THE ISOLATION OF CHOLINERGIC SYNAPTIC VESICLES FROM BOVINE SUPERIOR CERVICAL GANGLION AND ESTIMATION OF THEIR ACETYLCHOLINE CONTENT

Abstract— A method is described for the isolation of relatively pure cholinergic synaptic vesicles by hypo-osmotic shock and density gradient centrifugation of a synaptosome fraction prepared from bovine superior cervical ganglia. Vesicles from this source were found to sediment to a density equivalent to 0·3–0·41 M-sucrose. The vesicle subfraction from the gradient had an acetylcholine (ACh) content of 4.4 nmol/mg of protein and were subject to leakage of ACh. By a 'tagging' technique, the vesicles were counted under the electron microscope and their numbers related to their ACh concentration. After correction for leakage, an ACh content of 1630 molecules/vesicle was estimated.     

SOLUBLE AND PARTICLE-BOUND ACETYLCHOLINESTERASE AND ITS ISOENZYMES IN PERIPHERAL NERVES

Abstract— The distribution of AChE (EC 3.1.1.7) in soluble and particulate fractions of the peripheral nerves of dogs, cats, rabbits and frogs was examined. About 20–30% of the total AChE activity was found in the supernatant fluid after centrifugation (100,000 g for 90 min) of iso-osmotic sucrose homogenates. The effect of different media on the extent of solubilization of the enzyme was studied and Triton X-100 (0.2%) was found to be the most effective. The electrophoretic pattern of AChE in peripheral nerves was also investigated. The 2–3 types of AChE observed previously were found in both particulate and soluble fractions, but the proportions of these forms were different. The most slowly migrating form of AChE is the most firmly bound to nerve membranes. A very small but consistent proportion (3%) of AChE escaped into the medium from surviving dog nerves kept in aerated Ringer solution. It was calculated that the possible contribution of blood AChE contained in the nerve is negligible. Electrophoretograms of AChE released during incubation into Ringer solution were similar in pattern to those found for the soluble fraction.     

CHARACTERIZATION OF HYPOTHALAMIC SUBCELLULAR PARTICLES CONTAINING LUTEINIZING HORMONE RELEASING HORMONE AND THYROTROPIN RELEASING HORMONE

Abstract— The 900 g supernatant fluid prepared from male rat hypothalamic homogenates was fractionated by means of continuous sucrose density gradient centrifugation. Thyrotropin releasing hormone and luteinizing hormone releasing hormone in the gradient fractions were quantified by radioimmunoassays. TRH was associated with two populations of particles separable by means of nonequilibrium density centrifugation (100,000 g for 30min). However, after'equilibrium'centrifugation (100,000 × g for 180 min), a single peak of TRH was observed at 1.07 M-sucrose. Hypo-osmotic shock as well as treatment with 0.1% Triton X-100 or 0.1% deoxycholate (DOC) released TRH from both sets of particles. LRH, as TRH, was associated with two populations of particles which were separable by means of nonequilibrium density gradient centrifugation. After'equilibrium'centrifugation, both sets of LRH-containing particles banded at 1.27M-sucrose as a single symmetrical peak. Although 0.1% Triton X-100 released LRH from both populations of particles, hypo-osmotic shock or 0.1% DOC released LRH only from the large LRH-containing particles. The small LRH-containing particles were resistant to hypo-osmotic shock and to 0.1% DOC. Based on these criteria, it is concluded that in hypothalamic homogenates the TRH-containing particles and the large LRH-containing particles are synaptosomes. The small LRH-containing particles may be of different cellular and/or subcellular origin.     

