In vivo incorporation of l-[14C]serine into phospholipids and proteins of the subcellular fractions of developing rat brain

A study was conducted on the in vivo incorporation of l -[¹⁴C]-serine into the lipids and proteins of the various subcellular fractions of the developing rat brain before and during the stage of active myelination. The total radioactivity in the various fractions at 12 days of age was higher than that at 3 days, while the radioactive specific activity was reversed. The specific activities of the proteins and lipids were higher at 3 days of age with the exception of the subcellular fraction containing myelin. At both ages the lipids of the various cellular fractions had similar specific activities, a finding that suggests a common source for lipid biosynthesis. Incorporation of radioactivity into the various phospholipids was in the following order: phosphatidyl serine > phosphatidyl ethanolamine > phosphatidal serine > sphingomyelin and phosphatidyl choline. Of all the phospholipids, the plasmalogens increased most in total radioactivity during the period when meylination was most active. Serine-containing phospholipids appear to be most tightly bound to proteins. The brain mitochrondrial fraction contained most of the phosphatidyl serine decarboxylase activity with some activity in the nuclei. Biosynthesis of phosphatdyil ethanolamine through decarboxylation of phosphatidyl serine could take place in rat brain. Four unidentified radioactive metabolites were found in the acid-soluble fraction in addition to l -[¹⁴C]serine.     

Phosphoprotein phosphatase activity in the thyroid.

Phosphoprotein phosphatase activity in the calf thyroid was found in various subcellular fractions. The relative amount in each fraction varied according to the substrate used: The 500g fraction had the highest specific activity when protamine was used, while the 5000g fraction was highest when histone was used. Triton X-100 tended to increase activity in all the particulate fractions, the greatest change being found in the 105,000g pellet. DEAE chromatography of the 105,000 g supernatant resolved at least three peaks of phosphoprotein phosphatase activity.     

Experimental alcoholism in rats. Effect of acute ethanol intoxication on the in vitro incorporation of ( 3 H)leucine into neuronal and glial proteins

Abstract— Ethanol administered in vivo or in vitro during incubation of brain slices was studied with respect to its effect on brain protein synthesis. In the in vivo series the rats were given a single intraperitoneal injection of ethanol 3 h before death. Slices of cerebral cortex and liver were incubated in isotonic saline media containing [³H]leucine. Amounts of free and protein-bound radioactivity were determined. Subcellular fractions and fractions enriched in neuronal perikarya and in glial cells were prepared from cortical slices subsequent to incubation, and the specific radioactivity determined for each cell type. The incorporation of [³H]leucine into brain proteins was inhibited while incorporation into liver proteins was stimulated in ethanol-treated rats. The levels of TCA-soluble radio-activity, however, did not differ between the ethanol group and the controls. In the fractionated material from cerebral cortex, the specific radioactivity in the neuronal fraction was unaffected by ethanol, while the radioactivity in the glial fraction was significantly depressed. In vitro administration of ethanol induced a non-linear response in both brain and liver, with depression of leucine incorporation into proteins of cerebral cortex at all concentrations used. When brain slices were exposed to ethanol in vitro, in concentrations corresponding to the in vivo experiments, a similar reduction of the leucine incorporation into the glial fraction was obtained. Incorporation of leucine into subcellular fractions from whole brain cortex was also investigated. The specific sensitivity of the glial fraction to ethanol is discussed in relation to the involvement of the different cell types with transport processes in the brain.     

