Search

Duchenne muscular dystrophy represents one of the most common hereditary diseases. Abnormal ion handling is believed to render dystrophin-deficient muscle fibres more susceptible to necrosis. Although a reduced Ca2+ buffering capacity has been shown to exist in the dystrophic sarcoplasmic reticulum, surprisingly no changes in the abundance of the main luminal Ca2+ reservoir protein calsequestrin have been observed in microsomal preparations. To address this unexpected finding and eliminate potential technical artefacts of subcellular fractionation protocols, we employed a comparative subproteomics approach with total mouse skeletal muscle extracts. Immunoblotting, mass spectrometry and labelling of the entire muscle protein complement with the cationic carbocyanine dye‘Stains-All’ was performed in order to evaluate the fate of major Ca2+-binding proteins in dystrophin-deficient skeletal muscle fibres. In contrast to a relatively comparable expression pattern of the main protein population in normal vs. dystrophic fibres, our analysis showed that the expression of key Ca2+-binding proteins of the luminal sarcoplasmic reticulum is drastically reduced. This included the main terminal cisternae constituent, calsequestrin, and the previously implicated Ca2+-shuttle element, sarcalumenin. In contrast, the‘Stains-All’-positive protein spot, representing the cytosolic Ca2+-binding component, calmodulin, was not changed in dystrophin-deficient fibres. The reduced 2D‘Stains-All’ pattern of luminal Ca2+-binding proteins in mdx preparations supports the calcium hypothesis of muscular dystrophy. The previously described impaired Ca2+ buffering capacity of the dystrophic sarcoplasmic reticulum is probably caused by a reduction in luminal Ca2+-binding proteins, including calsequestrin. [ABSTRACT FROM AUTHOR]

The halophilic archaeon Haloferax volcanii is surrounded by a protein shell solely comprised of the S-layer glycoprotein. While the gene sequence and glycosylation pattern of the protein and indeed the three-dimensional structure of the surface layer formed by the protein have been described, little is known of the biosynthesis of the S-layer glycoprotein. In the following, pulse-chase radiolabeling and cell-fractionation studies were employed to reveal that newly synthesized S-layer glycoprotein undergoes a maturation step following translocation of the protein across the plasma membrane. The processing step, detected as an increase in the apparent molecular mass of the S-layer glycoprotein, is unaffected by inhibition of protein synthesis and is apparently unrelated to glycosylation of the protein. Maturation requires the presence of magnesium ions, involved in membrane association of the S-layer glycoprotein, and results in increased hydrophobicity of the protein as revealed by enhanced detergent binding. Thus, along with protein glycosylation, additional post-translational modifications apparently occur on the external face of the haloarchaeal plasma membrane, the proposed topological homologue of the lumenal face of the eukaryal endoplasmic reticulum membrane. [ABSTRACT FROM AUTHOR]

We report the first characterization of plasma-membrane-bound tyrosine phosphatase activity in the haemoprotozoan, Trypanozoma brucei. Several enzymic properties of the membrane fraction were identical to other protein tyrosine phosphatase (PTPases), such as (a) insensitivity to inhibitors of other protein phosphatases, including tetramisole, sodium tartrate and okadaic acid, (b) inhibition of other vanadate, and (c) activation by spermidine. Additionally, T. brucei PTPase activity presented two novel features, an acidic pH optimum at pH 4.0–5.0 and a very low Km value (2.5 nM) for the specific synthetic substrate, Tyr(P)Raytide. Higher Km values of 170 nM for Tyr(P)-RCML (RCML, reduced, carboxamido-methylated and maleylated lysozyme) and of 3 mM for the non-specific inorganic substrate p-nitrophenyl phosphate, suggested that the PTPase activity of T. Brucei was substrate specific. Reconstitution experiments on bloodstream-stage membrane proteins revealed that three polypeptides of 148, 115 and 72 kDa contained vanadate­inhibitable PTPase activity. Modulator assays revealed that the 72-kDa protein was responsible for the observed spermidine stimulation, but indicated that the modulator profile of the 148-kDa protein was most similar to the whole membrane fraction. Furthermore, the PTPase activity of T. brucei was life-cycle-stage regulated. Neither the whole membrane fraction nor the reconstituted proteins of the procyclic insect stage dephosphorylated tyrosine residues. [ABSTRACT FROM AUTHOR]

