Subcellular and regional distribution of 125I-labeled alpha-bungarotoxin binding in rat brain and its relationship to acetylcholinesterase and choline acetyltransferase.

1. The subcellular distribution of binding sites for 125I-labeled alpha-bungarotoxin was studied in rat cerebral cortex. Primary fractions showing higher specific activity than homogenate were P2 (crude mitochondria and nerve endings) and P3-P2 was subfractionated on a Ficoll gradient with the P2B (nerve ending) subfraction exhibiting the greatest recovery (65%) and enrichment of toxin binding. Toxin binding showed a distribution similar to that of acetylcholinesterase, choline acetyltransferase, and sodium and potassium ion-activated ATPase. 2. P2B and P3 were subfractionated on five-step discontinuous sucrose gradients. The highest specific activity of toxin binding and acetylcholinesterase was associated with fractions of relatively low buoyant density, while choline acetyltransferase activity was associated with fractions of higher density. 3. Toxin binding, acetylcholinesterase, and choline acetyltransferase activities were relatively high in olfactory lobes, cerebral cortex, thalamic region, caudate nucleus, and brain stem; intermediate in hippocampus; low in cerebellum. 4. The relationship of toxin binding to the putative acetylcholine receptor in brain is discussed.     

Separation of canine epididymal spermatozoa by Percoll gradient centrifugation

The objective was to characterize the separation of canine epididymal spermatozoa on a Percoll gradient. Epididymal spermatozoa were overlaid on a 45 and 90% discontinuous Percoll gradient and centrifuged at 700 x g for 20 min. The Percoll column was separated into six fractions (top to bottom, A-F) after centrifugation. Fractions A-C contained few spermatozoa. Spermatozoa with bent or folded tails and a large amount of granular debris were observed in Fraction B. Fraction D contained many nonmotile spermatozoa, erythrocytes and round epithelial cells. Spermatozoa in Fraction E had significantly lower motility than those in the initial layer. Spermatozoa in Fraction F had motility similar to those before separation. Fraction F contained 40.6% of the motile spermatozoa layered and 67.5% of all motile spermatozoa recovered. There was no significant difference between Fraction F and the initial layer in sperm membrane integrity. In the sperm-oocyte penetration assay, spermatozoa from Fraction F had a significantly higher penetration rate into the immature homologous oocytes than those from Fraction E. Although the recovery rate of the motile spermatozoa was low, the canine epididymal spermatozoa with motility, membrane integrity and penetrating capability could be separated by two-layer discontinuous Percoll gradient centrifugation.     

A comparison of binding properties and structure of NGF receptor on PC12 pheochromocytoma and A875 melanoma cells

Rat PC12 pheochromocytoma and human A875 melanoma cells express nerve growth factor (NGF) receptors on their surfaces. Covalent crosslinking of bound 125I-NGF to PC12 or A875 intact cells or plasma membrane-enriched fractions resulted in labelling of a peptide doublet at Mr = 110,000 and a single labelled peptide at Mr = 200,000 for each of the cell and membrane preparations. However, a difference between equilibrium binding properties of NGF-receptor on PC12 and A875 cells was observed. PC12 cells exhibited biphasic binding properties with two apparent binding sites: KD = 5.2 nM sites and KD = 0.3 nM sites. The high-affinity PC12 binding sites were trypsin resistant, and 125I-NGF dissociated slowly from them. A875 cells exhibited sites with homogeneous properties (KD = 1.0 nM), all binding sites were trypsin sensitive, and 125I-NGF dissociated rapidly in the presence of unlabelled NGF. Membrane-enriched fractions from either cell type contained binding sites with a uniform low affinity (KD = 3 nM) that were trypsin sensitive, and 125I-NGF rapidly dissociated from them. Sixty to 80 percent of binding sites in membranes could be converted to the high-affinity, trypsin-resistant state by addition of wheat germ agglutinin (WGA). The loss of high-affinity, trypsin-resistant sites from PC12 cells during preparation of plasma membrane fractions does not appear to be the result of selective isolation of low-affinity sites or proteolytic degradation since there is a loss of 125I-NGF binding immediately after cell lysis which is not blocked by protease inhibitors. Also, high-affinity, trypsin-resistant binding sites are not found associated with other cell fractions. The differences between receptor properties on PC12 cells and on A875 cells apparently are the result of differences in the respective intracellular environments. Thus, significant structural homology exists between receptors on A875 and PC12 cells. Cell components other than the binding unit of the NGF receptor may be responsible for the different properties of receptor.     

