The subcellular localization of cerebral phosphoproteins sensitive to electrical stimulation

1. On incubating cerebral-cortex slices at 37° in an oxygenated medium marked changes resulted in the subcellular distribution of proteins and phosphoproteins in the tissue. The protein content of the nuclear fraction more than doubled, whereas the yields of microsomal and supernatant proteins were both markedly decreased. The amount of phosphoprotein/mg. of protein decreased in the microsomal and supernatant fractions, but showed little change in the nuclear and mitochondrial fractions. The loss of microsomal protein could be partly prevented by rinsing the slices briefly in cold sucrose solution before dispersion; the altered subcellular distribution was apparently related to contamination of the dispersing solution with traces of salts from the medium. 2. The subcellular location of the phosphoprotein sensitive to the effects of electrical pulses applied to cerebral slices in vitro has been reinvestigated by two different procedures. Comparison between unstimulated and stimulated slices after incubation in the presence of [³²P]orthophosphate showed that phosphoprotein radioactivity increased on stimulation to a greater extent in a membrane-rich fraction than in a mitochondria-rich fraction, these being obtained by immediate density-gradient fractionation of the tissue dispersion. With fractions isolated by differential centrifuging the percentage increase in a combined mitochondrial and nuclear fraction was 5% as compared with 24% (P<0·02) in the microsomal fraction and 30% in the original dispersion before fractionation. The sensitive phosphoprotein therefore appears to be located in structures sedimenting with the microsomal fraction, rather than with the nuclear fraction as previously claimed.     

Subcellular localization of 3-hydroxy-3-methylglutaryl coenzyme A reductase in Pisum sativum seedlings

3-Hydroxy-3-methylglutaryl coenzyme A reductase from seedlings of Pisum sativum L. is localized in the plastids, mitochondria, and microsomes. Separation of the microsomal fraction into heavy and light subfractions shows that 95% of the microsomal activity is associated with the light subfraction. Definitive localization was achieved by showing that reductase activity comigrated with organelle markers on sucrose density gradients. Differential centrifugation studies showed that the microsomal fraction contained 80% of the total cellular activity, and the mitochondrial and plastid fractions each contained about 10%.The results suggest the existence of three parallel biosynthetic pathways which may be important in regulating the synthesis of isoprenoids characteristic of the individual organelles.     

Characteristics of mitochondrial and microsomal monoacyl- and diacyl-glycerol 3-phosphate biosynthesis in rabbit heart

Acylation of sn-glycerol 3-phosphate by heart subcellular fractions was characterized. The enzyme kinetics revealed that the rate of reaction of acylation by mitochondria was slower, but constant for a longer period (up to 20min), than that by the microsomal fraction. The range of palmitate, oleate and linoleate concentrations yielding optimal sn-glycerol 3-phosphate acylation was broader for mitochondria than for the microsomal fraction, the latter showing a preference for linoleate. The mitochondrial fraction synthesized a relatively large quantity of monoacyl-sn-glycerol 3-phosphate, reaching 135% of the microsomal biosynthesis during an assay period of 15min. By contrast, the microsomal fraction formed considerably more diacyl- than monoacyl-sn-glycerol 3-phosphate, except with linoleate as the acyl donor, in which case approximately equal quantities of the two products were produced. The biosynthesis of monoacyl-sn-glycerol 3-phosphate was also observed in experiments in which hepatic subcellular fractions were used to provide supporting evidence. Cardiac mitochondrial diacyl-sn-glycerol 3-phosphate formation was less than 17% of the microsomal formation. However, evidence is presented to exclude the possibility that monoacyl-sn-glycerol 3-phosphate in the mitochondrial fraction is formed by deacylation of the contaminating microsomal diacyl-sn-glycerol 3-phosphate. The participation of the dihydroxyacetone phosphate pathway in the biosynthesis of these substances was minimal. The addition of CTP and the fatty acid specificity of the reaction both provided results that reinforced the postulate that mitochondrial differs from microsomal acylation. Thus our findings demonstrate that the characteristics of acyl-CoA-sn-glycerol 3-phosphate O-acyltransferase (EC 2.3.1.15) in rabbit heart mitochondria are distinct from those of cardiac microsomal enzyme and hepatic enzymes.     

