Tubulin and Neurofilament Proteins Are Transported Differently in Axons of Chicken Motoneurons

SUMMARY 1. We previously showed that actin is transported in an unassembled form with its associated proteins actin depolymerizing factor, cofilin, and profilin. Here we examine the specific activities of radioactively labeled tubulin and neurofilament proteins in subcellular fractions of the chicken sciatic nerve following injection of L-[³⁵S]methionine into the lumbar spinal cord.2. At intervals of 12 and 20 days after injection, nerves were cut into 1-cm segments and separated into Triton X-100-soluble and particulate fractions. Analysis of the fractions by high-resolution two-dimensional gel electrophoresis, immunoblotting, fluorography, and computer densitometry showed that tubulin was transported as a unimodal wave at a slower average rate (2–2.5 mm/day) than actin (4–5 mm/day). Moreover, the specific activity of soluble tubulin was five times that of its particulate form, indicating that tubulin is transported in a dimeric or small oligomeric form and is assembled into stationary microtubules.3. Neurofilament triplet proteins were detected only in the particulate fractions and transported at a slower average rate (1 mm/day) than either actin or tubulin.4. Our results indicate that the tubulin was transported in an unpolymerized form and that the neurofilament proteins were transported in an insoluble, presumably polymerized form.     

Properties of a 110000 molecular weight protein in rat liver hnRNP particles

Particles carrying heterogeneous nuclear RNA (30–40 S-particles) were isolated from rat liver nuclei and the particle proteins separated by sodium dodecylsulfate gel electrophoresis. Some properties of a 110000 molecular weight component (P 110/103) were studied in detail: (i) P 110/103 was labeled to a 4–5 times higher specific activity than the major particle proteins in the presence of [¹⁴C]-amino acidsin vivo. (ii) In nuclei incubated with [³H]- or [³²P]-nicotinamide adenine dinucleotide P 110/103 was labeled presumably by ADP-ribosylation. (iii) A protein with the same molecular weight as P 110/103 and isolated from the nuclear extract by affinity chromatography was phosphorylated in vitro.Abbreviations hnRNA heterogeneous nuclear RNA - hnRNP and mRNP ribonucleoproteins which contain hnRNA respectively mRNA     

Metabolic studies in vitro of the CNS cytoskeletal proteins: Synthesis and degradation

General aspects of metabolic features of the most prominent CNS intermediate filament proteins, the 200,000 (200K), 150,000 (150K), and 70,000 (70K) dalton proteins of the neuron, and the glial fibrillary acidic protein (GFAP) have been explored using the incubated spinal cord slice from the rat. Measurement of shortterm uptake of³H-labeled amino acids into the individual proteins separated on polyacrylamide gels revealed that of the three neurofilament proteins, 200K was most metabolically active, 150K was less active, and 70K contained very little incorporated radioactivity. Glial fibrillary acidic protein based on Coomassie blue stain affinity showed less metabolic activity than any of the neurofilament proteins. Those relationships were constant at all ages, but the metabolic activity of all CNS intermediate filaments decreased with age. When Ca²⁺ was present in the medium of the incubated slices, the intermediate filaments were rapidly destroyed, but GFAP was more resistant to degradation than the neurofilament proteins. GFAP and probably the neurofilament proteins also were relatively resistant to Ca²⁺-activated degradative mechanisms in spinal cords of rats at younger ages (15 day) than in those of older animals (10–18 months). It is likely that the Ca²⁺ activated protease is less active in developing animals in which the nerve tracts are still elongating, than in adults. These results suggest that GFAP is less active metabolically and more resistant to degradation than the neurofilament proteins at all stages of maturation, but that metabolic activity of all CNS intermediate filaments decreases with age while the susceptibility to degradation increases.Special Issue dedicated to Dr. Elizabeth Roboz-Einstein.     

