Electrophoretic analysis of axonally transported proteins in rabbit vagus nerve

Proteins synthesized in the nodose ganglia of rabbits were radiolabeled with 35S-methionine and the proteins present in the vagus nerve, at various times later, were analyzed by SDS (sodium dodecyl sulfate)-polyacrylamide gel electrophoresis. Three major groups of proteins were transported as waves of radioactivity within the nerve at rates of 15-17 mm/h, 12-15 mm/day, and 25-30 mm/day. The front of the fastest wave was composed of two proteins only, of apparent molecular weights 21,000 and 24,000. These were followed after a delay by a number of proteins of higher molecular weight, traveling at the same fast rate. The 25-mm/day wave contained several proteins including a major one of molecular weight 43,000 while the 12-mm/day wave was composed entirely of two proteins of molecular weights 54,000 and 56,000. These groups of slowly transported proteins are therefore similar to those transported much more slowly in other mammalian nerves, with the exception that no proteins with molecular weight similar to the neurofilament proteins could be detected. We have confirmed the dependence of slow transport for both groups of proteins on contact between cell body and axon and suggest that it may be a general phenomenon in all mammalian nerves.     

Formation, size, and solubility in chloroform/methanol of products of protein synthesis in isolated mitochondria of rat liver and Zajdela hepatoma.

The number, size, solubility in chloroform/methanol and some aspects of the formation of the components labeled by radioactive amino acids in isolated mitochondria of rat liver and Zajdela hepatoma were studied. Isolated mitochondria were labeled with radioactive amino acids under various conditions, and the distribution of radioactivity in sodium dodecylsulfate-polyacrylamide gels after electrophoresis of mitochondrial membrane fraction was analysed. 1. Isolated mitochondria of rat liver and Zajdela hepatoma incroporated radioactive amino acids almost exclusively into the membrane fraction. Electrophoretic analysis of this fraction revealed the presence of 15 distinct peaks of radioactivity with corresponding apparent molecular weights of 10 000 to 58 000. The electrophoretic mobility of the labeled components was identical and the general pattern of the radioactivity distribution in the gel for the rat liver and the tumour mitochondria was very similar. 2. Components of the membrane fraction of rat liver mitochondria labeled in vitro displayed an unequal solubility in acidic (2 mM HC1) chloroform/methanol (2/1) mixture; as detected by sodium dodecylsulfate-polyacrylamide gel electrophoresis a single labeled component with apparent molecular weight of 10 000 was soluble in neutral chloroform/methanol. 3. Inverse relation was observed between amino acid incorporation activity of isolated mitochondria and the portion of the label incorporated into the component with apparent molecular weight 10 000. The identity of this component with that soluble in neutral chloroform/methanol mixture has been indicated. 4. The rate of incorporation of [3H]leucine by isolated Zajdela hepatoma mitochondria into the components with lower (10 000-25 000) apparent molecular weights decreased with time, whereas that into components with higher (above 25 000) apparent molecular weight remained approximately constant within the time interval tested (30 min). 5. From the total radioactivity incorporated into the membrane fraction during 5-min pulse labeling of isolated Zajdela hepatoma mitochondria by [3H]leucine up to 25% was recovered in the region of the gel corresponding to a component with apparent molecular weight 10 000. After 25 min chase the radioactivity in this region decreased about 3.5 times while the specific radioactivity of the total membrane fraction did not change significantly. The pattern of radioactivity distribution observed after the pulse was preserved by chloramphenicol. 6. Unlabeled sonicated mitochondria or postribosomal supernatant from rat liver regenerating in the presence of chloramphenicol were incubated with neutral chloroform/methanol extract of in vitro with [14C]leucine labeled rat liver mitochondria. After this incubation several labeled components with apparent molecular weights above 10 000 were recovered in the electrophoreograms of the originally unlabeled fractions.     