Release of the phosphodiesterase activator by cyclic AMP-dependent ATP:protein phosphotransferase from subcellular fractions of rat brain.

The subcellular distribution of the endogenous phosphodiesterase activator and its release from membranes by a cyclic AMP-dependent ATP:protein phosphotransferase was studied in fractions and subfractions of rat brain homogenate. These fractions were obtained by differential centrifugation and sucrose density gradient; their identity was ascertained by electron microscopy and specific enzyme markers. In the subcellular particulate fractions, the concentration of activator is highest in the microsomal fraction, followed by the mitochondrial and nuclear fractions. Gradient centrifugation of the main mitochondrial subfraction revealed that activator was concentrated in those fractions containing mainly synaptic membranes. Activator was releasted from membranes by a cyclic AMP-dependent phosphorylation of membrane protein. The release of activator occurred mainly from the mitochondrial subfractions containing synaptic membranes and synaptic vesicles. The data support the view that a release of activator from membranes may be important in normalizing the elevated concentration of cyclic AMP following persistent transsynaptic activation of adenylate cyclase.     

Subcellular distribution of cytochrome c in rat liver. Methods for its extraction and purification

1. A method for the extraction and purification of cytochrome c from rat liver is described. The method depends on multiple chromatography on Amberlite IRC-50 with elution with ammonium phosphate buffers of differing ionic composition and pH, interspersed with gel filtration with Sephadex G-25. Conditions leading to denaturation are avoided and the product is chromatographically pure. 2. The method may be used for the quantitative analysis of cytochrome c either in unfractionated liver or in subcellular fractions. 3. Two pools of cytochrome c were detected, one extractable at pH4.0 with distilled water and the other extracted from the residues of the first extraction with 0.15m-sodium chloride. 4. For subcellular distribution studies the liver was homogenized in 0.3m-sucrose and a nuclear fraction (washed thoroughly to remove trapped mitochondria), a mitochondrial fraction, a heavy microsomal fraction, a standard microsomal fraction and the cell sap were isolated. The mitochondrial fraction was subfractionated further by density-gradient centrifugation. Each fraction was analysed for protein, RNA, DNA, succinate-neotetrazolium oxidoreductase and glucose 6-phosphatase. 5. A total of 123mug. of cytochrome c was obtained/g. wet wt. of rat liver. 6. Values for the percentage subcellular distribution of cytochrome c are: nuclear fraction, 24.4; mitochondrial fraction, 57.2; heavy microsomal fraction, 5.2; standard microsomal fraction, 10.6; cell sap, 2.7. 7. Three out of the eight mitochondrial subfractions separated by gradient centrifugation contained 76% of the cytochrome c and 85% of the succinate-neotetrazolium oxidoreductase present in the mitochondrial fraction. 8. In unfractionated liver 94% of the cytochrome c was extracted at pH4.0 with water whereas in most of the subcellular fractions the corresponding value was approx. 75-80%.     

Release of the phophodiesterase activator by cyclic AMP-dependent ATP:protein phosphotransferase from subcellular fractions of rat brain

The subcellular distribution of the endogenous phosphodiesterase activator and its release from membranes by a cyclic AMP-dependent ATP:protein phosphotransferase was studied in fractions and subfractions of rat brain homogenate. These fractions were obtained by differential centrifugation and sucrose density gradient; their identity was ascertained by electron microscopy and specific enzyme markers.In the subcellular particulate fractions, the concentration of activator is highest in the microsomal fraction, followed by the mictochondrial and nuclear fractions. Gradient centrifugation of the main mitochondrial subfraction revealed that activator was concentrated in those fractions containing mainly synaptic membranes.Activator was released from membranes by a cyclic AMP-dependent phosphorylation of membrane protein. The release of activator occurred mainly from the mitochondrial subfractions containing synaptic membranes and synaptic vesicles.The data support the view that a release of activator from membranes may be important in normalizing the elevated concentration of cyclic AMP following persistent transsynaptic activation of adenylate cyclase.     