Evidence for the presence and role of tightly bound adenine nucleotides in phospholipid-free purifiedMicrococcus lysodeikticus adenosine triphosphatase

[³²P]-labeled ATPase was isolated in a highly purified state fromMicrococcus lysodeikticus strain PNB grown in medium supplemented with [³²P]orthophosphate. Selective extraction procedures allowed us to determine that at least 25% of the firmly bound label belonged to adenine nucleotides, ATP and ADP being present in equimolar amounts. However, no³²P label was found to be part of phospholipids. This was confirmed by purification of the ATPase from cells fed with [2—³H]glycerol. Using the luciferin-luciferase assay we estimated that ATPase freshly isolated by Sephadex chromatography (specific activity 10–14 µmole substrate transformed · min^–1 · mg protein^–1) contained 2 moles ATP/mole of enzyme. The ratio fell with the age of enzyme and its purification by gel electrophoresis and this was paralleled by a loss of ATPase activity. The endogenous nucleotides were readily exchanged by added ADP or ATP. This result suggests that the sites for tight binding of adenine nucleotides are equivalent, although ADP seems to have a higher affinity for them. The last properties represent a peculiar characteristic of this bacterial ATPase as compared with other bacterial and organelle energy-transducing proteins.     

[32P]orthophosphate and [35S]methionine label separate pools of neurofilaments with markedly different axonal transport kinetics in mouse retinal ganglion cells in vivo

Newly synthesized neurofilament proteins become highly phosphorylated within axons. Within 2 days after intravitreously injecting normal adult mice with [³²P]orthophosphate, we observed that neurofilaments along the entire length of optic axons were radiolabeled by a soluble³²P-carrier that was axonally transported faster than neurofilaments.³²P-incorporation into neurofilament proteins synthesized at the time of injection was comparatively low and minimally influenced the labeling pattern along axons.³²P-incorporation into axonal neurofilaments was considerably higher in the middle region of the optic axons. This characteristic non-uniform distribution of radiolabel remained nearly unchanged for at least 22 days. During this interval, less than 10% of the total³²P-labeled neurofilaments redistributed from the optic nerve to the optic tract. By contrast, newly synthesized neurofilaments were selectively pulse-labeled in ganglion cell bodies by intravitreous injection of [³⁵S]methionine and about 60% of this pool translocated by slow axoplasmic transport to the optic tract during the same time interval. These findings indicate that the steady-state or resident pool of neurofilaments in axons is not identical to the newly synthesized neurofilament pool, the major portion of which moves at the slowest rate of axoplasmic transport. Taken together with earlier studies, these results support the idea that, depending in part on their phosphorylation state, transported neurofilaments can interact for short or very long periods with a stationary but dynamic neurofilament lattice in axons.Special issue dedicated to Dr. Sidney Ochs.     

Synthesis and processing of mammalian protamines and transition proteins

Mouse and rat seminiferous tubule fragment cultures were used to examine synthesis and processing of mammalian protamines and transition proteins. The tubule fragments were incubated with [³H]-arginine, [³H]-histidine, [³⁵S]-cysteine, or [³²P]-PO₄, and radiolabeled proteins were analyzed by acid/urea polyacrylamide gel electrophoresis and fluorography or autoradiography. Newly synthesized protamines were recovered from sonication-resistant nuclei (SRN) and could not be detected in cytoplasmic fractions, indicating that protamines are deposited into nuclei immediately after synthesis. Newly synthesized mouse protamine 1 (mP1) and the precursor to mouse protamine 2 (pre-mP2) migrated more slowly during electrophoresis than their predominant testicular forms, identified by staining with Coomassie blue R-250. Within 1 hour of synthesis, the electrophoretic mobilities of mP1 and pre-mP2 increased to match those of their predominant forms. These changes are consistent with initial charge-neutralizing modifications of the newly synthesized protamines, followed by removal of at least some of the modifying ligands, to unmask protamine basicity. Steady-state phosphorylation rates were high for rat protamine 1 (rP1) and were independent of phosphate content; both rP1 molecules of low and high phosphate content were rapidly phosphorylated. Pre-mP2-3, a major processing intermediate derived by proteolysis of pre-mP2, was also rapidly phosphorylated. Like the protamines, transition protein 2 (TP2) was rapidly phosphorylated and increased in electrophoretic mobility soon after synthesis. In contrast, transition protein 1 (TP1) was not phosphorylated and did not exhibit multiple electrophoretic forms. © 1994 Wiley-Liss, Inc.     