In order to investigate regulatory mechanisms and to identify potential substrates of a novel member of the protein kinase C (PKC) family, PKCμ, specific antibodies have been raised against unique amino- and carboxy-terminal regions. PKCμ kinase activity was studied upon immunoprecipitation from stably transfected cell lines as well as from the A549 carcinoma cell line expressing the endogeneous PKCμ gene. Cell fractionation revealed that PKCμ is predominantly found in the particulate fraction, suggesting an association with the membrane or membrane-bound structures. In vitro kinase assays with immunoprecipitated PKCμ demonstrated a Ca2+ independent enhancement of constitutive autophosphorylation activity by phosphatidylserine. Despite a limited in vitro phorbol ester response, an apparent phorbol ester activation of PKCμ was observed when cell cultures, instead of immunoprecipitated enzyme, were treated with either phorbol 12-myristate 13-acetate or 1,2 dioleoyl-sn-glycerol. Both in vitro autophosphorylation and substrate phosphorylation of myelin basic protein and hi stone Ill were enhanced under these conditions. However, long-term treatment with the phorbol ester did not result in downregulation of PKCμ protein levels and kinase activity. Studies with several protein kinase inhibitors revealed a novel sensitivity profile of PKCμ, with no inhibition by calphostin C, reduced sensitivity to staurosporine but, compared to other PKCs, an approximately 60-fold higher sensitivity to the selective PKA inhibitor H89. Together, the data presented here show that localization of PKCμ and regulation of its kinase activity differ from thin of other PKCs suggesting a novel function of PKCμ in intracellular signal pathways. [ABSTRACT FROM AUTHOR]

Recombinant wild-type β1γ1 dimmers of signal-transducing guanine nucleotide-binding proteins (G proteins) and β1γ1 dimmers carrying a mutation known to block γ-subunit isoprenylation (β1γ1 C71S) were expressed in baculovirus-infected insect cells. Both wild-type and mutant β1γ1 dimmers were found in soluble fractions of infected cells upon subcellular fractionation. Anion exchange chromatographic and metabolic-radiolabeling studies revealed that the soluble β1γ1 preparation contained approximately equal amounts of non-isoprenylated and isoprenylated β1γ1 dimmers. Soluble β1γ1 dimmers and native β1γ1 dimmers purified from bovine retina were reconstituted with recombinant phospholipase C-β2. Only isoprenylated β1γ1 dimmers were capable of stimulating phospholipase C-β1. The results show that γ-subunit isoprenylation and/or additional post-translational processing of the protein are required for βγ subunit stimulation of phospholipase C. [ABSTRACT FROM AUTHOR]

Proteins similar to the bacterial Dnaj protein have been implicated as molecular chaperones in different compartments of eukaryots. A plant equivalent is now described in tissues of dark-grown cucumber seedlings. Using a cucumber Dnaj protein produced by expression in bacteria, we raised polyclonal antibodies against the protein and used them for localization studies. In etiolated cucumber seedlings, both cotyledons and hypocotyledons were found to contain Dnaj proteins. Cell fractionation of etiolated cotyledons showed that Dnaj proteins were detectable mainly in the postnuclear cell fraction after sedimentation at 10000Xg, and in the microsomes. Following subfractionation by sucrose density gradient centrifugation and analysis by immunoblotting, a 53-kDa protein was attributed to the glyoxysomal fraction and an 80-kDa protein to the mitochondrial fraction. The glyoxysomal Dnaj protein behaved as a membrane-bound form. Upon heat shock, a slight increase in the content of the glyoxysomal Dnaj protein was found. When glyoxysomes were treated with protease and subsequently isolated by gradient centrifugation, virtually all immunologically detectable Dnaj protein was removed. Administration of radiolabeled mevalonic acid to cotyledons and isolation of glyoxysomes yielded labeled Dnaj protein which remained membrane bound during the purification of glyoxysomal membranes by floatation in a density gradient. [ABSTRACT FROM AUTHOR]

The partial sequence of a novel PtdIns-specific phospholipase C of the β subfamily (PtdIns-PLCβ3) is described. Based upon the predicted protein sequence, monospecific antibodies have been raised and used to identify a suitable source for purification of the protein. Fractionation of HeLa S3 cells revealed that immunoreactive PtdIns-PLCβ3 is membrane associated; purification (≈ 1000-fold) from this fraction yielded a single immunoreactive protein of 158 kDa, with a specific activity of 136 µmol · min-1 · mg-1, with PtdIns 4,5-bisphosphate as substrate. Substrate specificity and Ca2+ dependence of this purified PtdIns-PLC are characteristic of the PtdIns-PLCβ subfamily. [ABSTRACT FROM AUTHOR]