The Synthesis and Possible Transport of Specific Proteins by Cells Associated with Brain Capillaries

Abstract: Vinblastine causes alterations in the subcellular distribution of certain proteins synthesized by telencephalon slices. Proteins in various subcellular fractions were separated according to their molecular weight by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and radioactive proteins were determined by autofluorography. A microvascular fraction contained very high amounts of radioactivity in proteins with a molecular weight of 71,000. At least one of these proteins accumulated in the microvascular fraction when the telencephalon slices were incubated in vinblastine. At the same time these proteins became depleted in a myelinated axon fraction, microsomal fraction, and soluble/cytosol fraction. Vinblastine also affected the subcellular distribution of some proteins with a molecular weight below 27,000, but unlike the proteins of mol. wt. 71,000 none of these were synthesized at very high rates. Vinblastine did not effect the synthesis of protein in telencephalon slices, nor did it alter the subcellular fractionation of particles and organelles from slices. It is suggested that a non-neuronal vinblastine-sensitive protein translocation system is functioning within the cells of the microvascular network in telencephalon slices, and that at least one protein of 71,000 molecular weight and one protein with a molecular weight below 27,000 are transported on this system.     

The Posttranslational Arginylation of Proteins in Different Regions of the Rat Brain

The posttranslational incorporation of arginine into proteins catalyzed by arginyl-tRNA protein transferase was determined in vitro in different rat brain regions. The incorporation was found in all the regions studied, although with different specific activities (pmol [14C]arginine incorporated/mg protein). Of the regions studied, hippocampus had the highest specific activity followed by striatum, medulla oblongata, cerebellum, and cerebral cortex. Electrophoretic analysis of the [14C]arginyl proteins from the different regions followed by autoradiography and scanner densitometry showed at least 13 polypeptide bands that were labeled with [14C]arginine. The radioactive bands were qualitatively coincident with protein bands revealed by Coomassie Blue. There were peaks that showed different proportions of labeling in comparison with peaks of similar molecular mass from total brain. Most notable because of their high proportions were those of molecular mass 125 kDa in hippocampus, striatum, and cerebral cortex; 112 and 98 kDa in striatum and cerebellum; and 33 kDa in hippocampus and striatum. In lower proportions than in total brain were the peaks of 33 kDa in medulla oblongata and cerebral cortex and of 125 kDa in medulla oblongata.     

Different Polypeptides Are Rapidly Transported in Auditory and Optic Neurons

Abstract: Rapidly transported proteins and glycoproteins in the auditory and optic nerves of the guinea pig were analyzed by electrophoresis and two-dimensional electrofocusing/electrophoresis. Proteins transported in the auditory nerve were analyzed in the cochlear nucleus 3 h after cochlear injection of radioactive precursor, and proteins transported in the optic nerve were analyzed in the superior colliculus 6 h after intraocular injection of radioactive precursor. Two-dimensional analysis showed that several rapidly transported polypeptides were present in one system, but not in the other. By use of [³H]fucose as a precursor or by separating [³⁵S]methionine-labeled polypeptides on immobilized concanavalin A or wheat germ agglutinin, it was shown that most of the proteins transported in only one system are glycoproteins. As previously reported a polypeptide of molecular weight 140,000 was a major labeled species in the auditory nerve. This polypeptide was also found in the optic nerve, but only as a minor species. Two other polypeptides with molecular weights and isoelectric points similar to those of the 140,000 molecular weight polypeptide were present in both systems, but were much more abundant in the optic nerve. The major labeled polypeptide in both systems had a molecular weight of 25,000.     

Characterization of proteins transported at different rates by axoplasmic flow in the dorsal root afferents of rats.