Subcellular distribution and properties of carbonyl reductase in guinea pig lung

On subcellular fractionation, carbonyl reductase (EC 1.1.1.184) activity in guinea pig lung was found in the mitochondrial, microsomal, and cytosolic fractions; the specific activity in the mitochondrial fraction was more than five times higher than those in the microsomal and cytosolic fractions. Further separation of the mitochondrial fraction on a sucrose gradient revealed that about half of the reductase activity is localized in mitochondria and one-third in a peroxidase-rich fraction. Although carbonyl reductase in both the mitochondrial and microsomal fractions was solubilized effectively by mixing with 1% Triton X-100 and 1 M KCl, the enzyme activity in the mitochondrial fraction was more highly enhanced by the solubilization than was that in the microsomal fraction. Carbonyl reductases were purified to homogeneity from the mitochondrial, microsomal, and cytosolic fractions. The three enzymes were almost identical in catalytic, structural, and immunological properties. Carbonyl reductase, synthesized in a rabbit reticulocyte lysate cell-free system, was apparently the same in molecular size as the subunit of the mature enzyme purified from cytosol. These results indicate that the same enzyme species is localized in the three different subcellular compartments of lung.     

Distribution and nonphotochemical transformation of phytochrome in subcellular fractions from pisum epicotyls

In etiolated pea (Pisum sativum L. cv. Alaska) shoots about 3% of the total extractable phytochrome was found in the mitochondrial fraction and about 4.5% in the microsomal fraction, while over 70% was soluble in the 105,000g supernatant. The value of Δ(ΔA) per milligram of protein was significantly higher in the 105,000g supernatant than in these particulate fractions. The percentage conversion of Pr to Pfr was approximately proportional to the total dose of red light in every subcellular fraction tested, unless the dose approached a saturation level. After a brief irradiation of intact shoots with red light at 26 C, each subcellular fraction showed different patterns of dark transformation in vivo at 26 C; that is, the amount of the particulate-bound phytochrome increased immediately after the irradiation, and a reversion of Pfr to Pr was indicated for the first 2 hr in the 12,000g supernatant, but not at all in the mitochondrial and microsomal fractions. The amounts of Pr in the mitochondrial and microsomal fractions did not change during the dark incubation, while those in the 12,000g supernatant increased with time. Similar results were obtained with apical shoot segments after exposure to red light at 0 C and a subsequent dark incubation on moist filter paper at 26 C.     

STUDIES ON NICOTINIC ACETYLCHOLINE RECEPTORS IN MAMMALIAN BRAIN. CHARACTERIZATION OF A MICROSOMAL SUBFRACTION ENRICHED IN RECEPTOR FUNCTION FOR DIFFERENT NEUROTRANSMITTERS

Abstract— A subfraction, derived from the microsomal fraction of rat cerebral cortex, with a buoyant density of 1.112 g μ ml⁻¹ appears to be enriched in receptor sites for a number of potential neurotrans-mitters. These include the cholinergic (nicotinic and muscarinic) and ß-adrenergic receptors. This microsomal subfraction (P₃B₂) has been isolated on a preparative scale by two sequential isopycnic sedimentations in discontinuous sucrose gradients.
We have studied the morphology, enzymatic markers and protein composition of this fraction and have compared them with the properties of other subcellular fractions from the same source. Synaptic plasma membranes resembled P₃B₂ by exhibiting the same high extent of enrichment in receptors. However, the synaptic membranes appear to contain more mitochondrial and presynaptic (axonal and cell surface) membranes than does P₃B₂, and the postsynaptic membranes in the two fractions appear morphologically distinct since P₃B₂ does not contain the characteristic postsynaptic densities. Thus these membranes may be derived from Gray's type II synapses.     