Microinjection of ubiquitin: intracellular distribution and metabolism in HeLa cells maintained under normal physiological conditions

Radioiodinated ubiquitin was introduced into HeLa cells by erythrocyte-mediated microinjection. Subsequent electrophoretic analyses revealed that the injected ubiquitin molecules were rapidly conjugated to HeLa proteins. At equilibrium, 10% of the injected ubiquitin was conjugated to histones and 40% was distributed among conjugates of higher molecular weight. Although the remaining ubiquitin molecules appeared to be unconjugated, the free pool of ubiquitin decreased by one-third and additional conjugates were present when electrophoresis was performed at low temperature under nonreducing conditions. Molecular weights of these labile conjugates suggest that they are ubiquitin adducts in thiolester linkage to activating enzymes. Despite the fairly rapid degradation of injected ubiquitin (t1/2 approximately 10-20 h), the size distribution of ubiquitin conjugates within interphase HeLa cells remained constant for at least 24 h after injection. The intracellular locations of ubiquitin and ubiquitin conjugates were determined by autoradiography, by differential sedimentation of subcellular fractions in sucrose, and by extraction of injected cells with buffer containing Triton X-100. Free ubiquitin was found mostly in the cytosolic or Triton X-100-soluble fractions. As expected, histone conjugates were located predominately in the nuclear fraction and exclusively in the Triton X-100-insoluble fraction. Although high molecular weight conjugates were enriched in the Triton X-100-insoluble fraction, their size distribution was similar to that of soluble conjugates. When injected HeLa cells were exposed to cycloheximide to inhibit protein synthesis, the size distribution of ubiquitin conjugates was similar to that found in untreated cells. Moreover, high molecular weight conjugates decreased less than 20% after inhibition of protein synthesis. These results indicate that most ubiquitin conjugates are not newly synthesized proteins which have been marked for destruction.     

Guanine-deaminase activity in rat brain and liver

1. Guanine deaminase in rat brain and liver was distributed among all the subcellular fractions: nuclei, `heavy' mitochondria, `light' mitochondria, microsomes and the supernatant fluid. The greater part of the activity passed into the soluble fraction. Among the particulate components, the `light' mitochondria constituted the richest fraction. 2. The sum of the enzymic activities of the component fractions obtained on differential centrifugation was considerably greater than the activity of guanine deaminase in the whole homogenate. 3. The `heavy'-mitochondrial fraction had a powerful inhibitory effect on the guanine-deaminase activity of the supernatant fraction. 4. All the sedimented fractions, except the microsomes, gave rise to higher guanine-deaminase activity on treatment with Triton X-100. 5. The inhibitory capacity of the `heavy' mitochondria increased on treatment with Triton X-100; the detergent-treated nuclear fraction also brought about inhibition of the 5000g supernatant. 6. Guanine-deaminase inhibitor from the `heavy' mitochondria was solubilized by high-speed grinding of the particles, followed by treatment with Triton X-100. The inhibitor appeared to be protein in nature, since it was precipitated by trichloroacetic acid and by half-saturation with ammonium sulphate, and was non-diffusible. It was inactivated by heating at 50° for 5min. 7. It is possible that the guanine deaminase associated with particles differs from the soluble enzyme in its response to inhibitor.     

Evidence for Membrane-Associated Choline Kinase Activity in Rat Striatum

The distribution of choline kinase (EC 2.7.1.32) activity was investigated in subcellular fractions of rat striatum. Enzyme activity in the crude mitochondrial fraction, determined after dissolution in Triton X-100, was 5.90 mumol/g initial wet weight/h. When a crude mitochondrial preparation was hypoosmotically shocked and fractionated, followed by the addition of Triton X-100, choline kinase activity in the soluble and particulate fractions was 4.58 and 1.40 mumol/g initial wet weight/h, respectively. Enzyme activity in the particulate fraction was not detected in the absence of Triton X-100 or in the presence of NaCl (up to 1.5 M). Subcellular enzyme markers indicated that the membrane-associated activity was not attributable to mitochondrial or microsomal contamination. Kinetic analysis of the activity of soluble and membrane-solubilized choline kinase indicated Km values of 0.74 mM and 0.68 mM, respectively. Results indicate that choline kinase activity may be measured in both the soluble and the particulate fractions of rat striatum, the latter most likely involving enzyme associated with membrane through hydrophobic or covalent interactions. The specific function of the membrane-associated enzyme has not yet been determined.     