Proteins synthesized by soma located in L4 dorsal root ganglia and supplied to the axonal branches extending centrally in the dorsal root and peripherally towards the sciatic nerve were analyzed for radioactivity following injections of [3H] leucine into the L4 dorsal root ganglia. All proteins located in the dorsal root and sciatic nerve were analyzed by SDS acrylamide gel electrophoresis at various times post injection. The differences in radioactivity between the dorsal root and sciatic nerve proteins were mainly quantitative and not qualitative, with many proteins of various molecular weight ranges being transported into both segments. Generally, it appears that in both axonal branches the high molecular weight proteins are transported at the highest rate, medium weights slower and low molecular weight proteins slowest. More proteins of high and low molecular weights are transported into the dorsal root whereas more of those of medium molecular weight are transported towards the sciatic nerve.     

ELECTROPHORETIC ANALYSES OF PROTEINS TRANSPORTED TO THE RAT POSTERIOR PITUITARY

Abstract— [³⁵S]cysteine, [³H]methionine, or [³H]fucose were injected into the supraoptic nuclei (SON) of rats, and the labelled proteins that were transported to and accumulated in the posterior pituitary 24h post-injection were analyzed electrophoretically. The transported, labelled proteins which were soluble in 0.1 m -HCl were primarily of low molecular weight (about 12,000 on SDS gels). However, the selectivity of labelling of these proteins by the three different labelled precursors could be revealed by isoelectric focusing. The 0.1 m -HCl insoluble labelled proteins, presumably reflecting membrane proteins transported from the SON to the pituitary, were more diverse and generally of higher molecular weight (> 43,000 on SDS gels).     

Changes in the pattern of RNA synthesis in different tissues of Xenopus larvae during induced metamoprhosis

The pattern of newly synthesized RNA in liver, tail muscle, brain and muscle of hindlimbs during induced metamorphosis was analysed by electrophoretic separation of RNA extracts on exponential polyacrylamide gels. During the first 24 h of thyroxine treatment the distribution of labelled RNA was identical in treated animals and controls. Four days after induction of metamorphosis relatively more radioactivity was observed in stable RNA, especially rRNA, whereas unstable RNA, mainly HnRNA, was relatively less labelled. Apart from these general effects, in tail muscle thyroxine treatment caused the disappearance of a definite high molecular weight RNA. Moreover maturation of 28 S rRNA was accelerated and degradation of newly synthesized rRNA was no more detectable.     

SDS GEL ELECTROPHORESIS OF RAPIDLY TRANSPORTED PROTEINS IN GARFISH OLFACTORY NERVE

Abstract— Proteins undergoing rapid axonal transport in the garfish olfactory nerve were examined by sodium dodecyl sulphate gel electrophoresis. The distribution of polypeptides and the extent of their labeling by transported molecules was determined in several nerve subfractions including: total particulate, total membrane, mitochondrial and two membrane subfractions rich in axolemma. The polypeptide composition of the various fractions was found to be relatively similar, with each showing a major protein with an estimated MW of 58,000. Specific differences in the concentrations of certain proteins were noted between fractions, including differences between the lower and higher density axolemma rich subfractions. Axonally transported radioactivity was predominantly localized among high molecular weight proteins, with all fractions, except mitochondrial pellet, displaying a major peak of radioactivity centered at 126,000-MW. Several major proteins including the 58,000-MW band were labeled by rapid transport to a much smaller extent. Certain labeled peaks were found to be concentrated in individual fractions, particularly a polypeptide (MW 35,000) more predominantly found in the lower density axolemma rich fraction.
Systemic labeling of the nerve is found to give a general distribution of radioactivity on gels, which is clearly different from the pattern obtained after axonal transport labeling.     

Characterization of proteins transported at different rates by axoplasmic flow in the dorsal root afferents of rats

Proteins synthesized by soma located in L₄ dorsal root ganglia and supplied to the axonal branches extending centrally in the dorsal root and peripherally towards the sciatic nerve were analyzed for radioactivity following injections of [³H] leucine into the L₄ dorsal root ganglia. All proteins located in the dorsal root and sciatic nerve were analyzed by SDS acrylamide gel electrophoresis at various times post injection. The differences in radioactivity between the dorsal root and sciatic nerve proteins were mainly quantitative and not qualitative, with many proteins of various molecular weight ranges being transported into both segments. Generally, it appears that in both axonal branches the high molecular weight proteins are transported at the highest rate, medium weights slower and low molecular weight proteins slowest. More proteins of high and low molecular weights are transported into the dorsal root whereas more of those of medium molecular weight are transported towards the sciatic nerve.     