A Synaptosomal Preparation from the Guinea Pig Ileum Myenteric Plexus

Abstract: Our interest in investigating the presynaptic modulation of acetylcholine release led to the development of a synaptosomal preparation from the guinea pig ileum myenteric plexus-longitudinal muscle. A crude synaptosomal fraction (P₂) was obtained by homogenization and differential centrifugation. The preparation exhibited a specific uptake system for choline and for nor-adrenaline (NA), but not for 5-hydroxytryptamine (5-HT). Synaptosomes were isolated from this P₂ fraction by an isoosmotic density gradient prepared from sucrose and metrizamide. The resultant synaptosomal fraction was enriched about sevenfold in both choline uptake and in choline acetyltransferase (ChAT). Choline was transported by a high-affinity system with a K_m of 6.5 × 10⁻⁷ M and a V_max of 41 pmol/mg protein/min. Electron microscopy confirmed the synaptosomal nature of the gradient fraction. Some synaptosomal profiles contained only small, translucent vesicles whereas others also contained large (approx. 100 nm diameter) electron-opaque vesicles. The crude synaptosomal fraction synthesized acetylcholine (ACh) from exogenous choline and it released the synthesized ACh in a calcium-dependent manner.     

Partial Isolation of Two Classes of Dopamine β-Hydroxylase-Containing Particles Undergoing Rapid Axonal Transport in Rat Sciatic Nerve

The rapid bidirectional transport of dopamine beta-hydroxylase (DBH) in adrenergic axons provides a means of analyzing the life cycle of adrenergic storage vesicles. We compared the physical characteristics of DBH-containing particles traveling to or returning from the terminal varicosities of ligated rat sciatic nerves. Density gradient centrifugation and Sephacryl S1000 gel-permeation chromatography were used to fractionate extracts from nerve segments proximal or distal to the ligatures. A series of experiments indicated the existence of at least two populations of rapidly transported DBH-containing particles, a "light" 85-nm particle and a larger "dense" 120-nm particle. The 85-nm particles were prevalent in unligated nerve, but accounted for only one-third of the total anterogradely transported DBH activity accumulated after 18 h. The 120-nm particles were barely detectable in the unligated nerve, but they accumulated at twice the rate of the 85-nm particles and accounted for the rest of the anterogradely transported particulate DBH activity. These two populations of particles were readily isolated from proximal nerve extracts by sucrose density gradient centrifugation. Similar-appearing dense and light peaks of particulate DBH activity were obtained from distal nerve extracts. Much of the retrogradely transported DBH of the extracts, however, was associated with large particles (greater than 300 nm) not resolved by Sephacryl S1000. Retrogradely transported exogenous NGF was found only in the dense sucrose gradient peak. We propose that the 85-nm DBH-containing particles correspond to "large dense-cored vesicles," and that the 120-nm particles are derived from the dense tubules visualized in adrenergic nerves by the chromaffin reaction.     

The Glycoprotein Composition of Peripheral Nervous System Myelin Subfractions

Two fractions were isolated by continuous density gradient centrifugation from total particulate matter of rabbit sciatic nerves: a minor fraction, B, consisting of small-sized membrane fragments and a major fraction, C, of characteristic multilayered myelin figures, with maxima at 0.33 and 0.58 M-sucrose, respectively. In comparison with C, fraction B was enriched in CNPase and alkaline phosphatase activities and the P0, 23K and Z proteins, but was virtually devoid of basic protein. The glycoprotein composition of all fractions was examined with four fluorescein isothiocyanate-labelled lectins (WGA, Con A, RCA-60, U.E.). These revealed the presence of six glycoproteins in all fractions with similar lectin binding capacities and molecular weights ranging from 35,500 to 16,000, of which P0 was the predominant component. Material found on the heavy side of fraction C was characterized by the presence of a multitude of glycoproteins which bound variable proportions of the four different lectins, suggesting substantial variations in their carbohydrate moieties. Their absence from the central portion of fraction C points to a location other than that of compact PNS myelin.     