Protein synthesis in neurons and glial cells of the developing rat brain: an in vivo study

Abstract— A technique for the isolation of pure neuronal perikarya and intact glial cells from cerebral cortex has been developed for routine use. The yield of neuronal perikarya and glial cells was greater from highly immature (5–10 days) rat cerebral cortex than from the cortex of older rats (18–43 days). The perikarya/glia yield ratio decreased with age indicating that, as the glial population matured, the procedure succeeded in isolating a gradually smaller proportion of the existing neurons. The perikarya/glia ratio was highest for the 5-day-old cortex in which no mature glial cells could be identified. After a 10-min pulse in vivo of intrathecally injected [¹⁴C]phenylalanine, the specific radioactivity of the neuronal proteins was higher than that of the glial proteins in the 5-, 10- and 18-day-old rat but was lower in the 43-day-old rat. The values for absolute specific radioactivity of the ¹⁴C-labelled proteins in both cell types were greater, the younger the brain. The ¹⁴C-labelling of neuronal and glial proteins in the 18-day-old rat was assessed in vivo as a function of time by determining the incorporation of [¹⁴C]phenylalanine into such proteins at 5, 10, 20 and 45 min after administration of the amino acid. The rate of incorporation of [¹⁴C]phenylalanine into the glial cells was faster than into the neurons since higher specific radioactivities of the glial proteins could be achieved at earlier times. Also, a biphasic pattern of ¹⁴C-labelling of the glial proteins was noted, suggesting, perhaps, a sequential involvement of the oligodendrocytes and astrocytes. Homogenates of prelabelled neuronal perikarya were fractionated into the nuclear, mitochondrial microsomal and soluble cell sap fractions. In the 18-day-old cerebral cortex, the proteins of the microsomal fraction exhibited the highest specific radioactivity at the end of 10 min, whereas by 20 min proteins of the mitochondrial fraction were most highly labelled. The specific radioactivity of the nuclear proteins increased over the entire 45-min experimental period. On the contrary, the proteins of the soluble cell sap, in which the specific radioactivity was at all times by far the lowest, were maximally labelled by 5 min. Examination of the labelling of the neuronal subcellular fractions as a function of age revealed that at 10 min after administration of [¹⁴C]phenylalanine, the specific radioactivities of all ¹⁴C-labelled proteins were highest in the youngest (5-day-old) neurons. The proteins of the microsomal fraction were most rapidly labelled at all ages. During this interval the proteins of the soluble cell sap were only moderately labelled in the 5-day-old neurons and were totally unlabelled in the 43-day-old neurons, indicating age-dependent differences in the rate of utilization of the amino acid precursor by the neurons.     

Identification of the protein producing transmembrane diffusion pores in the outer membrane of Pseudomonas aeruginosa PA01

The outer membrane of Pseudomonas aeruginosa PA01 is permeable to saccharides of molecular weights lower than about 6000. Triton X-100/EDTA-soluble outer membrane proteins were fractionated by ion-exchange chromatography in the presence of Triton X-100 and EDTA, and the protein contents of the various fractions analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis. Each of the major protein bands present in the Triton X-100/EDTA soluble outer membrane was separated from one another. Adjacent fractions were pooled, concentrated and extensively dialyzed to reduce the Triton X-100 concentration. Vesicles were reconstituted from lipopolysaccharide, phospholipids and each of these dialyzed fractions, and examined for their ability to retain [¹⁴C]sucrose. Control experiments indicated that the residual levels of Triton X-100 remaining in the dialyzed fractions had no effect on the formation or permeability to saccharides of the reconstituted vesicles. It was concluded that a major outer membrane polypeptide with an apparent weight of 35 000 is a porin, responsible for the size-dependent permeability of the outer membrane.     

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.     