(51CR) sodium chromate incorporation ito the soluble protein fractin of the human erythrocyte: binding not associated with the hemoglobin monomeric subunit.

The distribution of radioactive chromium on proteins isolated in the soluble fraction of the human erythrocyte was studied utilizing sodium dodecyl sulfate polyacrylamide gel electrophoresis. The radioactive label was associated with the hemoglobin monomeric subunit and, unexpectedly, with another soluble fraction protein. This protein was approximately 6% of the total isolated protein, contained approximately 20% of the radioactivity, and had an estimated molecular weight of 26,700 daltons.     

Poly-ADP-ribosylation of nuclear proteins in differentiating Friend cells

Isolated nuclei from exponentially growing Friend cells and from cells that had been induced to synthesize hemoglobin were incubated with radioactive NAD under conditions favourable for poly-(ADP-ribose) synthesis. Both in control and induced cells between 5 and 10 % of the total nuclear acid-insoluble radioactivity was found in the histone fraction. Specific activity remained unaltered in non-histone proteins but increased 2–2.5 fold in the histone fraction during induction. Incubation of nuclei from control and from induced cells with radioactive NAD resulted in the appearance of new protein bands in the SDS-polyacrylamide gel electrophoretic pattern of the acid-extracted nuclear proteins. The majority of radioactivity from NAD comigrated with these bands.     

ASSOCIATION OF AXONALLY TRANSPORTED PROTEINS WITH GOLDFISH BRAIN MYELIN FRACTIONS

—The presence of rapidly transported axonal proteins in purified preparations of myelin has been investigated in the goldfish visual system. Fish were injected intraocularly with ³H proline and contralateral optic tecta were pooled 8–12 h later for purification of myelin. Three purification procedures were employed using continuous and discontinuous gradients of sucrose and continuous gradients of CsCl. All of the myelin preparations were found to have physical, chemical and enzymatic properties attributable to relatively pure preparations of myelin. The goldfish myelin differed from mammalian preparations in having a slightly lower density and in containing an additional major protein of approx. 45,000 mol. wt. All of the myelin preparations retained relatively high levels of axonally transported radioactivity with specific radioactivities which ranged from 70 to 80 per cent of that of the whole tectal homogenate. Acrylamide gel analysis showed the myelin-associated radioactivity to be confined to the higher molecular weight proteins with very little radioactivity associated with basic protein or proteolipid protein. Both the axonally transported radioactivity and the group of higher molecular weight proteins were found to be more concentrated in a myelin subfraction of relatively high density than in a subfraction of low density. The possible significance of the association of axonally transported proteins with myelin is discussed.     