Axonal transport of muscarinic receptors in vesicles containing noradrenaline and dopamine-β-hydroxylase

Presynaptic muscarinic receptors labeled with [³H]dexetimide and noradrenaline in dog splenic nerves accumulated proximally to a ligature at the same rate of axonal transport. After fractionation by differential centrifugation, specific [³H]quinuclidinyl benzilate or [³H]dexetimide binding revealed a distribution profile similar to that of dopamine-β-hydroxylase and noradrenaline. Subfractionation by density gradient centrifugation showed two peaks of muscarinic receptors; the peak of density 1.17 contained noradrenaline and dopamine-β-hydroxylase whereas that of density 1.14 was devoid of noradrenaline. Therefore the foregoing experiments provide evidence that presynaptic muscarinic receptors are transported in sympathetic nerves in synaptic vesicles which are similar to those containing noradrenaline and dopamine-β-hydroxylase. This suggests a possible coexistence of receptor and neurotransmitter in the same vesicle.     

Density distribution of 2′,3′-cyclic nucleotide 3′-phosphodiesterase and myelin proteins in particulate material from myelin deficient (mld) mutant and control brains

Total particulate material from control and myelin deficient (mld) brains was subjected to density centrifugation on a continuous sucrose gradient. Particles from control brains distributed in a bell-shaped mode with a peak density near 0.64 M-sucrose. In mld material only a slight elevation of optical density was observed near 0.8 M-sucrose. The highest specific activities of 2′,3′-cyclic nucleotide 3′-phosphodiesterase were observed at densities of 0.63 and 0.71 M-sucrose for mld and control brains, respectively. The peak of myelin basic protein in control fractions was near 0.60 M-sucrose. In mld fractions no peak was observed. Proteolipid and Wolfgram proteins had a maximum near 0.65 and 0.73 M-sucrose in control and mld fractions, respectively. The absence of myelin basic proteins in all the fractions makes it unlikely that, in mld mice, myelin basic proteins are synthesized but not incorporated into myelin.     

Mechanism of conjugation and recombination in bacteria. XVII. Further evidence for single-stranded regions in recipient DNA during conjugation in Escherichia coli K-12.

The secondary structure of recipient DNA mated with Hfr strain was investigated by CsCl density gradient fractionation. After 45 min of HfrH64 X 3h-f-ab1157 mating one-fourth of the radioactive recipient DNA was recovered as a single-strand but only after shearing of cell lysates prior to centrifugation. This heavier than native DNA fraction of radioactive material (obtained after the first centrifugation) was degraded by single-strand specific nuclease S from Aspergillus oryzae. These findings thus confirm the authors' earlier results suggesting that in the course of mating are generated local single-stranded regions in recipient DNA.     

Subcellular distribution of dopamine in substantia nigra of the rat brain: effects of gamma-butyrolactone and destruction of noradrenergic afferents suggest formation of particles from dendrites.

Abstract— The subcellular distribution of dopamine (DA) in substantia nigra from individual male rats was studied with a fractionation procedure on microscale. After differential centrifugation the distribution of DA coincided with that of noradrenaline (NA) which can serve as a marker for synaptosomes in this area. The proportion of DA/NA concentrations was about 1–2 in most fractions. Sixty per cent of nigral DA was found in P₂ (17,000 g). When P, was layered on a continuous density gradient, DA and NA peaked at the density of 1.0–1.2 M-sucrose. Since DA-containing particles covered a relatively broad range on this gradient, particles between 0.7 and 1.3 M-sucrose were collected with a discontinuous density gradient. Sixty per cent of DA from P₂, was found in this subfraction. The particles containing DA could have been derived from dendrites or axon collaterals of nigrostriatal neurones or represent precursor DA in noradrenergic (NA) terminals. The role of collaterals was investigated by comparing the effect of γ-butyrolactone (GBL, 750 mg/kg, 1 h) on DA concentrations in subcellular fractions from substantia nigra and caudate-putamen. In caudate-putamen, GBL produced a marked increase of DA in total homogenates and subcellular fractions except P₃, whereas DA concentrations remained unchanged in all fractions from substantia nigra. This speaks against a contribution from DA terminals. The proportion of DA contained as precursor in NA terminals was analysed after destruction of the NA input to substantia nigra by two methods. A single injection of 6-hydroxydopamine into the IV ventricle decreased nigral NA by 5574, DA only by 17%. Unilateral electrolytic lesions in the pontine tegmentum affected NA concentrations in homogenates and fraction P₂ of the ipsilateral substantia nigra to a much greater extent than DA. From the results obtained with the two approaches, it is estimated that precursor DA in particulate fractions does not exceed 10%. Our observations indicate that dendrites of the DA neurones in substantia nigra can form particles which behave like synaptosomes on density gradients centrifugation; they may be termed ‘dendrosomes’. According to the proportion of DA found in the particulate fractions at least 4040% of nigral DA appear to be localised in dendrites.     