Recently, the purification to homogeneity was reported of a novel broad-specificity α-mannosidase from rat liver microsomal membranes [P. Bonay and R. C. Hughes (1991) Eur. J. Biochem. 197,229– 238]. The enzyme catalyzed the ordered removal of α1 ↵2-, α1 ↵ 3- and α1 ↵6-linked mannose residues from MannGlcNAc oligosaccharide substrates where n = 4–9 . We now show by subcellular fractionation and immunocytochemistry that the novel mannosidase is present in the endoplasmic reticulum, Golgi apparatus and endosomes. Enzyme activity is enriched in a heavy Golgi membrane fraction and to lesser extent in an intermediate density Golgi membrane fraction containing GlcNAc transferase I activity and in a ‘late’ endosomal fraction. Low levels of enzyme activity were detectable in endoplasmic reticulum membranes and in ‘early’ endosomes but not in receptor-enriched and ligand-free endosomes. Assays of enzymic activity using Golgi membrane fractions in the absence and presence of Triton X-100 showed that the active site of the enzyme faces the lumen of the membrane vesicles. Antibodies directed against the purified mannosidase showed no immunological cross-reaction to known endoplasmic reticulum and Golgi mannosidases. Conversely, the purified mannosidase was not recognized by antibodies directed against endoplasmic reticulum mannosidase nor Golgi mannosidase IA. [ABSTRACT FROM AUTHOR]

The cell-free forms of the multiple cellulase-containing protein complex (cellulosome), isolated from the cellulolytic bacterium Clostridium thermocellum strains YS, ATCC 27405 and LQRI, have a total carbohydrate content of 5-7% (by mass), consisting of O-linked oligosaccharide chains, The carbohydrate chains were liberated by alkaline-borohydride treatment and fractionated as oligosaccharide alditols via gel-permeation chromatography and HPLC. The fractions were investigated by 500-MHz ¹H-NMR spectroscopy in combination with monosaccharide and methylation analysis and with fast-atom-bombardment mass spectrometry (FAB-MS). In addition to the previously described major oligosaccharide, Multiple line equations cannot be represented in ASCII text. [Gerwig, G. J., de Waard, P., Kamerling, J. P., Vliegenthart, J. F. G., Morgenstern, E., Lamed, R. & Bayer, E. A. (1989) J. Biol. Chem. 264, 1027-1035], the following partial structures of this compound could be established: Multiple line equations cannot be represented in ASCII text. I Cell-free and cell-associated forms of the cellulosome of C. thermocellum, as determined for strain YS, have the same oligosaccharide pattern. Based on the oligosaccharide structures, a biosynthetic pathway is suggested. [ABSTRACT FROM AUTHOR]

The presence of acetylcholinesterase (AChE) in chromaffin granules has been controversial for a long time. We therefore undertook a study of AChE molecular forms in chromaffin cells and of their distribution during subcellular fractionation. We characterized four main AChE forms, three amphiphilic forms (G1a, G2a and G4a), and one non-amphiphilic form (G4na). Each form shows the same molecular characteristics (sedimentation, electrophoretic migration, lectin interactions) in the different subcellular fractions. All forms are glycosylated and seem to possess both N-linked and O-linked carbohydrate chains. There are differences in the structure of the glycans carried by the different forms, as indicated by their interaction with some lectins. Glycophosphatidylinositol-specific phospholipases C converted the G2a form, but not the other amphiphilic forms, into non-amphiphilic derivatives. The distinct patterns of AChE molecular forms observed in various subcellular compartments indicate the existence of an active sorting process. G4na was concentrated in fractions of high density, containing chromaffin granules. We obtained evidence for the existence of a lighter fraction also containing chromogranin A, tetrabenazine-binding sites and G4na ACHE, which may correspond to immature, incompletely loaded granules or to partially emptied granules. The distribution of G4na during subcellular fractionation suggested that this form is largely, but not exclusively, contained in chromaffin granules, the membranes of which may contain low levels of the three amphiphilic forms. [ABSTRACT FROM AUTHOR]