SUBCELLULAR DISTRIBUTION OF BIOGENIC MONOAMINES IN THE CENTRAL NERVOUS SYSTEM OF ANODONTA CYGNEA L. AS REVEALED BY DENSITY GRADIENT CENTRIFUGATION

Abstract— Differential and gradient centrifugation revealed that 90 per cent of the 5-HT, dopamine and noradrenaline in the CNS of the fresh water mussel was bound to particles; 60-70 per cent of the bound monoamines appeared in the mitochondrial and 15-20 per cent in the microsomal fraction. Spectrofluorimetric assay and electron microscopic analysis of the subfractions obtained by separation of the mitochondrial fraction on sucrose density gradients showed that the nerve endings and their dense-core vesicles were concentrated in fractions with high relative specific activity of the three monoamines. This supports the proposed function of these monoamines as interneuronal mediators. Osmotic shock treatment resulted in the formation of a synaptosomal subfraction of low density displaying a high relative specific activity for 5-HT. From the results obtained one cannot draw clear-cut conclusions regarding the participation of subcellular particles in the storage of serotonin detectable in the perikarya by means of histochemical methods.     

Inhibition and stimulation of rat luteal protein phosphorylation by protein kinase effectors

Estradiol-17 beta (E2) predetermined protein phosphorylation systems have been identified recently in midpregnant rat corpus luteum. Major type protein kinase activities in these systems were explored here using as probes protein kinase inhibitors. Luteal nuclear, mitochondrial, microsomal and cytosolic fractions were obtained from rats hysterectomized and hypophysectomized on day 12 of pregnancy and then treated for 72 h with E2. In vitro phosphate transfer from [gamma-32P]ATP was monitored by SDS-PAGE followed by autoradiography. Polymyxin B (PMB), 1-200 microM, a PKC inhibitor, completely blocked, in a dose dependent manner, the Ca2+ phospholipid (PL) stimulated radiolabeling of nuclear fraction Mr 79,000 substrate(s) as expected. Similarly, the calmodulin (CaM) antagonist compound 48/80, 1-20 micrograms/ml, inhibited the Ca2+/CaM-dependent phosphorylation of the microsomal fraction Mr 60,000 and Mr 56,000 proteins. The Ca2+ PL-enhanced labeling of mitochondrial fraction Mr 76,000 substrate(s) was only partially susceptible to inhibition by PMB or compound 48/80. Studies of microsomal fraction phosphoprotein bands not stimulated by added cofactors indicated that the radiolabeling of Mr 75,000 protein(s) was partially blocked by compound 48/80 but not by PMB. Phosphate transfer to Mr 41,000 protein(s) was inhibited by the cAMP-dependent kinase protein inhibitor (PKI), while the phosphorylation of Mr 31,000 protein(s) was refractory to all inhibitors employed here. Surprisingly, regardless of hormonal pretreatment, PMB and compound 48/80 activated in every subcellular fraction the cofactor independent appearance of at least one phosphoprotein band, between Mr 87,000-99,000. This novel observation should be instrumental in understanding the actions of these compounds towards living cells.     

A rapid method for the fractionation of isolated hepatocytes

The fractionation of rat liver hepatocytes using a mechanical disruption technique followed by centrifugation is reported; the whole procedure requires approximately 10 min. Marker enzyme distribution data are in good agreement with distribution data from standard techniques connected with the production of three subcellular fractions—cytoplasmic, mitochondrial, and microsomal. Electrophoretic analysis of the mitochondrial and microsomal fractions show total band correspondence between the fractions produced by the method and traditional techniques. Examination of the fractions by electron microscopy supports the view that the mitochondrial fraction is comprised of both intact mitochondria and mitochondria from which the outer membrane has been removed. The microsomal fraction contains discrete vesicles derived from both rough and smooth endoplasmic reticulum.     