CYTOPLASMIC SYNTHESIS OF NUCLEAR PROTEINS : Kinetics of Accumulation of Radioactive Proteins in Various Cell Fractions after Brief Pulses

The synthesis of cytoplasmic and nuclear proteins has been studied in HeLa cells by examining the amount of radioactive protein appearing in the various subcellular fractions after labeling for brief periods. Due to the rapid equilibration of the amino acid pool, the total radioactivity in cytoplasmic protein increases linearly. The radioactivity observed in the cytoplasm is the sum of two components, the nascent proteins on the ribosomes and the completed proteins. At very short labeling times the specific activity of newly formed proteins found in the soluble supernatant fraction (completed protein) increases as the square of time, whereas the specific activity of the ribosomal fraction (nascent protein) reaches a plateau after 100 sec. The kinetics of accumulation of radioactive protein in the nucleus and the nucleolus is very similar to that of completed cytoplasmic protein, which suggests that the proteins are of similar origin. The rate of release and migration of proteins from the ribosomes into the nucleus requires less time than the synthesis of a polypeptide, which is about 80 sec. The uptake of label into nucleolar proteins is as rapid as the uptake of label into proteins of the soluble fraction of the cytoplasm, while nuclear proteins, including histones, tend to be labeled more slowly. The same results are obtained if protein synthesis is slowed with low concentrations of cycloheximide. The kinetics of incorporation of amino acids into various fractions of the cell indicates that the nucleus and the nucleolus contain few if any growing polypeptide chains, and thus do not synthesize their own proteins.     

Synaptic junctional glycoconjugates from chick brain

Forebrains from day-old chicks were homogenized and fractionated by differential sedimentation and density gradient centrifugation to yield subcellular fractions. The synaptosomal plasma membrane fraction was further treated with Triton X-100 to yield subsynaptic membrane fractions including synaptic junctions. Glycoproteins from these subsynaptic membrane fractions were identified after separation by SDS-polyacrylamide gel electrophoresis by incubating the gel slabs with radioiodinated concanavalin A. Two lectin-binding proteins were discerned in the synaptic junction fraction while none were observed in the Triton-soluble portion of the synaptic plasma membrane. The carbohydrate content of the glycoproteins from each subcellular fraction was quantitated after methanolysis and derivatization aso-methyl-trifluoroacetyl analogs by gas-liquid chromatography. The lowest concentration of glycoprotein sugars was found in the synaptic junction, mitochondrial, and soluble fractions while the greatest concentration was found in the myelin, light-synaptic plasma membrane, and the Triton-soluble portion of the synaptic plasma membrane. Of the subcellular fractions, the synaptic junction contained the highest porportion of mannose and lowest proportion of sialic acid. Moreover, this fraction's content of galactose andN-acetylglucosamine, relative to mannose was the lowest while its content of fucose was low. The oligosaccharide chains extending into the synaptic cleft therefore are predominantly of the neutral, mannose-rich type and are attached to a limited number of high-molecular-weight glycoproteins.     

The fate of an N-formylated chemotactic peptide in stimulated human granulocytes. Subcellular fractionation studies

Experiments were performed to examine how human granulocytes process the chemotactic peptide N-formyl-Met-Leu-Phe after stimulation by the same peptide. Purified human granulocytes were stimulated with 50 nM N-formyl-Met-Leu-[3H]Phe at 37 degrees C for various times, washed, lysed by N2 cavitation, and fractionated by isopycnic sucrose density gradient sedimentation. The major subcellular fractions identified were plasma membrane, Golgi, granules, endoplasmic reticulum, and mitochondria. After 1 min of stimulation, radioactivity was found only in the plasma membrane (sedimentable) and cytosol (soluble) fraction. At 5, 10, and 25 min, radioactivity also appeared in a sedimentable, low density fraction (25-28% sucrose) enriched in galactosyl transferase activity and containing Golgi structures. The accumulation in the sedimentable fractions was maximal after 5 min but continued to increase linearly in the cytosol fraction. Incorporation of radioactivity into cells or membrane and soluble fractions was 60 to 85% specific and was inhibited if incubation with N-formyl-Met-Leu-[3H]Phe was performed at 4 degrees C. 80-90% of the radiolabel in the plasma membrane or Golgi-containing fractions remained sedimentable despite freeze thawing or sonication. Solubilization of these fractions in Triton X-100 followed by Sepharose 4B column chromatography revealed that the radiolabel eluted in the void volume. Our results are consistent with internalization which proceeds by passage of an occupied receptor in a high affinity, supramolecular complex from the plasma membrane to the Golgi followed by accumulation of peptide in the cytosol.     