Carnitine acetyltransferase was purified from the cytoplasmic fraction of Candida tropicalis grown on alkanes in continuous culture. By ion-exchange chromatography the enzyme was resolved in two fractions with the same specific activity of 80 U/rag. The molecular mass of both enzyme forms, determined by non-denaturing gradient gel electrophoresis, was 540 kDa. After SDS electrophoresis only one band of 64 kDa was detected indicating that both enzymes are oligomers each containing eight subunits. Isoelectric focusing in agarose under non-denaturing conditions demonstrated the presence of at least four different charged species in the pH range between 5.6 and 6.7. After isoelectric focusing in 9 M urea/1% Nonidet P-40 gels, both enzyme forms were resolved into four bands. Peptide mapping, performed by cyanogen bromide cleavage of polypeptides separated by denaturing isoelectric focusing followed by second-dimension SDS electrophoresis, revealed a very high degree of homology between these polypeptides. The presence of the octameric form of carnitine acetyltransferase already in the starting material was demonstrated by non-denaturing gradient gel electrophoresis and immunoblotting. Antibodies against carnitine acetyltransferase from C. tropicalis ATCC 32113 formed precipitation lines with extracts from several Candida species but not with extracts of Candida utilis, Candida ethanothermophilum and an another strain of C. tropical& [ABSTRACT FROM AUTHOR]

A recombinant gene comprising phoS (the gene for the phosphate-binding protein PhoS) fused to a synthetic gene for a modified human growth-hormone-releasing factor (rahGRF) has been constructed. This gene was highly expressed in cells growing under conditions of phosphate starvation. Various conditions of continuous culture, varying in phosphate concentrations and dilution rates, have been tested to optimize the expression of the hybrid gene product (PhoS-mhGRF). Conditions were obtained such that a large amount of the hybrid protein was no longer exported as a result of saturation of export sites, which also induce the inhibition of processing of pre-PhoE and pre-OmpA. The pre-PhoS-mhGRF, accumulated in the cell, was recovered mainly in the particulate fraction after cell fractionation. This protein was purified. Besides the methionine residues located within the signal sequence, the only other one is located in the fusion joint of the hybrid protein. Thus cyanogen bromide treatment allowed the isolation of pure mhGRF. The yield obtained is about of 1 mg/1 culture. [ABSTRACT FROM AUTHOR]

In the present work chromatography on phosphocellulose and blue Sepharose have been used to fractionate the different phosphorylated forms of the low-molecular-mass high-mobility-group (HMG) proteins from metaphase arrested HeLa cells. The proteins in the different fractions from the blue Sepharose column were analysed by acetic acid/urea gel electrophoresis. Aliquots from the same fractions were also treated with alkaline phosphatase and the dephosphorylated and phosphorylated proteins were then compared by electrophoresis to identify the phosphorylated proteins. It was found that HMG 14 consisted of a mixture of an unphosphorylated and two phosphorylated forms, while HMG Y existed as one homogeneous superphosphorylated form. These findings remove previous uncertainty about phosphorylation of HMG Y and HMG 14. The presence of HMG M and phosphorylated forms of HMG 17 was confirmed. Peptide mapping of HMG I and HMG M gave further evidence that HMG M is a superphosphorylated form of HMG I, and it is suggested that the term HMG Im be used instead of HMG M. The results suggested that HMG I and Y from HeLa cells contained at least three and two metaphase-specific phosphate groups respectively, while HMG 14 and 17 both consisted of an unphosphorylated form and two phosphorylated forms. A protein corresponding to HMG Im from HeLa cells was also found to be present in metaphase-arrested human lymphocytes, while HMG I and from two different rodent species seemed to be less phosphorylated then their counterparts from HeLa metaphase cells. [ABSTRACT FROM AUTHOR]

Subcellular fractionation techniques have been used to assess the localization of injected 125I-labeled cholera toxin (125I-CT) taken up by rat liver in vivo, and to determine whether internalization of the toxin is required for the generation of the active A1 peptide. The uptake of injected 125I-CT into the liver is maximal at 5 rain (about 10% injected dose/g). At this time the radioactivity is for the most part recovered in the microsomal (P) fraction, but later on it progressively associates with the mitochondrial-lysosomal (ML) and supernatant fractions. The radioactivity is enriched 7-fold in plasma membranes at 5-15 min, and 15-60-fold in Golgi-endosome (GE) fractions at 15-60 min. On analytical sucrose gradients the radioactivity associated with the P fraction is progressively displaced from the region of 5′-nucleotidase (a plasma membrane marker) to that of galactosyltransferase (a Golgi marker). On Percoll gradients, however, it is displaced towards acid phosphatase (a lysosomal marker). Density-shift experiments, using Triton WR 1339, suggest that some radioactivity associated with the P fraction (at 30 min) and all the radioactivity present in the ML fraction (at 2 h) is intrinsic to acid-phosphatase-containing structures, presumably lysosomes. Comparable experiments using 3,3′-diaminobenzidine cytochemistry indicate that the radioactivity present in GE fractions is separable from galactosyltransferase, and thus is presumably associated with endosomes. The fate of injected 125I-labeled cholera toxin B subunit differs from that of the whole toxin by a more rapid uptake (and/or clearance) of the ligand into subcellular fractions, and a greater accumulation of ligand in the ML fraction. Analysis of GE fractions by SDS/polyacrylamide gel electrophoresis shows that, up to 10 rain after injection of 125I-CT, about 80% of the radioactivity is recovered as A subunit ... [ABSTRACT FROM AUTHOR]