INTRACELLULAR TRANSPORT OF SECRETORY PROTEINS IN THE PANCREATIC EXOCRINE CELL : I. Role of the Peripheral Elements of the Golgi Complex

It has been established by electron microscopic radioautography of guinea pig pancreatic exocrine cells (Caro and Palade, 1964) that secretory proteins are transported from the elements of the rough-surfaced endoplasmic reticulum (ER) to condensing vacuoles of the Golgi complex possibly via small vesicles located in the periphery of the complex. To define more clearly the role of these vesicles in the intracellular transport of secretory proteins, we have investigated the secretory cycle of the guinea pig pancreas by cell fractionation procedures applied to pancreatic slices incubated in vitro. Such slices remain viable for 3 hr and incur minimal structural damage in this time. Their secretory proteins can be labeled with radioactive amino acids in short, well defined pulses which, followed by cell fractionation, makes possible a kinetic analysis of transport. To determine the kinetics of transport, we pulse-labeled sets of slices for 3 min with leucine-¹⁴C and incubated them for further +7, +17, and +57 min in chase medium. At each time, smooth microsomes ( = peripheral elements of the Golgi complex) and rough microsomes ( = elements of the rough ER) were isolated from the slices by density gradient centrifugation of the total microsomal fraction. Labeled proteins appeared initially (end of pulse) in the rough microsomes and were subsequently transferred during incubation in chase medium to the smooth microsomes, reaching a maximal concentration in this fraction after +7 min chase incubation. Later, labeled proteins left the smooth microsomes to appear in the zymogen granule fraction. These data provide direct evidence that secretory proteins are transported from the cisternae of the rough ER to condensing vacuoles via the small vesicles of the Golgi complex.     

Proteins of axonal transport: investigation of solubility characteristics and behaviour in gel filtration

Rapid axonal transport of proteins in retinal ganglion cells of the rabbit was studied following intraocular injections of labelled amino acids. Approximately 10% of the transported radioactivity was found in the supernatant following homogenization and high-speed centrifugation of the nerve terminal region. Relatively simple manipulations with ionic strength, pH and the presence of a chelating agent could solubilize an equivalent amount of radioactivity from the pellet. Lithium diiodosalicylate solubilized most rapidly transported membrane proteins. Gel filtration of readily soluble rapidly transported radioactivity gave a main macromolecular radioactive peak with an approximate mol. wt. of 500,000 dalton as determined on Sephadex G-200. However, gel filtration on Sepharose CL-6B gave a mol. wt. of about 160,000 for the same radioactive peak. SDS polyacrylamide gel electrophoresis of rapidly transported soluble proteins and fractions derived from these proteins via gel filtration and ion exchange chromatography revealed in all cases a very complex picture of labelled polypeptides. Thus rapid axonal transport of soluble proteins in this system seems to involve many different macromolecules.     