The disc electrophoretic separation of proteins from various parts of the guinea pig brain

—Disc electrophoresis was used to study the saline-soluble and detergent-soluble proteins of various parts of the auditory pathway in the guinea pig brain. The five areas studied were the cochlear nucleus, olivary complex, inferior colliculus, medial geniculate and the auditory cortex. A study was also made of the proteins extracted from subcellular fractions obtained from guinea pig cerebral cortex. The electrophoretic patterns showed small but significant differences in the five areas of the brain, both in the fast-running acidic proteins and in the very slow-moving proteins. The higher levels of the auditory pathway, to which the more complex information processing and storage is normally attributed, showed more complex electrophoretic patterns than the lower areas. Gross differences also occurred in the patterns of both the saline-soluble and T-X-100-soluble proteins of the subcellular fractions obtained by gradient density centrifugation. The simplest patterns were obtained from the myelin-rich fraction and the mitochondrial fraction whilst the most complex patterns were given by the proteins of the nerve ending fraction and the cell soluble fraction.     

ISOLATION AND CHARACTERIZATION OF SOLUBLE ACIDIC LIPOPROTEINS FROM RAT BRAIN SYNAPTIC VESICLES

—Highly purified fractions of synaptic vesicles were prepared from rat cerebrum or cerebral cortex by density gradient centrifugation. Treatment of synaptic vesicle fractions by autoincubation, freeze-thawing and sonication in an isotonic alkaline-salt medium or in 0·1-0·3% (v/v) Triton X-100 released increasing quantities of synaptic vesicle protein and phospholipid into solution. When the soluble synaptic vesicle proteins were extracted with 0·1% (v/v) Triton X-100, the insoluble residue consisted mostly of 5–8 nm-thick membranes resembling the limiting membranes of intact synaptic vesicles. This finding, together with other considerations, suggested that the soluble proteins and accompanying phospholipids originated from the interior of the synaptic vesicles. A 0·3% (v/v) Triton X-100 extract of synaptic vesicle was fractionated by ultracentrifugal flotation and dialysis into three lipoprotein fractions: a low density lipoprotein (d < 1·21 g/ml), a high density lipoprotein (d = 1·21–1·35 g/ml) and a very high density lipoprotein (d > 1·35 g/ml). The phospholipid contents of the low, high and very high density lipoprotein fractions were 0·74, 0·38 and 0·20 mg/mg of protein, respectively. All three apolipoproteins had a high ratio of acidic to basic, and of polar to nonpolar, amino acids, and were rich in glycine, alanine and serine. Polyacrylamide gel electrophoresis of the alkaline-salt and Triton X-100 extracts of synaptic vesicles at pH 8·8 resolved a single anionic component which stained for protein, lipid (Sudan black B; iodine) and anionic groups (acridine orange). Polyacrylamide gel electrophoresis of synaptic vesicle extracts at pH 2·7 in 5 m urea and 0·25% (v/v) Triton X-100 resolved about 20 protein components. However, the protein profiles of electropherograms of the Triton X-100 and alkaline-salt extracts differed in certain respects, suggesting that these media to some extent solubilized different proteins. However, most of the protein bands in electropherograms of the Triton X-100 and alkaline-salt extracts also stained for lipid and anionic groups. In addition, two lipoprotein components in the alkaline-salt extract and four in the Triton X-100 extract contained carbohydrate. Isoelectric focusing of synaptic vesicle extracts resolved 6–8 protein fractions. The major fraction in Triton X-100 and alkaline-salt extracts had an apparent isoelectric point of approximately 4·2 and contained 0·24 mg of phospholipid per mg of protein. Soluble synaptic vesicle proteins released by incubating, freeze-thawing and sonicating in the alkaline-salt medium, and protein fractions of the latter obtained by electrofocusing had an absorption maximum of 260–265 nm which was enhanced in a cold 0·5 n perchloric acid extract, an observation suggesting the presence of a bound nucleotide. These findings demonstrate that rat brain synaptic vesicles contain a heterogenous array of soluble acidic lipoproteins which vary in buoyant density, lipid content, amino acid and carbohydrate composition and electrophoretic mobility in polyacrylamide gels. These acidic lipoproteins apparently comprise the bulk of the macromolecular contents of synaptic vesicles and probably serve as ‘carrier’ proteins for the binding and sequestration of the neurotransmitters.     