The rate of exocellular levansucrase synthesis in an overproducing (sacUh) strain of Bacillus subtilis was shown to be directly proportional to the amount of two different transient forms of this enzyme located within the membrane fraction of the cells. The apparent Mr of the larger membrane form was 53 000, and that of the smaller form 50000; the half-life time of each form was estimated in vivo to be 4-6 s and 32-42 s, respectively. Ethanol treatment of the cells lead to the accumulation of the 53000-Mr form which may represent 1.5% of total membrane proteins. This latter form, partially purified, was transformed in vitro into the 50000-Mr form by the action of the Escherichia coli leader peptidase. These enzyme forms were quite different from the exocellular levansucrase since they showed a weak affinity for hydroxyapatite and needed complexed iron to display enzyme activity. Assuming the membrane forms were precursors of exocellular levansucrase, we propose a two-step mechanism for the secretion process of levansucrase. The number of exoprotein synthesis/secretion sites in a B. subtilis cell is estimated to 2.5 x 104. [ABSTRACT FROM AUTHOR]

The stability of the higher-order structure of chromatin containing high-mobility-group (HMG) proteins has been studied in rat liver nuclei by mild micrococcal nuclease digestion at low temperature and fractionation by sucrose gradient centrifugation. Nuclei preparation and digestion, chromatin solubilization and analysis have been carried out in two ionic conditions, 140 mM and 40 mM monovalent cation concentration, avoiding drastic changes in ionic conditions and temperature during preparation and analysis. During the time course of digestion at 140 mM ionic strength a material stable at 80 S appears, whose DNA is cleaved at values around 12 nucleosomes. The distribution of HMG proteins in different chromatin fractions was analyzed by immunodot using antibodies elicited against proteins HMG-1, HMG-2, and HMG-14 and 17. It appears that these proteins have a distribution distinctly different from the bulk of chromatin. They are never found in the chromatin fragments that keep their internucleosomal interactions, indicating that these proteins tend to accumulate in points where the chromatin has a less stable structure. [ABSTRACT FROM AUTHOR]

Investigates the effect of two agents of erythroid cell differentiation on the adenylate cyclase activity of fractionated rabbit bone marrow erythroblasts. Stimulation of the adenylate cyclase activity of rabbit bone marrow immature erythroblasts by erythropoietin and haemin; Effect of the addition of erythropoietin to cell cultures; In vitro effect of erythropoietin; Loss of hormonal responsiveness due to desensitization or receptor down-regulation.

Examines the effect of buffer volume and ionic strength on apparent cytosolic versus nuclear distribution of unoccupied Ah receptor during fractionation of liver from mice, Sprague-Dawley rats and Hepa1c1c9 cells in culture. Distribution of the Ah receptor; Distribution of each of three standard cytosolic marker enzymes, namely aldolase, glucose-6-phosphate dehydrogenase and lactate dehydrogenase; Fractionation at various buffer volumes and ionic strengths.

1. Neutrophils, isolated in large quantities from porcine blood were disrupted by nitrogen cavitation and separated by differentiaI centrifugation into a nuclear fraction and a post-nuclear supernatant. The latter was subfractionated by sucrose density gradient centrifugation into cytosol, a fraction consisting of membrane vesicles and two granule-rich fractions. 2. The membrane fraction accounted for 1.9% of the protein in the post-nuclear supernatant, the light granule fraction for 2.2% and the dense granule fraction for 4.2%. 3. Catalase, lactate dehydrogenase and malate dehydrogenase were largely confined to the cytosol. The dense granule fraction contained the highest quantities of the hydrolytic enzymes, although the membrame fraction was also rich in alkaline and acid phosphatase and γ-glutamyl transpeptidase activities. 4. Electron microscopy of the membrane fraction showed intact membrane vesicles, whereas the granular fractions consisted of electron-dense, membrane-bound granules. Two granular fractions were isolated which contained granules of differing size and density. 5. 3H-labeled wheat germ agglutinin bound to the surface of intact neutrophils and when these were disrupted and fractionated the membrane fraction showed a specific binding activity 16-times greater than that of the cavitated sample. 6. The membrane fraction interacted with the detergent digitonin and as a result underwent density perturbation increasing from 1.13 g · cm-3 to 1.18 g · cm-3. 7. Dodecylsulphate-polyacrylamide gel electrophoresis showed the membrane fraction to consist of at least 40 protein bands, with relative molecular masses ranging from 200000–16000. The granule fractions contained less protein bands, with a protein composition quite distinct from that of the membrane fraction. [ABSTRACT FROM AUTHOR]