Uptake and metabolism of female sex steroids by isolated small neurons and other cell fractions from the rat medial basal hypothalamus

Rat medial basal hypothalami (MBH) and sections of cerebral cortex (CC) were dissociated with trypsin to prepare single cells and subcellular fractions. They were then separated into four fractions on a discontinuous sucrose gradient. The small neurons in Fraction D were highly purified. Fraction A had synaptosomes, myelin and other cell particulates. Fraction B had glial cells, neurons and a few synaptosomes. Fraction C had large neurons and red blood cells. All four fractions contained LHRH, but most (62.5%) of this hormone was present in Fraction A. Dissociated cell suspensions were incubated with [³H]-steroids, with and without a 100-fold excess of unlabeled steroids, then separated on sucrose gradients. In most fractions the total uptake and specific uptake of [³H]-progesterone, [³H]-5α-pregnane-3,20-dione (5α-dihydroprogesterone) and [³H]-l7β-estradiol were greater for the dissociated cells from the MBH than the CC. The dissociated cells and cell particulates in all four fractions from the MBH and CC metabolized progesterone, 5α-dihydroprogesterone and l7β-estradiol.These results indicate that hypothalamic neurons contain small amounts of LHRH and retain the ability to take up and metabolize progesterone, 5α-dihydroprogesterone and 17β-estradiol.     

Specific binding sites for 125I-nerve growth factor in peripheral tissues and brain

Specific binding of ¹²⁵I-nerve growth factor (NGF), defined as that part of the total binding of the iodinated derivative displaced by 15–30 μg/ml native NGF, is found at significant levels in many peripheral tissues of chick embryos and rats. Destruction of the sympathetic innervation of tissues by treatment of newborn rats with guanethidine does not materially alter the ¹²⁵I-NGF specific binding capacity of tissues, indicating that these binding sites for NGF are part of the tissues themselves and not a property of the sympathetic nerve terminals which innervate them. Specific binding of ¹²⁵I-NGF which is also resistant to guanethidine treatment exists in chick embryonic and rat brain. The time course of the development of this specific binding in chick embryonic heart and brain suggests a developmental role for these peripheral and central nervous system NGF binding sites.     

Isolation of a porcine liver plasma membrane fraction that binds low density lipoproteins.