Solubilization of the Cytoplasmic Membrane of Escherichia coli by Triton X-100

Treatment of a partially purified preparation of cell walls of Escherichia coli with Triton X-100 at 23 C resulted in a solubilization of 15 to 25% of the protein. Examination of the Triton-insoluble material by electron microscopy indicated that the characteristic morphology of the cell wall was not affected by the Triton extraction. Contaminating fragments of the cytoplasmic membrane were removed by Triton X-100, including the fragments of the cytoplasmic membrane which were normally observed attached to the cell wall. Treatment of a partially purified cytoplasmic membrane fraction with Triton X-100 resulted in the solubilization of 60 to 80% of the protein of this fraction. Comparison of the Triton-soluble and Triton-insoluble proteins from the cell wall and cytoplasmic membrane fractions by polyacrylamide gel electrophoresis after removal of the Triton by gel filtration in acidified dimethyl formamide indicated that the detergent specifically solubilized proteins of the cytoplasmic membrane. The proteins solubilized from the cell wall fraction were qualitatively identical to those solubilized from the cytoplasmic membrane fraction, but were present in different proportions, suggesting that the fragments of cytoplasmic membrane which are attached to the cell wall are different in composition from the remainder of the cytoplasmic membrane of the cell. Treatment of unfractionated envelope preparations with Triton X-100 resulted in the solubilization of 40% of the protein, and only proteins of the cytoplasmic membrane were solubilized. Extraction with Triton thus provides a rapid and specific means of separating the proteins of the cell wall and cytoplasmic membrane of E. coli.     

Ontogeny of cyclic AMP-dependent protein phosphokinase during hepatic development of the rat.

The ontogeny of protein kinase (ATP: protein phosphotransferase, EC 2.7.1.37) and cyclic AMP-binding activity in subcellular fractions of liver was examined during prenatal and postnatal development of the male rat. 1. Protein kinase activity and cyclic AMP-binding activity were found in the nuclear, microsomal, lysosomal-mitochondrial, and soluble liver fractions. 2. The protein kinase activity of the soluble (105 000 X g supernatant) fraction measured with histone F1 as substrate was stimulated by cyclic AMP. Cyclic AMP did not stimulate the protein kinase activity of the particulate fractions. 3. The protein kinase activity of all subcellular fractions increased rapidly from the activity observed in prenatal liver (3-4 days before birth) to reach maximal activity in 2-day-old rats. Thereafter, the protein kinase activity declined more slowly and regained the prenatal levels at 10 days after birth. 4. Considerable latent protein kinase activity was associated with liver microsomal fractions which could be activated by treatment of microsomes with Triton X-100. The latent microsomal protein kinase activity was highest in prenatal liver, at the time of birth, and 2 days after birth. During the subsequent postnatal development the latent microsomal protein kinase activity gradually declined to insignificantly low levels. 5. During the developmental period examined (4 days before birth to age 60-90 days) marked alterations of the cyclic AMP-binding activity were determined in all subcellular fractions of rat liver. In general, cytosol, microsomal, and lysosomal-mitochondrial cyclic AMP-binding activity was highest in 10-11 day-old rats. Nuclear cyclic AMP-binding activity was highest 3-4 days before birth and declined at birth and during the postnatal period. There was no correlation between the developmental alteration of cyclic AMP-binding activity and cyclic AMP dependency of the protein kinase activity in any of the subcellular fractions. This suggests that the measured cyclic AMP-binding activity does not reflect developmental alterations of the cyclic AMP-binding regulatory subunit of cyclic AMP-dependent protein kinase.     