1. Chemical extractions are proposed as a major tool for a fractionation of cellular proteins. As a model system, proteins from cultured hamster lens cells have been divided by independent extractions into seven subcellular fractions, corresponding to water-soluble proteins and the proteins from membranes, microfilaments (and other deoxycholate-soluble proteins), intermediate filaments, microtubules, polysomes and nuclei respectively. The latter two fractions have been subfractionated yielding ribosomal proteins, the elongation and initiation factors of the protein-synthesis machinery, chromatin proteins and non-chromatin proteins. 2. The protein compositions of the fractions have been analyzed by one-dimensional and two-dimensional Gel electrophoresis. This resulted in an almost complete topography of the proteins detected on two-dimensional gels of total-cell lysates. 3. Comparison of two-dimensional patterns of proteins from the total-cell lysate and proteins from hamster erythrocytes or from liver, muscle or brain tissue showed that the different cell types have only few proteins in common. Two proteins are common to all of these cell types, namely actin and a 68-kDa protein. The latter protein was, like actin, vimentin and the tubulin subunits, also present in most cell fractions. Evidence is presented that this protein is identical to a 68-kDa heat-shock protein. [ABSTRACT FROM AUTHOR]

When isolated liver cells from starved rats were incubated with fatty acids, the rates of O2 uptake and ketone body production in the presence of hexanoate were somewhat greater than with added palmitate. However, the 3-hydroxybutyrate/acetoacetate ratio was consistently higher in the presence of palmitate. Moreover, palmitate was much more effective than hexanoate in slowing the restoration to normal of an elevated lactate/pyruvate ratio. This action of palmitate was counteracted by inhibitors of long-chain fatty acid oxidation to ketone bodies. Palmitate, but not hexanoate, partially inhibited lactate-stimulated ethanol oxidation. These effects of palmitate were in most instances exaggerated in cells from clofibrate-treated rats, which had somewhat higher rates of ketogenesis. When cells were incubated with rotenone, ketone bodies were produced at about 30% of control rates and accumulated almost entirely as 3-hydroxybutyrate. In the presence of antimycin, ketone body accumulation from added palmitate again occurred at about 30% of control rates, but ketogenesis from endogenous substrates or hexanoate was inhibited to a greater extent. Cells from clofibrate-treated rats were equally sensitive to antimycin but considerably less inhibited by rotenone than control cells, although the 3-hydroxybutyrate/acetoacetate ratio remained very high. The depression of fatty acid oxidation in cells incubated with rotenone could be relieved to a considerable degree by coupling the dehydrogenation of hydroxyacyl-CoA to the reduction of acetoacetate. This observation was found to be valid only for longer chain fatty acids (C10–C18). The coupling of octanoate oxidation to acetoacetate reduction was promoted by the addition of carnitine. Carnitine did not increase the coupling of the oxidation of other fatty acids tested to acetoacetate reduction. The formation of labelled ketone bodies from [1-14C]palmitate and [1-14C]hexanoate was also examined. When the two fatty acids were presented together to rotenone-poisoned cells in the presence of acetoacetate, hexanoate caused some inhibition of 14CO2 formation and ketone body production from [1-14C]palmitate, but the converse did not apply. In fact palmitate stimulated the oxidation of [1-14C]hexanoate to 14CO2 and labelled ketone bodies. This stimulation was abolished by low levels of 2,4-dinitrophenol. The results of these experiments are explained on the basis of compartmentation of fatty acid oxidation between mitochondrial matrix and peroxisomes. Whereas both long-chain and short-chain fatty acids can be oxidized within the mitochondrial matrix, an additional site of long-chain acyl-CoA oxidation exists in the peroxisomes. Thus, peroxisomes and mitochondria co-operate in the oxidation of long-chain fatty acids. The oxidation of long-chain acyl groups within the peroxisomes and the subsequent transfer of reducing equivalents to the mitochondria, probably via a glycerol 1-phosphate shuttle, explains why long-chain fatty acids induce a more reduced ‘redox state’ than short-chain species in isolated liver cells. [ABSTRACT FROM AUTHOR]