Axonal transport of proteoglycans to the goldfish optic tectum

The study addressed the question of whether³⁵SO₄ labeled molecules that the have been delivered to the goldfish optic nerve terminals by rapid axonal transport include soluble proteoglycans. For analysis, tectal homogenates were subfractionated into a souluble fraction (soluble after centrifugation at 105,000g), a lysis fraction (soluble after treatment with hypotonic buffer followed by centrifugation at 105,000g) and a final 105,000g pellet fraction. The soluble fraction contained 25.7% of incorporated radioactivity and upon DEAE chromatographys was resolved into a fraction of sulfated glycoproteins eluting at 0–0.32 M NaCl and containing 39.5% of total soluble label and a fraction eluting at 0.32–0.60 M NaCl containing 53.9% of soluble label. This latter fraction was included on columns of Sepharose CL-6B with or without 4 M guanidine and after pronase digestion was found to have 51% of its radioactivity contained in the glycosaminoglycans (GAGs) heparan sulfate and chondroitin (4 or 6) sulfate in the ratio of 70% to 30%. Mobility of both intact proteoglycans and constituent GAGs on Sepharose CL-6B indicated a size distribution that is smaller than has been observed for proteoglycans and GAGs from cultured neuronal cell lines. Similar analysis of lysis fraction, containing 11.5% of incorporated³⁵SO₄, showed a mixture of heparan sulfate and chondroitin sulfate containing proteoglycans, apparent free heparan sulfate and few, if any, sulfated glycoproteins. Overall, the result support the hypothesis that soluble proteoglycans are among the molecules axonally transported in the visual system.     

A COMPARISON OF THE AXONAL TRANSPORT OF TAURINE AND PROTEINS IN THE GOLDFISH VISUAL SYSTEM

Abstract— Radioactive cystathionine, a metabolic precursor of taurine, was injected into the right eye of goldfish. At various times after injection the retina and both optic tecta were extracted with trichloroacetic acid (TCA) and the amount and nature of the radioactivity was determined. Radioactive taurine and inorganic sulfate were present in the TCA-soluble extract of retina and radioactive taurine and a small amount of inorganic sulfate was found in the contralateral optic tectum. That taurine is migrating intraaxonally and is not diffusing in extraaxonal spaces is suggested from experiments in which the migration of taurine was compared with that of [¹⁴C]mannitol, used here as a marker of extracellular diffusion. In the time studied (up to 15 h) mannitol did not migrate to the tectum, whereas taurine was detectable in the tectum as early as 8 h after injection. Since intra-axonal diffusion of amino acids and other small molecules in this system has been ruled out, it is likely that taurine is being transported axonally. The axonal transport of taurine was found to be similar to the fast component of protein transport because: (1) their rates of transport are similar, (2) the transport of both is blocked by the protein synthesis inhibitor cycloheximide, (3) vinblastine, which disrupts neurotubules, appears to have similar effects on both protein and taurine transport, and (4) both rapidly transported proteins and taurine remain mostly intra-axonal once they have been transported to the tectum. Taurine and proteins differ in that rapidly transported proteins are primarily paniculate in nature and localized to a large extent in nerve endings, while taurine is primarily in a soluble fraction and is present in nerve endings only in trace amounts. We suggest that taurine may be loosely linked to a newly synthesized protein in the soma and is then transported along with that protein on a similar conveying mechanism in the axoplasm.     

COMPARATIVE STUDIES IN SYNAPTOSOME FORMATION: THE PREPARATION OF SYNAPTOSOMES FROM THE HEAD GANGLION OF THE SQUID, LOLIGO PEALII

Abstract— Relatively high concentrations of ACh have been found in the head ganglion of the squid ( Loligo pealii ) and the identity of the ACh has been verified by ion-exchange chromatography. Following homogenization in media iso-osmotic with sea water about 40 per cent of the ACh survives in particle-bound form. Experiments using media of varying osmolarity suggest that this bound ACh is osmotically sensitive. A study has been made of the subcellular fractionation of squid head ganglion using sucrose homogenates. A rapid and novel method is described for the preparation of a synaptosome fraction freed from mitochondria. This preparation contains synaptosomes of well-preserved morphology with occluded cytoplasm and a high specific content of ACh. The synaptosomes are osmotically sensitive and when suspended in water they burst, releasing cytoplasmic constituents and ACh-containing synaptic vesicles. The synaptic vesicles can be separated from other sub-synaptic constituents by density gradient centrifugation.     