Identification of the protein producing transmembrane diffusion pores in the outer membrane of Pseudomonas aeruginosa PA01.

The outer membrane of Pseudomonas aeruginosa PA01 is permeable to saccharides of molecular weights lower than about 6000. Triton X-100/EDTA-soluble outer membrane proteins were fractionated by ion-exchange chromatography in the presence of Triton X-100 and EDTA, and the protein contents of the various fractions analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis. Each of the major protein bands present in the Triton X-100/EDTA soluble outer membrane was separated from one another. Adjacent fractions were pooled, concentrated and extensively dialyzed to reduce the Triton X-100 concentration. Vesicles were reconstituted from lipopolysaccharide, phospholipids and each of these dialyzed fractions, and examined for their ability to retain [14C]sucrose. Control experiments indicated that the residual levels of Triton X-100 remaining in the dialyzed fractions had no effect on the formation or permeability to saccharides of the reconstituted vesicles. It was concluded that a major outer membrane polypeptide with an apparent weight of 35,000 is a porin, responsible for the size-dependent permeability of the outer membrane.     

Distribution of protein kinase activities in subcellular fractions of rat brain

The subcellular distribution of histone and phosvitin kinase activities in brain has been studied and the ability of the various fractions to catalyse the phosphorylation of their endogenous proteins (intrinsic protein kinase activity) also examined. Synaptosome membrane fragments have little or no histone or phosvitin kinase activity but contain the highest concentration of cyclic AMP-stimulated intrinsic protein kinase activity. Homogenisation of the membrane fragments in Triton X-100 increased the histone kinase activity but on centrifugation it was all recovered in the supernatant, while the insoluble material contained all the intrinsic protein kinase activity. These results indicate that the intrinsic protein kinase activity of cerebral membrane fragments is due to the presence of a kinase enzyme which is specific to certain membrane proteins. The intrinsic protein kinase activity of synaptosome membrane fragments is a rather slow reaction which takes several minutes to saturate all the acceptor proteins.     

Distribution of protein kinase activities in subcellular fractions of rat brain.

The subcellular distribution of histone and phosvitin kinase activities in brain has been studied and the ability of the various fractions to catalyse the phosphorylation of their endogenous proteins (intrinsic protein kinase activity) also examined. Synaptosome membrane fragments have little or no histone or phosvitin kinase activity but contain the highest concentration of cyclic AMP-stimulated intrinsic protein kinase activity. Homogenisation of the membrane fragments in Triton X-100 increased the histone kinase activity but on centrifugation it was all recovered in the supernatant, while the insoluble material contained all the intrinsic protein kinase activity. These results indicate that the intrinsic protein kinase activity of cerebral membrane fragments is due to the presence of a kinase enzyme which is specific to certain membrane proteins. The intrinsic protein kinase activity of synaptosome membrane fragments is a rather slow reaction which takes several minutes to saturate all the acceptor proteins.     

RNA metabolism in rabbit brain: study with neuron-glia and subcellular fractions

—Uridine incorporation into RNA of rabbit brain was studied by using an in vitro system for incubation of brain slices for up to 180 min. Neuron-enriched and glia-enriched fractions were prepared by ficoll density gradient centrifugation, and various subcellular fractions were prepared by sucrose density gradient centrifugation. Although the difference was not as great as in the case of l -leucine incorporation into protein, the neuron-enriched fraction consistently showed a higher specific radioactivity than the glia-enriched fraction. The specific radioactivity of the nuclei increased promptly and remained high at 180 min; the increase in the microsomes was gradual. Comparison of these data suggests that both neuron-enriched and glia-enriched fractions retain high radioactivities in their nuclei at 180 min when a considerable portion of the ribosomal RNA in these fractions is not labeled. The sharp diffusion gradient of nucleotides is discussed in relation to the acid-soluble radioactivity.