Large amounts of injected radiolabeled low density lipoproteins have been found by others to accumulate primarily in the liver and studies in various types of isolated cells, including hepatocytes, have indicated the presence of specific cell membrane recognition sites for lipoproteins. In the present studies, the high affinity binding of radiolabeled low density lipoproteins ([125I]LDL, d 1.020--1.063 g/mL) was measured in the major subcellular fractions of porcine liver homogenates. The nuclear and mitochondrial fractions were 1.9- and 1.4-fold enriched in binding activity with respect to unfractionated homogenates and contained 15% and 12% of the total binding activity, respectively. The microsomes, which contained most of the plasma membranes and endoplasmic reticulum, were approximately 4-fold enriched in binding and contained 73% of the binding activity. Microsomal subfractions obtained by differential homogenization and centrifugation procedures were 5.6--7.0-fold enriched in LDL binding and contained 54--58% of the homogenate binding activity. They were separated by discontinuous sucrose density gradient centrifugation into fractions which contained "light" and "heavy" plasma membranes and endoplasmic reticulum. The heavy membrane fraction was 2--4 fold in binding with respect to the parent microsomes (16--22 fold with respect to the homogenate). There was no enrichment of binding activity in the other two fractions. Two plasma membrane "marker" enzymes, nucleotide pyrophosphatase and 5'-nucleotidase, were also followed. Of the two, binding in the sucrose density gradient subfractions most closely followed nucleotide pyrophosphatase, which was also most highly enriched (3.2--3.3-fold) in the heavy membrane fraction, but did not follow it exactly. The enzyme was 2-fold richer in the light membranes than in the parent microsomes, though the light membrane binding activity was only 0.4--1.4 times that of the parent microsomes. High affinity binding was time and temperature dependent, saturable, and inhibited by unlabeled low density lipoproteins but not by unrelated proteins. Binding was stimulated 2--3 fold Ca2+, was not affected by treatment with Pronase or trypsin and was inhibited by low concentrations of phospholipids and high density lipoproteins (HDL). Heparin-Mn2+ treatment of HDL did not affect its ability to inhibit [125I] LDL binding. The LDL recognition site was distinct from the liver membrane asialoglycoprotein receptor; LDL binding was not inhibited by desialidated fetuin. We conclude that porcine liver contains a high affinity binding site that recognizes features common to both pig low density and high density lipoproteins. Further studies may elucidate the significance of this binding site in lipoprotein metabolism.     

THE ASSOCIATION OF ACETYLCHOLINESTERASE AND MEMBRANE IN SUBCELLULAR FRACTIONS OF THE ELECTRIC TISSUE OF ELECTROPHORUS

Subcellular fractions of the electric tissue of the main organ of the eel Electrophorus electricus were prepared in sucrose media by differential centrifugation and differential discontinuous gradient centrifugation. The distributions of acetylcholinesterase, cytochrome oxidase, DNA, and protein were determined. The appearance of the fractions was determined by phase contrast microscopy and by electron microscopy. A fraction prepared by differectial centrifugation at 30,000 g for 20 minutes in 0.89 M sucrose contained 63 per cent of the total acetylcholinesterase activity at 4 times the specific activity of that of the tissue homogenate. A subfraction prepared by centrifugation in a discontinuous density gradient showed a peak of total and relative specific acetylcholinesterase activity of 35 per cent and 1.9, respectively. The average over-all purification was 7 times. The acetylcholinesterase peak was below the cytochrome oxidase peak and above the DNA peak in the density gradient. The presence of acetylcholinesterase in the fractions was correlated with the presence of large fragments of the cell membrane; however, the presence of other tissue components was noted. The acetylcholinesterase associated with membrane was found to be activated by incubation with sodium deoxycholate. The possible use of the peak fraction containing membranes rich in acetylcholinesterase for the investigation of other components of the acetylcholine system and of other properties of the membrane is discussed.     

Isolation of peroxisomes from the dog kidney cortex.

The present study was undertaken to separate peroxisomes of the dog kidney cortex by the methods of discontinuous sucrose density gradient and zonal centrifugation. The separation of subcellular particles was evaluated by measuring the activities of reference enzymes, beta-glycerophosphatase for lysosomes, succinate dehydrogenase for mitochondria, glucose-6-phosphatase for microsomes, and catalase and D-amino acid oxidase for peroxisomes. The activities of D-amino acid oxidase and catalase were mainly observed in fractions 1 and 2 (1.6 and 1.7 M sucrose) obtained by discontinuous sucrose density-gradient centrifugation. Small amounts of acid phosphatase and succinate dehydrogenase contaminated these fractions. Considerably higher activity of catalase was determined in the supernatant, while D-amino acid oxidase showed a lower activity. By the method of zonal centrifugation, the highest specific activities of catalase and D-amino acid oxidase were found in fraction 50 (1.73 M sucrose) with no succinate dehydrogenase, acid phosphatase or glucose-6-phosphatase activity. These results suggested that peroxisomes of dog kidney cortex were clearly separated in 1.73 M sucrose from mitochondria, lysosomes and microsomes by zonal centrifugation.     