A liver plasma-membrane fraction capable of Ca2+ uptake was isolated. The fraction exhibited high Na+, K+-ATPase, low glucose-6 phosphatase activity, and transported alanine in a Na+-dependent fashion. The uptake of Ca2+ was ATP-dependent; UTP, GTP, or CTP did not substitute for ATP. The presence of oxalate did not significantly alter the rate of uptake. The pH optimum of the reaction was basic (no uptake was visible at pH 6.8). These properties are at variance with those of the endoplasmic reticulum Ca2+ uptake system, which is oxalate-dependent. and has an acid pH optimum. The ATP-dependent Ca2+ uptake has a Km(Ca2+) of 1.4 × 10-8 M and a Kmax of transport of 30 nmol × mg protein-1 × min-1. No conclusive results were obtained on the calmodulin-sensitivity of the process: addition of calmodulin to the vesicles did not stimulate uptake, and the anti-calmodulin drug trifluoperazine had no inhibitory effect. However, another anti-calmodulin drug (R24571) had a limited, but statistically significant, inhibitory action. A partial release of the accumulated Ca2+ from the vesicles could be induced by the addition of Na+, and incubation of the vesicles in a high Na+ medium (as compared to high K+ medium) resulted in lower (about 25%) calcium uptake. Partial release of the accumulated Ca2+ could be induced also by the addition of H+. The releasing effect of H+, taken together with the absence of Ca2+ uptake at acid pH, suggests the possibility of a H+/Ca+ exchange. [ABSTRACT FROM AUTHOR]

Cyanophora paradoxa, a unicellular flagellate, contains cyanelles which are supposed to be cyanobacterial origin. DNA was isolated from subcellular fractions and separated according to density components in CsCl density gradients. The main DNA component, comprising more than 90% of the total DNA, has a buoyant density of 1.724g · cm&sup-3;. Several subsfractions m the range from 1.718 g · cm&sup-3; to 1,735 g · cm&sup-3; are contained in this component. This DNA of high complexity was considered to be host nuclear DNA. The DNA from the endosymbiotic cyanelles, which were isolated, treated with DNase, and purified by sucrose density gradient centrifugation exhibited a buoyant density of 1.692 g · cm&sup-3; in one strain and 1.695 g · cm&sup-3; in a second strain. Both cyanelle DNAs (cyDNA) have a complexity of approximately 126 × 10³ base pairs and comprise about 5% of the total cellular DNA content. Two additional DNA components of low complexity were isolated from crude cyanelle pellets obtained without DNase treatment. The larger of these, approximately 48 × 10³ base pairs in size, had a density of approximately 1.688 g · cm&sup-3;. The second component, about 15 × 10³ base pairs in size, banded in the density range between 1.710 g · cm&sup-3; and 1.720 g · cm&sup-3;. The latter is associated with nuclear DNA, The 48 × 10³-base-pair component was located in the cytosol and could be obtained after CsCl/ethidium bromide density gradient centrifugation at the position of covalently closed circular DNA, Both these components amounted to approximately 0.5–1% of total DNA. A further DNA component with a complexity of more than 150 × 10³ base pairs, enriched in fractions where mitochondria are expected, was not characterized further. The density was intermediate between cyDNA and nuclear DNA (1.710–1.720 g ·cm&sup-3;) and it amounted to 1–2% of the total DNA. Our results indicate that the DNA from cyanelles, believed to be endosymbiotic cyanobacteria, is not more complex than higher plant chloroplast DNAs. [ABSTRACT FROM AUTHOR]

We describe a sensitive immunological procedure for the detection of specific proteins in total cell extracts and for the comparison of antigenically related polypeptides. Proteins are fractionated in polyacrylamide gels and transferred electrophoretically to diazophenylthioether paper, to which they bind covalently. Specific proteins are identified by incubation with specific antibody and ⊃125 -labeled protein A from Staphylococcus aureus, followed by autoradiography. High-resolution separation of proteins prior to transfer is achieved by polyacrylamide gradient gel electrophoresis in the presence of sodium dodecyl sulfate or by nonequilibrium pH gradient electrophoresis, followed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Further information can be obtained by limited enzymatic proteolysis of the proteins in the gel following polyacryllamide gel electrophoresis in the presence of sodium dodecyl sulfate and analysis of the cleavage products by gel electrophoresis at right angles to the first gel. We show the application of this technique to the detection and comparison in extracts from infected cells of proteins related immunologically to the simian virus 40 capsid proteins VP1 and VP3. [ABSTRACT FROM AUTHOR]

