, except succinate dehydrogenase, was demonstrated.