DNA POLYMERASES IN FRACTIONATED RAT BRAIN NUCLEI

Abstract— —Adult rat brain nuclei were separated by discontinuous sucrose gradient centrifugation into astrocyte enriched, neuron enriched, and oligodendrocyte/microglia fractions. Nuclear fractions were subjected to velocity sucrose gradient centrifugation and gradient fractions assayed using relatively specific reaction mixtures for DNA polymerase-α, -β and TdT. NEM resistant DNA polymerase activity (DNA polymerase-β) was detected in equivalent amounts in all nuclear fractions. High molecular weight NEM sensitive activity (DNA polymerase-α) was found primarily in the neuron enriched fraction. The significance of the presence of DNA polymerase-α, an enzyme thought to be involved in DNA replication, in a cell incapable of cell division is unknown. TdT was detected in all fractions with increased activity in the neuron enriched fraction. The finding of TdT in thymocytes and neurons further supports the hypothesis that this enzyme is involved in the storage of noninherited information.     

A comparative study of RNA fractions mediating delayed sensitivity to a chemically defined antigen in vitro.

Hot-cold phenol extracts of RNA prepared from guinea pigs sensitized to mono (p-azobenzene-arsonate)-N-chloroacetyl-l-tyrosine (ARSNAT) contains limited but distinct fractions able to transfer ARSNAT or KLH sensitivity to guinea pig peritoneal exudate cells in vitro. Each of these fractions have been compared by oligo(dT) affinity chromatography and sucrose density gradient analysis. One RNA fraction initially obtained from a sucrose density gradient (designated as B fraction) possessed two separate peaks and contained polyadenylic acid sequences as evidenced by its ability to bind to an oligo (dT) column. Another fraction (Fraction II) initially isolated by oligo (dT) affinity chromatography possessed two peaks after sucrose density gradient analysis, contained poly-A sequences, and had an S-value range approximating the B fraction. RNA fractions prepared from the liver or skeletal muscle of sensitized guinea pigs fails to transfer ARSNAT sensitivity and all fractions are completely inactivated by bovine pancreatic RNase. The results suggest that portions of density gradient prepared B fraction and Fraction II binding to oligo (dT) cellulose may represent the same and/or similar moieties of immunobiologically active RNA.     

Isolation and partial characterization of chick brain synaptic plasma membranes

An isolation procedure for synaptic plasma membranes from whole chick brain is reported that uses the combined flotation-sedimentation density gradient centrifugation procedure described by Jones and Matus (Jones. D. H. and Matus. A. I. (1974) Biochim. Biophys. Acta 356, 276–287) for rat brain. The particulate of the osmotically shocked and sonicated crude mitochondrial fraction was used for a flotation-sedimentation gradient step. Four fractions were recovered from the gradient after 30 min centrifugation. The fractions were identified and characterized by electron microscopy and by several markers for plasma membrane and other subcellular organcelles. Fraction 2 was recovered from the 28.5–34% (w/v) sucrose interphase and contained the major part of the activities of the neuronal plasma membrane marker enzymes. The specific activities of the (Na⁺+K⁺)-activated ATPase (EC 3.6.1.3), acetylcholinesterase (EC 3.1.1.7) and 5′-nucleotidase (EC 3.1.3.5) were, respectively, 4.5. 2.0 and 1.2 times higher than in the homogenate. However, Fraction 2 also contained considerable amounts of activities of putative lysosomal and microsomal markers in addition to lower amounts of mitochondrial and myelin markers. Although no prepurification of synaptosomes from the crude mitochondrial fraction was performed, the synaptic plasma membranes obtained showed many properties analogous to similar preparations from rat brain described in recent years.     