Putative histidin-rich proteins in the epidermis of lizards

In the stratum granulosum of mammalian epidermis, histidin-rich proteins (filaggrins) determine keratin clumping and matrix formation into terminal keratinocytes of the stratum corneum. The nature of matrix, interkeratin proteins in the epidermis of nonmammalian vertebrates, and in particular in that of reptilian, mammalian progenitors are unknown. The present biochemical study is the first to address this problem. During a specific period of the renewal phase of the epidermis of lizards and during epidermal regeneration, keratohyalin-like granules are formed, at which time they take up tritiated histidine. The latter also accumulate in cells of the alpha-keratin layer (soft keratin). This pattern of histidine incorporation resembles that seen in keratohyalin granules of the stratum granulosum of mammalian epidermis. After injection of tritiated histidine, we have analysed the distribution of the radioactivity by histoautoradiography and electrophoretic gel autoradiography of epidermal proteins. Extraction and electrophoretic separation of interfilamentous matrix proteins from regenerating epidermis 3-48 hours post-injection reveals the appearance of protein bands at 65-70, 55-58, 40-43, 30-33, 25-27, and 20-22 kDa. Much weaker bands were seen at 100, 140-160, and 200 kDa. A weak band at 20-22 kDa or no bands at all are seen in the normal epidermis in resting phase and in the dermis. In regenerating epidermis at 22 and 48 hours post-injection, little variation in bands is detectable, but low molecular weight bands tend to increase slightly, suggesting metabolic turnover. Using anti-filaggrin antibodies against rat, human, or mouse filaggrins, some cross-reactivity was seen with more reactive bands at 40-42 and 33 kDa, but it was reduced or absent at 140, 95-100, 65-70, 50-55, and 25 kDa. This suggests that different intermediate degradative proteins of lizard epidermis may share some epitopes with mammalian filaggrins and are different from keratins with molecular weight ranging from 40 to 65-68 kDa. The immunocytochemical observation confirms that a weak filaggrin-like immunoreactivity characterizes differentiating alpha-keratogenic layers in normal and regenerating tail. A weak filaggrin labeling is discernable in small keratohyalin-like granules but is absent from the larger granules and from mature keratinocytes. The present results indicate, for the first time, that histidine-rich proteins are involved in the process of alpha-keratinization in reptilian epidermis. The cationic, interkeratin matrix proteins implicated may be fundamentally similar in both theropsid-derived and sauropsid amniotes.     

Distribution of radioactive silver in the subcellular fractions of various tissues of the rat and its binding to low molecular weight proteins

In view of the electron microscopic evidence that silver does not penetrate cellular barriers, the distribution of radioactive silver in rat blood and subcellular fractions of liver, kidneys, spleen, and forebrain was studied. It was found that 24 h after a single intraperitoneal injection high levels of radioactivity were reached which decreased at different rates in the various tissues studied. In plasma, liver, and kidneys there was an initial rapid loss of radioactivity which was followed by a slower rate of loss. In the blood, forebrain, and spleen the loss of radioactivity was linear and somewhat slower than in the other three tissues. The cytosols of the liver and kidneys contained 60% while those of the forebrain and spleen contained 30% of the total radioactivity found in the tissue homogenates. Gel filtration on Sephadex G-75 showed that all cytosols contained two peaks of radioactivity; a high molecular weight peak which eluted just after the void volume and a low molecular weight peak. The amount of radioactivity in both peaks was, however, much lower in the chromatographic peaks of the forebrain and spleen than that found in those of the liver and kidneys. Furthermore, the spleen had a comparatively very small low molecular weight radioactive peak. In vitro experiments with liver cytosol showed similar results to those found in vivo in that the high molecular weight radioactive peak could be removed by heat. It is concluded that silver does enter cells and that silver thionein exists in the cytosols of forebrain, spleen, kidney, and liver.     

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.     

SUBCELLULAR DISTRIBUTION OF RADIOACTIVITY IN NEURONAL AND GLIAL-ENRICHED FRACTIONS AFTER INCORPORATION OF [3H]LEUCINE IN VIVO AND IN VITRO

Abstract— The distribution of protein-bound radioactivity among subcellular organelles of cerebral cortex was followed after intravenous administration of [3H]leucine and after incubation of brain slices in the presence of [3H]leucine. Neuronal and glial cell-enriched fractions were prepared by discontinuous sucroseFicol1 gradient centrifugation of cerebral cortex cell suspensions. Subcellular fractions were obtained from each of the cell prepara- tions and the protein-bound radioactivity determined after in uiuo and in vitro incorporation of [3H]leucine. The unfractionated neuronal material had a considerably higher level of protein-bound radioactivity than the glial material. The most marked neuronal-glial dif- ferences were observed in microsomes and soluble proteins, while the radioactive labelling of the nuclear and mitochondria1 fractions was similar for the two cell types.     