Identification of Low Molecular Mass GTP-Binding Proteins in Membranes of the Halotolerant Alga Dunaliella salina

A family of specific guanine nucleotide-binding proteins in Dunaliella salina was studied. Polypeptides of different subcellular fractions were separated by electrophoresis and transferred to nitrocellulose or Immobilon membranes. Incubation of the transfer blots with [³⁵S]GTPγS or [α-³²P]GTP showed no evidence for GTP-binding proteins in the chloroplast and cytosol fractions. However, two GTP-binding proteins with molecular masses of 28 and 30 kilodaltons were present in the plasma membrane and microsomal fractions. An additional 29 kilodalton GTP-binding protein was detected in the plasma membrane. The mitochondrial fraction contained significant amounts of only the 28 kilodalton GTP-binding protein. Binding of [³²P]GTP to the protein blots was completely prevented by 10 micromolar GTP or guanosine 5′-O-(2-thiodiphosphate) (added in 3 × 10⁴-fold excess), whereas ATP or CTP had no effect on the binding. The 28 kilodalton GTP-binding protein was recognized by polyclonal antibodies to the ras-related YPT1 protein of yeast but not by the anti-ras Y13-259 monoclonal antibody. GTP-binding proteins present in the microsomal fraction could not be solubilized by incubation of microsomes with 1 molar NaCl or 0.2 molar Na₂CO₃, but some GTP-binding activity was solubilized when microsomes were treated with 6 molar urea. These results indicate that D. salina GTP-binding proteins are tightly associated with the membranes. The covalent attachment of fatty acids to these proteins was also investigated. Electrophoresis followed by fluorography of delipidated microsomal proteins extracted from [³H]myristic acid-labeled cells showed an intense labeling of a 28 kilodalton protein. We conclude that D. salina contains proteins resembling the ras-related proteins found in animal cells and higher plants.     

The preparation and use of pyridoxal [32P] phosphate as a labeling reagent for proteins on the outer surface of membranes

Pyridoxal [³²P] phosphate was prepared using [γ-³²P]ATP, pyridoxal, and pyridoxine kinase purified from Escherichia coli B. The pyridoxal [³²P] phosphate obtained had a specific activity of at least 1 Ci/mmol. This reagent was used to label intact influenza virus, red blood cells, and both normal and transformed chick embryo fibroblasts. The cell or virus to be labeled was incubated with pyridoxal [³²P] phosphate. The Schiff base formed between pyridoxal [³²P] phosphate and protein amino groups was reduced with NaBH₄. The distribution of pyridoxal [³²P] phosphate in cell membrane or virus envelope proteins was visualized by autoradiography of the proteins separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis.The labeling of the proteins of both influenza and chick cells appeared to be limited exclusively to those on the external surface of the virus or plasma membrane. With intact red blood cells the major portion of the probe was bound by external proteins, but a small amount of label was found associated with the internal proteins spectrin and hemoglobin.     

The effect of cyclic nucleotides and cholera toxin on in vivo and in vitro phosphorylation of small intestinal brush border membranes

The effect of cyclic nucleotides and cholera toxin on the phosphorylation of the brush border membrane proteins of the rat jejunum was studied. Phosphorylation was analyzed by autoradiography of brush border membrane proteins separated by SDS-polyacrylamide gel electrophoresis. Phosphorylation was performed either in vivo by perfusion of the jejunum with [³²P]orthophosphate followed by an analysis of the isolated membranes or in vitro by phosphorylation of isolated brush border membranes by [γ-³²P]ATP in the presence of saponin. The addition of cholera toxin (10 μg/ml) or dibutyryl-cAMP (5 mmol/l) to the perfusate was unable to produce significant changes in the phosphoprotein pattern. On the other hand, cAMP (at 5 μmol/l) induced an increase of the phosphorylation of a 86 kDa protein when freshly isolated brush border membranes were phosphorylated by [γ-³²P]ATP. However, the same effect could also be induced by low concentrations of cGMP (0.1 μmol/l). It is concluded that brush border membranes from rat jejunum do not contain cAMP-dependent protein kinase activity and that cAMP-dependent protein phosphorylation of this membrane does probably not represent the final event of cholera toxin-induced secretion.     