Presynaptic localization of histamine H3-receptors in rat brain.

The localization of histamine H3-receptors in subcellular fractions from the rat brain was examined in a [3H] (R) alpha-methylhistamine binding assay and compared with those of histamine H1- and adrenaline alpha 1- and alpha 2-receptors. Major [3H](R) alpha-methylhistamine binding sites with increased specific activities ([3H]ligand binding vs. protein amount) were recovered from the P2 fraction by differential centrifugation. Minor [3H](R)alpha-methylhistamine binding sites with increased specific activities were also detected in the P3 fraction. Further subfractionation of the P2 fraction by discontinuous sucrose density gradient centrifugation showed major recoveries of [3H](R)alpha-methylhistamine binding in myelin (MYE) and synaptic plasma membrane (SPM) fractions. A further increase in specific activity was observed in the MYE fraction, but the SPM fraction showed no significant increase in specific activity. Adrenaline alpha 2-receptors, the pre-synaptic autoreceptors, in a [3H] yohimbine binding assay showed distribution patterns similar to histamine H3-receptors. On the other hand, post-synaptic histamine H1- and adrenaline alpha 1-receptors were closely localized and distributed mainly in the SPM fraction with increased specific activity. Only a negligible amount was recovered in the MYE fraction, unlike the histamine H3- and adrenaline alpha 2-receptors.     

Appearance of nerve growth factor receptors on cultured neural crest cells

Light microscopic radioautography of differentiating quail neural crest cultures (1 to 2 weeks after explanation) incubated with Iodine-125-labeled nerve growth factor (125I-NGF) revealed that approximately 35% of the cells bound NGF. The binding was specific and saturable; it was blocked by an excess of nonradioactive NGF, and was not detected following incubation with biologically inactive 125I-NGF. In addition, the binding did not appear to be blocked or diminished by insulin. Cell cultures prepared from somites or notochord showed no specific binding of 125I-NGF. Melanocytes comprised approximately 10% of the cell population in these cultures and appeared to be unlabeled. The subpopulation of cells with NGF receptors that were morphologically similar to other non-melanocyte unlabeled cells present in the neural crest cultures are probably the targets of the factor during differentiation and development. In contrast, there was no evidence of 125I-NGF binding by premigratory neural crest (adherent to the isolated neural tube) or by early migratory neural crest cells (24 hr after explantation). Both of these types of neural crest cells are relatively undifferentiated. The cells of the neural tube were also unlabeled. The binding of 125I-NGF to differentiating neural crest cells was not noticeably diminished by a brief pretreatment with trypsin or Dispase, enzymes used in the isolation of neural tubes. Hence, the absence of NGF receptors on premigratory neural crest and early migratory neural crest cultures was not due to enzymatic alterations of the receptor. It seems, therefore, that receptors for NGF appear on neural crest cells during the time when these cells are acquiring their phenotypic characteristics.     

Synaptic and extra-synaptic distribution of histamine H1-receptors in rat and guinea pig brains

Localization of histamine H1-receptors in subcellular fractions from rat and guinea pig brains was examined in a [3H]mepyramine binding study. Major [3H]mepyramine binding sites with increased specific activities [( 3H]mepyramine binding vs. protein amount) were recovered from P2 fractions from both rat and guinea pig brains by differential centrifugation. Further subfractionation of both rat and guinea pig P2 fractions by a discontinuous sucrose density gradient centrifugation showed the highest recovery of [3H]mepyramine binding with further increased specific activities found in synaptic plasma membrane (SPM) fractions. Minor [3H]mepyramine binding sites with increased specific activities were detected in both rat and guinea pig P3 fractions. [3H]Mepyramine binding sites in SPM and P3 fractions showed identical Kd values in each species. These results indicate that histamine H1-receptors are located not only in synaptic but also in extra-synaptic membranes of both rat and guinea pig brains.     