Electrophoretic and immunological charactorization of rat neurophysin

1. The electrophoretic properties of rat posterior pituitary proteins have been compared on starch gel with those of bovine and porcine neurophysins. 2. [(35)S]-Cysteine was injected into the supraoptic nucleus of male rats and 16-24h later the distribution of labelled neural-lobe protein in starch and polyacrylamide gels was determined. In both systems a single major protein component was found to contain more than 80% of the total recovered radioactivity. Between 5 and 10% of the radioactivity was found in a minor component in polyacrylamide gel. 3. In agar, microimmuno-diffusion and -electrophoresis of the rat neural-lobe proteins gave a single arc with neurophysin antiserum, and after starch-gel electrophoresis this arc was shown to be due to the major labelled component. 4. The molecular weights of the rat neural-lobe proteins were estimated by polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. The molecular weight of the major labelled component was found to be 12000. 5. It is concluded that the rat neurophysin consists of one major and possibly one minor component.     

CBP-18, a Ca2+-Binding Protein in Rat Brain: Tissue Distribution and Localization

Abstract: The distribution of a novel calcium-binding protein with a molecular mass of 18 kDa (CBP-18) in the rat brain was studied by means of biochemical methods and immunohistochemistry on cryostat-sectioned tissue and compared with staining patterns of parvalbumin on adjacent sections. The biochemical analysis revealed high levels of CPB-18 in cortex and cerebellum, low levels in the lungs, and undetectable levels in all other tissues tested. Immunohistochemically, the polyclonal rabbit-derived antibody for CPB-18 showed selective affinity with periglomerular cells and dendrites in the olfactory bulb. Distinct immunostaining of scattered cells and their proximal dendrites was found in the anterior olfactory nuclei and in the perirhinal and entorhinal cortex. Strong staining of neuropil with recognizable but diffusely outlined cells was observed in the retrosplenial cortex, central amygdala, hippocampal rudiment, septum, area preoptica, hypothalamus, colliculus superior, and parabrachial nuclei. The cerebellum showed strong neuropil staining of both the molecular and the granule cell layer. Less intense neuropil staining and a few scattered cells were found in the neocortex, the remaining basal forebrain, and in the entire brainstem. Immunoreactivity was barely detectable or missing in the striatum, the hippocampus, the thalamus, and in the colliculus inferior. Thus, CPB-18 shows a unique staining pattern in the CNS, different from all other Ca²⁺-binding proteins studied so far.     

Axonal Transport, Deposition, and Metabolic Turnover of Glycoproteins in the Rat Optic Pathway

Abstract: Following intraocular injection of [³H]fucose in the rat, radioactive glycoproteins are rapidly transported to the nerve terminals in at least two waves, one with a peak at 8 h and a second with a peak at about a week. The molecular weight distribution of radioactive peptides in ach transport wave as determined by gel electrophoresis in buffers containing sodium dodecyl sulfate is very similar. Most of the many glycopeptides in the first wave of rapid transport pass through the optic tract in unison (apparent half-life of about 15 h) and are preferentially destined for the nerve endings. However, two proteins of apparent M. W. 28,000 and 49,000 are preferentially retained in the axons. The remaining proteins, after reaching the nerve endings (superior colliculus), decay with apparent half-lives ranging from 17 to 34 h. During the second wave a large amount of the 28,000 and 49,000 M. W. peptides are again preferentially retained in the axons. The remaining proteins, on reaching the nerve endings, decay with apparent half-lives ranging from 5 to 9 days. Subcellular fractionation of the superior colliculus supports the hypothesis that the 49,000 and 28,000 M. W. peptides are the predominantly labeled glycoproteins present in myelinated axons (representing over 50% of the radioactive glycoproteins 7 days following injection), although they are probably also present in membranes of the nerve endings. A comparison with glycoprotein transport in other tracts (geniculocortical and nigrostriatal tracts) suggests that glycoprotein transport in these pathways has many similarities to glycoprotein transport in the retinal ganglion cells, and that the optic system is a good general model for axonal transport in the CNS.