Protein phosphorylation during 1-methyladenine-induced maturation of Asterias oocytes

Maturation was induced in Asterias oocytes with 1-methyladenine (1-MA) at a final concentration of 2 μM. At 5, 10, and 30 min of treatment, oocytes were homogenized and the cytosolic fraction was prepared. The cytosol was incubated with [γ-³²P]ATP and [γ-³²P]GTP. The phosphorylated proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the radioactivity in the gels was determined by autoradiography. The cytosol prepared from 1-MA-treated oocytes incubated with [γ-³²P]ATP showed a marked increase in the radiolabeling of proteins with estimated molecular weights of 70,000 and 62,000 Da. With [γ-³²P]GTP a 56,000-Da protein showed increased radiolabeling. The present finding suggests that an early biochemical event of 1-MA-induced oocyte maturation in Asterias is the stimulation of phosphorylation of specific proteins.     

Quantitative evaluation of two pathways for phosphatidylcholine biosynthesis in rat brain in vivo

[Me-³H] choline and [³²P] orthophosphate were injected intraventricularly into adult female rats. After variable intervals from injection (1–10 min) the animals were sacrificed by means of a microwave apparatus, and phosphorylcholine and choline phosphoglycerides extracted from brain and counted for radioactivity content after separation. The kinetic constants (K) for phosphorylcholine incorporation into lipids were determined both for [³²P] and [³H] labeling. From the data obtained by these procedures it is concluded that base-exchange reactions for choline incorporation into lipids are operating in rat brain in vivo and that they represent a rapidly equilibrating system.     

Studies on the biosynthesis of neurofilament proteins

To determine whether the triplet polypeptides of neurofilaments arise by degradation of precursor, we studied the biosynthesis of neurofilament polypeptides both in vivo and in cell-free systems. Neurofilament-enriched fractions and polyribosomes were prepared from the same rabbit spinal cord homogenates. At 1 h after intracisternal administration of [34S]methionine, radiolabeled neurofilament proteins were detected in spinal cord homogenates as well as in isolated filaments. When polyribosomes from rabbit spinal cord were allowed to incorporate [35S]methionine into protein, triplet polypeptides were among the proteins labeled. Addition of spinal cord polyribosomes to rabbit reticulocyte lysates led to several cycles of translation of the spinal cord mRNA; the three neurofilament polypeptides were among the proteins synthesized in this system. The results demonstrate that the triplet polypeptides of neurofilaments are synthesized as such in the course of individual translational events and do not arise from degradation of P200 or a larger precursor.     

Regulation of endogenously catalyzed ADP-ribosylation in adipocyte plasma membranes by Ca and calmodulin

The effects of Ca²⁺ and calmodulin on endogenously catalyzed ADP-ribosylation were investigated in adipocyte plasma membranes. Four specific proteins of 70, 65, 61 and 52 kDa were labeled with [³²P]ADP-ribose and ADP-ribosylation of the proteins was highly dependent upon the conditions employed. ADP-ribosylation of the 70 kDa protein was observed only in membranes supplemented with Ca²⁺. Maximal incorporation of [³²P] into the protein was achieved with free Ca²⁺ concentrations of 90 μM. Calcium-stimulated ADP-ribosylation of the 70 kDa protein was inhibited by calmodulin. Half-maximal inhibition was observed in membranes incubated with 1.2 μM calmodulin. The effect of calmodulin was characterized by an inhibition of the incorporation of [³²P]ADP-ribose as opposed to a stimulation of its removal. ADP-ribosylation of the 61 kDa protein was not altered by added Ca²⁺ and/or calmodulin whereas ADP-ribosylation of the 65 kDa protein was partially (50%) inhibited by free Ca²⁺ concentrations between 10⁻⁶ – 10⁻⁵ M. These results provide evidence that the adipocyte plasma membrane contains ADP-ribosyltransferase activities and demonstrate that ADP-ribosylation of a 70 kDa protein is regulated by Ca²⁺ and calmodulin.     

An ATP pool with rapid turnover within the cell membrane

Seven-day-old cultures of rat leg muscle cells were double labelled by addition of [¹⁴C] adenine in the culture medium (2 hrs 15 mins) and followed by addition of [³²P] phosphate (15 min). The specific activity (S.A.) of the isolated cyclic [¹⁴C] adenine 3′ – 5′ monophosphate (cAMP) was similar to that of the bulk ATP. The S.A. of [³²P] from cAMP was, however, higher than that of bulk ATP. The S.A. of [³²P] from cAMP could be further modified by prevention of normal muscle cell fusion. It is probable that the cAMP with high [³²P] S.A. was synthesized from a cell membrane pool of ATP with rapid turnover.     