Interactions between nerve growth factor binding and estradiol in early development of the zebra finch telencephalon.

The zebra finch telencephalon exhibits rapid and substantial development in the first few weeks after hatching. In parallel, the rate of estradiol synthesis is very high in the zebra finch forebrain, and estradiol can have potent neurotrophic effects in specific telencephalic regions, including those that control the learning and production of song. In an attempt to elucidate mechanisms regulating telencephalic development, potentially including a role for the large capacity for estrogen production, (125)I-nerve growth factor (NGF) binding was measured in homogenates of telencephalon from zebra finches age 3, 15, 30, 60, and 120 days. The highest density of low- and high-affinity (125)I-NGF binding sites was observed in 3-day-old finches. Using an aromatase inhibitor, Fadrozole, to reduce estradiol levels in 1 to 4-day-old zebra finches significantly decreased both high- and low-affinity (125)I-NGF binding sites. Conversely, treating adult or 8 to 14-day-old hatchlings with estradiol increased high-affinity (125)I-NGF binding sites. These results are consistent with the hypothesis that estradiol influences the level of NGF receptors, and suggest one mechanism through which the steroid could affect brain development. The data also indicate that estradiol and NGF activity may be important for very early development of the telencephalon.     

Characterization of nerve growth factor binding to cultured neural crest cells: evidence of an early developmental form of the NGF receptor

Cultured neural crest cells undergoing differentiation have been shown to contain a subpopulation of cells with specific receptors for nerve growth factor (NGF). These cells are the potential targets of NGF during differentiation and development. This study was done to pharmacologically characterize the binding of NGF to long-term (1- to 3-week) cultures of quail neural crest cells. The data indicate that 125I-NGF binding was specific and saturable, with less than 20% nonspecific binding. Scatchard analysis revealed the presence of one type (class) of receptors with a binding constant (Kd) similar to that of the low-affinity binding site described for embryonic dorsal root and sympathetic ganglia (approximately 3.2 nM). This was corroborated by displacement experiments (Kd of 1.3 nM), in which 125I-NGF binding was measured in the presence of increasing concentrations of nonradioactive NGF. In addition, affinity labeling revealed that the 125I-NGF-receptor complex had a molecular weight of about 93K, characteristic of the low-affinity NGF receptor of PC12 cells. The NGF receptor of cultured neural crest cells was trypsin-sensitive, as is typical of the low-affinity NGF binding sites. These findings indicate that differentiating neural crest cells lack high-affinity 125I-NGF binding sites. In contrast, embryonic dorsal root and sympathetic ganglia cells, known NGF targets, have both high- and low-affinity receptors. Measurements of the differential release of surface-bound 125I-NGF indicated that a relatively small amount (about 14%) of NGF is internalized over a 1-hr period. Cultured neural crest cells which bear NGF receptors were also shown by light microscopic radioautographic techniques to incorporate [3H]thymidine. I suggest, therefore, that cultured neural crest cells which have not terminally differentiated, as judged by morphological criteria and continued proliferation, may express an early developmental form of the NGF receptor.     

Binding and degradation of nerve growth factor by PC12 pheochromocytoma cells

The specific binding of various concentrations of 125I-labeled nerve growth factor (NGF) to PC12 cells at 37 degrees C reached maxima after 90 min and then declined to 25% of maximal binding after 10 h. Decreased binding was accompanied by degradation of 125I-NGF and the appearance of acid-soluble biologically inactive 125I (mainly 125I-monoiodotyrosine) in the medium as well as a decrease in the number of surface NGF receptors. The time-dependent decrease in binding and the degradation of 125I-NGF were inhibited by low temperature and the lysosomotropic agent chloroquine while degradation was inhibited by metabolic energy inhibitors in the absence of glucose. Chloroquine also produced an increase in the accumulation of 125I-NGF which was not readily removed from the cells. These data suggest that 125I-NGF bound to PC12 cells is efficiently internalized by receptor-mediated endocytosis and degraded by the lysosomes. It appears from other data that this process does not produce the intracellular signals regulating neurite outgrowth.     