Studies on the biosynthesis of protein by lactating guinea-pig mammary gland: Characteristics of the synthesis of α-lactalbumin and total protein by slices and cell-free systems

1. Slices of lactating guinea-pig mammary gland were incubated with radioactive amino acids and the various subcellular fractions separated by centrifugation after disruption of the cells by mincing and homogenization. The most active fraction for protein synthesis appeared to be the `mitochondrial'. 2. When the subcellular fractions were prepared without previous incubation of the cells and were then incubated with radioactive amino acid and an energy-generating system, the `mitochondrial fraction' was at least as active for protein synthesis as the `microsomal fraction'. 3. The ribosomes in the microsomal fraction are mainly unattached to membrane whereas those in the mitochondrial fraction are probably attached to fragments of the rough-surfaced endoplasmic reticulum. This latter fraction contains few mitochondria. 4. The combined mitochondrial and microsomal fractions incorporated radioactive amino acids into α-lactalbumin. 5. The radioactive leucine isolated from tryptic and chymotryptic peptides of α-lactalbumin synthesized in the cell-free system was not of uniform specific radioactivity. This was consistent with the polypeptide being assembled by the sequential addition of amino acids. 6. Evidence is presented for the polypeptide chain of α-lactalbumin being assembled from the N-terminus and for chain initiation in the cell-free system. 7. It is concluded that cell-free extracts of lactating mammary gland synthesize α-lactalbumin.     

Studies on the fractionation of mucosal homogenates from the small intestine

1. Homogenates of the mucosa of the small intestine of the guinea pig were separated by fractional sedimentation into seven different fractions. The enzymic properties of some of these subcellular fractions were compared with those obtained from the mucosa of the small intestine of the rabbit and cat. 2. The enzymic properties of the low-speed sediment (15000g-min.) were investigated and it was shown that invertase and alkaline ribonuclease were predominantly located in this subcellular fraction, whereas alkaline phosphatase, aryl-amidase, acid phosphatase, acid ribonuclease and phosphoprotein phosphatase, though true constituents of this fraction, occurred to varying degrees in other subcellular structures also. 3. It was shown that the most probable source of the enzymic activities observed in the low-speed sediment was the brush border. Electron micrographs of the purified brush-border fraction indicated vesicles derived from the brush-border membrane. 4. A method is described for the fractionation of mucosal homogenates into a brush border-plus-nuclei fraction, a mitochondrial fraction, a microsomal fraction and a particle-free supernatant. The fractions were shown to be relatively pure, as indicated by the distribution of invertase, DNA, succinate dehydrogenase, glucose 6-phosphatase and 6-phosphogluconate dehydrogenase. 5. Most of the activity of four lysosomal enzymes present in the nuclei-free homogenate was sedimented at 375000g-min., suggesting the occurrence of lysosomal particles in mucosal homogenates. 6. Further fractionation of the microsomal membranes into three fractions is described. The enzymic composition of the membrane fractions is given and discussed in relation to their structure as seen in electron micrographs.     

The subcellular distribution and properties of hexokinases in the guinea-pig cerebral cortex

1. Hexokinase activities were estimated in primary subcellular fractions from guinea-pig cerebral cortex and in sucrose-density-gradient subfractions of the mitochondrial and microsomal fractions. 2. Appreciable activities were observed in mitochondrial, microsomal and soluble fractions. The activity in the mitochondrial fraction was associated with the mitochondria rather than with myelin or nerve endings and that in the microsomal fraction was associated with membrane fragments. 3. Most of the mitochondrial activity was extracted in soluble form by osmotic ;shock'. The activity of the mitochondrial extract differed from the soluble activity in kinetic properties and in electrophoretic behaviour. 4. No evidence was obtained for the presence of a high-K(m) glucokinase in the brain. 5. The results are discussed in terms of relevance to considerations of glucose utilization by the brain.     

The lipid composition of ovine placental tissue homogenate and its subcellular fractions

Homogenates of the placental tissue of near term sheep were separated by differential centrifugation into mitochondrial, microsomal and cytosolic fractions. The relative proportions of the major neutral lipids and phospholipids, together with their fatty acid compositions, were determined in the homogenates and in each subcellular fraction. The cytosolic fraction contained the highest proportion of cholesteryl esters (CEs) and these possessed a fatty acid composition markedly different from the total CEs extracted from the homogenate. Both the mitochondrial and microsomal fractions contained significant proportions of solvent front phospholipid (SFP) and whereas the mitochondrial SFP displayed the relatively unsaturated fatty acid composition characteristic of diphosphatidylglycerol (cardiolipin), the fatty acids of the microsomal SFP were distinctly more saturated. These results are compared with those obtained from other mammalian tissues, both ruminant and non-ruminant, and discussed in terms of the function of the components of the subcellular fractions.     