REGULATION OF THE LEVEL OF ENDOGENOUS PHOSPHORYLATION OF SPECIFIC BRAIN PROTEINS BY DIPHENYLHYDANTOIN1

Diphenylhydantoin (DPH) in therapeutic concentrations caused a decrease in the net level of endogenous phosphorylation of two specific proteins from rat brain cerebri, while not significantly affecting the phosphorylation of other protein substrates. The apparent molecular weights of the DPH- specific substrate proteins were 60-63,000 and 49-52,000, and were designated proteins DPH-L and DPH-M, respectively. DPH decreased both the initial rate and the net level of [³²P] phosphate incorporation from [γ-³²P] ATP into proteins DPH-L and DPH-M. The concentrations of DPH required to produce a half maximal decrease in the levels of phosphorylation of proteins DPH-L and DPH-M was 3 × 10^-4 and 8 × 10 ^-4 M, respectively. The effects of DPH on the incorporation of [³²P] phosphate into these specific brain proteins were independent of the concentration of ATP over a wide range of ATP concentrations. The DPH-specific proteins were demonstrated to be present in synaptosomal preparations. The results are compatible with the hypothesis that some of the stabilizing actions of DPH on neuronal tissue and seizure discharge may be mediated by the effect of this anticonvulsant on the phosphorylation of specific brain protein substrates.     

An easy and rapid method for the measurement of [γ-P]ATP specific radioactivity in tissue extracts obtained from in vitro rat heart preparations labeled with Pi

A rapid method for the measurement of [γ-³²P]ATP specific radioactivity in tissue extracts containing other ³²P-labeled compounds is described. The neutralized acid extract is incubated with cyclic AMP-dependent protein kinase, cyclic AMP and casein. The incorporation of ³²P into casein from [γ-³²P]ATP is measured by perchloric acid precipitation of the protein on filter paper. ³²P-Casein formation is linearly related to the specific radioactivity of the [γ-³²P]ATP. Separation of ATP from other ³²P-labeled compounds is not required for the assay. Application of this method in the evaluation of [γ-³²P]ATP specific radioactivity in two rat cardiac muscle preparations exposed to ³²P_i is demonstrated.     

Patterns of nutrient release from nutrient diffusing substrates in flowing water

As nutrient diffusing substrates age, the availability of nutrients to periphyton may decline with time either because of diffusion or dilution of nutrients into the water column or because of the effects of grazing by herbivores. Typically, large amounts of nutrients are added to nutrient diffusing substrates (NDS) to insure continuous enrichment throughout experimental periods of 2 to 8 weeks. This study examined the release of phosphates and nitrates from NDS exposed to three different current velocities (0.07 m s^–1, 0.11 m s^–1, 0.20 m s^–1) in recirculating laboratory flumes. Replicated agar samples from four treatments (control, nitrate (N), phosphate (P), and N+P) were sampled throughout 32 days (day 1, 2, 3, 6, 12, 18, 24, 32). Increasing concentrations of agar were required to solidify the P and N+P treatments.Nutrient release rates from NDS were independent of agar concentrations (with the exception of [PO₄] in the medium velocity flume). Nutrient concentrations in the agar of spiked samples declined substantially within a week when exposed to flowing water. Nitrates were retained in agar to a greater extent than phosphates particularly when NDS were exposed to low or medium flows. Although floods physically remove or abrade periphyton in natural streams, findings from this laboratory study suggest that ambient flows deplete the availability of nutrient concentrations to potential periphyton colonizers within the first week of incubation. Because of the rapid decline of nutrients from NDS, short incubation periods in natural running waters seem warranted.     