Isolation and fractionation of the photosynthetic membranous organelles from Rhodopseudomonas spheroides

Molecular sieve chromatography and sucrose gradient centrifugation were used to prepare large quantities of purified chromatophores from Rhodopseudomonas spheroides. Electron micrographs of these chromatophores revealed that the final preparations were very homogeneous and free of non-chromatophore particulate material. As an additional check on purity, (14)C-l-phenylalanine-labeled aerobic cells, devoid of chromatophores, were mixed with unlabeled photosynthetic cells. The resulting preparation contained less than 1% of the radioactivity, originally located in non-chromatophore protein. The purified chromatophores were solubilized in 2-chloroethanol and separated into two fractions. Fraction P(1) contained 3 to 5% of the total chromatophore protein and could be resolved into 10 electrophoretic components. The second fraction, P(II), contained five electrophoretic components. One of these components had associated with it all of the pigment and phospholipid present in P(II). Preliminary immunochemical studies on these fractions are also reported.     

Multiple molecular forms of phosphoprotein phosphatase. III. Phosphorylase phosphatase and phosphohistone phosphatase of rabbit liver.

1. Phosphoprotein phosphatase (phosphoprotein phosphohydrolase EC 3.1.3.16) in the soluble fraction of rabbit liver which catalyzes the dephosphorylation of muscle phosphorylase a and phosphohistone (P-histone) was resolved into three active fractions by NaCl gradient elution from a DEAE-cellulose column (Fraction I, 11 and III in order of elution). They have different relative reaction rates for the two substrates and different degrees of stimulation by Mn-2+. Apparent Km values of Fraction I, II and III were 15, 20 and 16 muM for phosphorylase a, and 6.9, 5.3 and 4.4 muM for P-histone, respectively (with Mn-2+ in the assay mixture). 2. On sucrose density gradient centrifugation Fraction I and II were revealed to contain a major peak (7.0 S and 7.8 S, respectively) and a minor peak (4.0 S) of activity, while Fraction III contained only one peak (5.8 S). Freezing and thawing in the presence of 0.2 M mercaptoethanol dissociated all three fractions into subunits of similar molecular size (3.4 S), with concomitant enhancement of phosphorylase phosphatase activity. The Km values all became essentially the same (20 muM for phosphorylase a and 16 muM for P-histone). 3. The phosphorylase phosphatase and P-histone phosphatase activities could not be separated with any of the procedures described. Competition between the two phosphoprotein substrates was observed with some of the fractions.?     

Density Gradient Study of Victorin-Binding Proteins in Oat (Avena sativa) Cells

Victorin-binding proteins (VBPs) in oat (Avena sativa) cells were identified using native victorin and anti-victorin polyclonal antibodies. Homogenates of oat tissues were fractionated in continuous or discontinuous sucrose density gradients or with an aqueous two-phase method, and covalent binding sites of victorin were detected by western blotting. In a 20 to 45% (w/w) sucrose continuous density gradient, the 100-kD VBP was located in fractions of 37 to 44% sucrose, with a peak at 39% sucrose. Based on marker enzyme assays, plasma membranes peaked at 39 to 41% sucrose, mitochondria peaked at 41%, but Golgi and endoplasmic reticulum were in lower density fractions, peaking at 28 to 29% and 22 to 24% sucrose, respectively. The 100-kD VBP was not found in plasma membranes purified by the aqueous two-phase method or in mitochondria purified by discontinuous density gradient centrifugation. Victorin binding to 65- and 45-kD proteins was detected in all fractions in the continuous sucrose density gradients. The 65- and 45-kD proteins were both detected in purified plasma membranes, but only the 65-kD protein was detected in purified mitochondria. The subcellular location of VBPs was the same in sensitive and resistant oat cells.