QUAKING MOUSE MYELIN: BIOCHEMICAL CHARACTERIZATION OF ZONAL GRADIENT SUBFRACTIONS

The brains of Quaking and littermate control mice were fractionated by differential and density gradient centrifugation into soluble, microsomal, myelin and related (SN 4) fractions. There were no apparent differences in protein composition between any Quaking and control fraction with the exception of myelin and SN 4. Analysis of CNP activity indicated that in Quaking animals a high proportion of the total activity was localized in microsomal fractions, while in controls a large percentage of activity was found in myelin and SN4; in contrast, there were no marhcd differences in the distribution of AChE activity between Quaking and control fractions. The yield of myelin isolated from Quaking animals was 3.6%, of that from controls by electron microscopy myelin fractions from both Quaking and controls consisted of compact myelin whorls. Zonal centrifugation on continuous sucrose gradients demonstrated that both control and Quaking myelin was distributed in a bell-shaped mode with peak densities at 0.66 0.68 and 0.71-0.75 M-sucrose, respectively. The specific activity of CNP was generally lower in mutant subfractions than in controls. Protein analysis revealed that there were similar qualitative trends between light and heay myelin subfractions from both mutant and control animals, although the levels of proteolipid and small basic proteins were substantially lower in all Quaking fractions. These results indicate that. although all mutant myelin subfractions are compositionally abnormal, the type of particle heterogeneity in Quaking myelin is similar to that observed in controls.     

Acetylcholine and its metabolic enzymes in developing antennae of the moth, Manduca sexta.

Antennae of the moth, Manduca sexta, are thickly populated with sensory neurons, which send axons through antennal nerves to the brain. These neurons arise by cell divisions and differentiate synchronously during the 18 days of metamorphosis from pupa to adult. Biochemical studies support the hypothesis that antennal neurons use acetylcholine (ACh) as a neurotransmitter: (1) Antennae incubated with [¹⁴C]choline synthesize and store [¹⁴C]ACh; several other transmitter candidates do not accumulate detectably when appropriate radioactive precursors are supplied; (2) antennae and antennal nerves contain endogenous ACh; and (3) extracts of mature antennae contain choline acetyltransferase (ChAc) and acetylcholinesterase (AChE) with properties similar to those reported for the enzymes from other arthropods. Levels of ACh, ChAc, and AChE begin to increase in antennae soon after the sensory neurons are “born.” Levels rise exponentially for over a week as the neurons differentiate and then reach a plateau, at about the time the neurons reach morphological maturity, that is maintained into adulthood. In contrast, levels of carnitine acetyltransferase, cholinesterase, and soluble protein, presumably not confined to nervous tissue, change little during metamorphosis. Levels of ACh, ChAc, and AChE rise in an intracranial segment of antennal nerve at about the same time as in the antenna, indicating that axons can transport neurotransmitter machinery at an early stage in their development.     

ANALYSIS OF PROTEINS UNDERGOING AXONAL TRANSPORT IN NIGRO-STRIATAL NEURONS IN THE RAT

Abstract— An analysis of proteins undergoing axonal transport in nigro-striatal neurons, after the stereotaxic injection of [³H]leucine into the substantia nigra of rat brain was performed. As early as 6 h after the injection [³H]proteins appeared in the caudate-putamen. The maximum accumulation was at 5 days and there was still residual protein radioactivity present at 30 days. About 70 per cent of the total radioactive protein in the caudate-putamen was solubilized by homogenization in 0–5%, (v/v) Triton X-100 and remained in the supernatant on centrifuging for 1 h at 100,000 g. The supernatant fraction, when chroma-tographed on a DEAE-cellulose column, was resolved into four protein peaks (A, B. C and D) which were found to be labelled differently as a function of time after the injection of [³H]leucine. Peak A was substantially labelled in a first phase (6–24 h) and reached its maximum in a second phase (5 days). The proteins comprising this peak appeared to undergo both fast and slow axonal transport. Although some labelling in peak B was evident at 6 h, maximal activity did not occur until 5 days. No radioactivity could be detected in peaks C and D at 6 h. Maximal labelling of these two peaks also occurred at 5 days. These data suggest that the proteins of peaks B, C and D were transported primarily by slow axoplasmic flow. The radioactive protein peaks A and B from the second phase of the transport were excluded from a Sephadex G-200 column, pointing to their high molecular weights (13,000–200,000). Peak B. which had the highest specific radioactivity (c.p.m./mg protein) at 5 days, contained a significant level of tyrosine hydroxylase, an important component of dopaminergic neurons.     