Phosphorylation of muscle plasma membrane protein by a membrane-bound protein kinase

Protein kinase activity has been demonstrated in purified plasma membranes from rat diaphragm by measuring the incorporation of ³²P from [³²P]-ATP into endogenous membrane proteins and into histone, in vitro. Histone appears to be a better substrate than the endogenous membrane proteins; however, the properties of the enzyme are similar when phosphorylating endogenous or exogenous proteins. The activity of this membrane-associated protein kinase is not significantly affected by cyclic adenosine 3′,5′-monophosphate or by cyclic guanosine 3′,5′-monophosphate, but is inhibited by theophylline. The ³²P incorporated into membrane proteins is alkali-labile and is released from the membrane by protease digestion, but it is not removed by phospholipase C, by hydroxylamine, or by chloroform—methanoll extraction. Solubilization of ³²P-labeled membranes by sodium dodecylsulfate and fractionation by sodium dodecylsulfate polyacrylamide gel electrophoresis reveals that the radioactivity is predominantly associated with a single protein band with an apparent molecular weight of about 51 000. The phosphoprotein is a minor membrane component as judged by Coomassie blue staining.     

Cyclic nucleotide- and calcium-independent phosphorylation of proteins in rat brain polyribosome: Effects of ACTH,spermine, and hemin

The incorporation of [-³²P]ATP into proteins of rat brain polyribosomes was studied in vitro. The effects of cyclic nucleotides, calcium, hemin, ACTH, GTP, and spermine were examined. The incorporation of phosphate into proteins increased with time and phosphatase activity was very low; thus, the extent of phosphorylation was predominantly a reflection of protein kinase activity. Phosphorylation of proteins was not sensitive to Ca²⁺ in the presence or absence of either calmodulin or phosphatidylserine. Phosphorylation was also unaffected by cyclic nucleotides in the absence of exogenous enzymes. However, addition of a cMAP-dependent protein kinase together with cAMP resulted in a stimulation of the incorporation of phosphate into 4 phosphoproteins (pp70, pp58, pp43, and pp32); phosphorylation of pp32 was completely dependent on the addition of the kinase. ACTH (1–24), (11–24), and spermine inhibited the endogenous phosphorylation of one protein band (pp30). The phosphorylation of this 30 kD band was also selectively increased by hemin (5 M). Higher concentrations of hemin exerted an inhibitory effect on the majority of the phosphoproteins. Protein phosphatase activity was not influenced by ACTH or spermine. The specific inhibition of pp30 phosphorylation by ACTH or spermine is most probably explained by an interaction with a cyclic nucleotide- and Ca²⁺-independent protein kinase.     

Phosphorylation in vivo of four basic proteins of rat brain myelin

When rat brain myelin was examined by sodium dodecyl sulphate/polyacrylamideslab-gel electrophoresis followed by fluorography of the stained gel, it was found that a host of proteins of rat brain myelin were labelled 2, 4 and 24h after the intracerebral injection of H₃³²PO₄. Among those labelled were proteins migrating to the positions of myelin-associated glycoprotein, Wolfgram proteins, proteolipid protein, DM-20 and basic proteins. The four basic proteins with mol.wts. 21000, 18000 (large basic protein), 17000 and 14000 (small basic protein) were shown to be phosphorylated after electrophoresis in both acid-urea- and sodium dodecyl sulphate-containing gel systems followed by fluorography. The four basic proteins imparted bluish-green colour, after staining with Amido Black, which is characteristic of myelin basic proteins. The four basic proteins were purified to homogeneity. Fluorography of the purified basic proteins after re-electrophoresis revealed the presence of phosphorylated high-molecular-weight `polymers' associated with each basic protein. The amino acid compositions of the phosphorylated large basic protein and small basic proteins are compatible with the amino acid sequences. Proteins with mol.wts. 21000 and 17000 gave the expected amino acid composition of myelin basic proteins. Radiolabelled phosphoserine and phosphothreonine were identified after partial acid hydrolysis of the four purified basic proteins. The [³²P]phosphate–protein bond in the basic protein was stable at an acidic pH but was readily hydrolysed at alkaline pH, as would be expected of phosphoester bonds involving both serine and threonine residues. Double-immunodiffusion analysis demonstrated that the four phosphorylated proteins showed complete homology when diffused against antiserum to a mixture of small and large basic proteins. Since the four basic proteins of rat brain myelin were phosphorylated both in vivo and in vitro it is postulated that the same protein kinase is responsible for their phosphorylation in both conditions.