Adenosine triphosphatases of cestodes Bothriocephalus scorpii

Activities and properties of adenosine triphosphatases (ATPases) were studied in mitochondrial and microsomal fractions of cestodes Bothriocephalus scorpii parasitizing in pyloric appendages of the Brandt’s bullhead Myoxocephalus brandti. The highest activity was revealed in the mitochondrial fraction. The mitochondrial and microsomal fractions of B. scorpii had the ATPase activity dependent on the presence of cations Mg²⁺, Mn²⁺, and Ca²⁺. Effects of ions and inhibitors on the B. scorpii ATPase activity with various cations were. Both subcellular fractions were able to hydrolyze, apart from ATP, also GTP, CTP, and UTP.     

THE BIOSYNTHESIS OF CHOLESTEROL AND OTHER STEROLS BY BRAIN TISSUE

Abstract— The distribution of [¹⁴C]-labeIled material into subcellular fractions of 15-day-old rat brain was studied as a function of time after intracerebral injection of [2-¹⁴C]mevalonic acid. As previously shown for adult brain, the data indicated the microsomal fraction to be the site of sterol biosynthesis. The synaptosomal fraction exhibited a marked early uptake of [¹⁴C]-nonsaponifiable material. Total radioactivity in both myelin and myelin-like fractions remained low in comparison to that in the other subcellular fractions at all time periods examined. At 2 h after injection, labelled digitonin-precipitable material was demonstrable in all subcellular fractions. Examination of the [¹⁴C]-labelled nonsaponifiable material by thin-layer chromatography indicated the rapid appearance of labelled 4-desmethyl sterol in all subcellular fractions, with the most rapid appearance in the myelin fraction, followed in decreasing order by microsomal, synaptosomal, and mitochondrial fractions. Examination of [¹⁴C] digitonin-precipitable material from each fraction by the dibromide method demonstrated that although 4-desmethyl sterol appeared quickly, the formation of cholesterol was slow in all fractions, an effect that had been reported earlier for adult brain.     

SUBCELLULAR DISTRIBUTION OF NORADRENALINE IN GUINEA-PIG CEREBRAL CORTEX AND SPLEEN

Abstract— The distribution of noradrenaline (NA) in subcellular fractions of guinea-pig cerebral cortex and spleen was determined by differential and density gradient centrifugation. Of the primary fractions, the microsomal fraction from both tissues was enriched in NA, that of the spleen having the higher specific activity. Microsomal fractions were therefore placed on gradients and NA determined in the subfractions since these fractions appeared suitable preparations in which to search for discrete populations of vesicles. So that the non-occluded micro-particulate bound noradrenaline (MPBNA) content of gradient subfractions could be measured, [³H]NA was used to control for the diffusion and or adsorption of free NA, and occluded lactate dehydrogenase was used to estimate the amount of entrapped MPBNA and soluble NA. Non-occluded MPBNA on gradients from microsomal fractions of cerebral cortex formed a single peak mainly in subfraction F (0.6-0.8 m -sucrose). Spleen microsomal fractions, however yielded two peaks of MPBNA. one in sub-fractions D to G (0.4-1.0 m -sucrose) and the other in sub-fraction J (1.4 m -sucrosc); electron microscopy showed that the latter subfraction contained large vesicles.
Since there were unexpectedly small amounts of MPBNA in microsomal subfractions D and E of cerebral cortex, the synaptosome fraction was investigated. Following water treatment of synaptosomes. MPBNA formed a peak in subfraction E (0.4-0.6 m -sucrose) with smaller amounts in subfractions D and F (0.4 and 0.6 0.8 m -sucrose).