STUDY OF RNA IN SUBCELLULAR FRACTIONS FROM RAT BRAIN: SIMULTANEOUS INCORPORATION OF [14C]URIDINE AND [3H]METHYL-l-METHIONINE

Abstract— By using a combination of subcutaneous and intraventricular injections of [¹⁴C]uridine and [³H]methyl- l -methionine we have obtained maximum incorporation in about 40 min of both radioactive precursors into nuclear RNA from rat brain. In this nuclear fraction we found at least two different types of RNA that were rapidly labelled. One of them incorporated both [¹⁴C]uridine and [³H]methyl groups and seemed to correspond to species of rRNA and their precursors. The other RNA fraction was less methylated or non-methylated and exhibited sedimentation coefficients distributed along a continuous 8–30 % sucrose density gradient. At least part of the latter type of RNA very probably was mRNA, but much of it must conespond to a different RNA similar to that recently described in HeLa cells by P enman , V esco and P enman (1968).
We also found that labelled 185 and 285 rRNA components began leaving the nucleus for the cytoplasm within 24 to 33 min after the radioactive precursors had been injected, and, in the cytoplasmic fraction, the patterns of incorporation for [¹⁴C]uridine and [³H]-methyl groups were similar for the 18S and 28S rRNA components. We estimate that in this fraction of rat brain the 18S rRNA component was 1·4 times more methylated than the 28S component. We also detected a lower sedimentation coefficient for the non- or slightly methylated, species of soluble RNA found in the cytoplasmic fraction.     

Comparison of Axonal Transport of Cytoplasmic- and Particulate-Associated Tubulin in Rat Optic System

Abstract: Adult rats were injected intraocularly with [³⁵S]methionine and killed from 1 to 10 weeks later. Optic nerves, optic tracts, and superior colliculi were dissected and then homogenized and separated into soluble and particulate fractions by centrifugation. Radioactivity coelectrophoresing with tubulin in buffers containing sodium dodecyl sulfate was determined (in cytoplasmic fractions, preliminary enrichment was achieved by vinblastine precipitation). Accumulation of radioactive tubulin along the optic pathway occurred in parallel (and in approximately equal amounts) in cytoplasmic and particulate fractions. Transported tubulin peaked at approximately 2 and 4 weeks in the optic nerve and tract, respectively, corresponding to a transport rate of ～ 0.4 mm/ day. There was little diminution in the amount of transported tubulin between optic nerve and tract, suggesting tubulin was not degraded in the axon. Accumulation in the superior colliculus reached a plateau by 4 weeks at less than 20% of the peak in the optic nerve, indicating turnover of tubulin at the nerve endings. The α/β subunit labeling ratio (radioactivity distribution between the tubulin subunits) was 0.57 for both cytoplasmic- and particulate-transported tubulin. In contrast, this ratio was 0.69 for whole brain tubulin prepared by vinblastine precipitation of soluble material. Isoelectric focusing and two-dimensional gel electrophoresis showed that the subunit compositions (microheterogeneity of the α and β bands) of transported tubulins in the cytoplasmic and particulate fractions were very similar. However, some differences relative to whole brain tubulin were noted; a tubulin subunit not identifiable in whole brain tubulin preparations but present in both soluble- and particulate-transported tubulin was observed. Because of the compositional and metabolic similarities of transported tubulin in the soluble and particulate fractions, we conclude that they form a common metabolic pool. This suggests either that, at least for some membranes, the well-characterized tight association between particulate tubulin and membranes may be artifactual or else that an equilibrium exists between soluble and particulate tubulin.