With tyramine as substrate, a considerable part of the amine oxidase activity of rat aorta was inhibited by 0.1 mM semicarbazide. The residual activity was little affected by 1 mM semicarbazide. Oxidation of 5-hydroxytryptamine was not inhibited by 0.1 mM semicarbazide. The subcellular location of the semicarbazide-sensitive and semicarbazide-resistant amine oxidases was investigated by analytical density gradient centrifugation. The .semicarbazide-resistant enzyme was identified with the mitochondrial monoamine oxidase, located in the outer envelope of mitochondria. The semicarbazide-sensitive amine oxidase was ascribed to the plasma membrane because it was distributed like 5′-nucleotidase and (oligomycin-insensitive) Mg2+-ATPase in various fractionation experiments, and markedly shifted by digitonin towards higher equilibrium densities in sucrose gradient. [ABSTRACT FROM AUTHOR]

Inside-out cell envelopes from Halobacterium halobium R1M1 have been isolated by sonication and subsequent sucrose-density-gradient fractionation. The inverted cell envelopes transport protons from the exterior of the vesicles by a light-dependent, uncoupler-inhibited process. Enzymes usually facing the inside of the cell are exposed to the external medium in this preparation. [ABSTRACT FROM AUTHOR]

The native ribosomal subunits of the mouse plasmacytoma cell line MPC-11 were fractionated by CsCl gradient centrifugation into seven classes of particles designated p1, p2, p3, p4, p5, p6 and p7, which possessed densities of approximately 1.38, 1.43, 1.46, 1.49, 1.50, 1.51 and 1.53 g/cm³ respectively. The RNA was analysed by oligo(dT)-cellulose chromatography and gel electrophoresis. In both well-fed and starved cells p3 was generally the dominating class of particles. During metabolic shift-up conditions, however, there was a great increase of p6 particles. Transfer of material containing rRNA and mRNA into this region of the CsCl gradient indicates the accumulation of an initiation complex containing mRNA. During metabolic shift-down conditions a decrease of p6 particles and an increase of p2 and p3 particles was observed. The p2 fraction was rich in mRNA and may contain considerable quantities of messenger ribonucleoprotein particles. A substantial fraction of newly synthesised mRNA-containing material in starved cells banded in fact in the p2 region of the CsCl gradients. Circumstantial evidence indicates the existence of a transient 40-S initiation complex which bands in the p2 — p3 region of the gradients, Double-labelling experiments demonstrated that radioactivity in 40-S subunits first appeared in precursor particles with a density of 1.49 – 1.50 g/cm³ and only later in p3 and p6 particles, p7 particles were detected in starved cells. They were slowly labelled and were presumably derived from old ribosomes following completion of the polysome cycle. [ABSTRACT FROM AUTHOR]

A method for the subcellular fractionation of rat liver using whole homogenates of rat liver and analytical sucrose density gradient centrifugation is presented. The distributions in the sucrose gradients of marker enzymes for all organelles have been determined for control homogenates and for homogenates prepared in the presence of selective membrane perturbants. This technique is not subject to potential loss of information inherent in the use of postnuclear supernatants as starting material for fractionation experiments. Particular attention has been paid to the distributions of putative plasma membrane marker enzymes, up to 50% of which may be found in the nuclear pellet. ), Γ-Glutamyltransferase has been found to be entirely plasma membrane in location but has a different distribution pattern when compared with other plasma membrane markers. Particulate alkaline phosphatase and alkaline phosphodiesterase are shown to have bimodal distribution, one peak of which is coincident with 5′-nucleotidase. The other peak is coincident with that of the golgi marker, galactosyltransferase, but the membrane structure containing these activities shows characteristics of plasma membrane rather than golgi apparatus. [ABSTRACT FROM AUTHOR]

Four hours after infection with Sindbis virus, chick embryo fibroblasts were studied by electron microscopy and cell fractionation. Electron microscopy of infected and non-infected cells revealed that infection produced a disaggregation of polyribosomes into monomers. Apart from this observation most cells appeared well preserved, and no degranulation of the rough endoplasmic reticulum was visible. Analysis of postnuclear supernatants by sucrose density gradients showed that no change in the relative proportions of free and membrane-bound ribosomes was produced by infection. Approximately 30 % of the ribosomes and 50 % of the viral RNA were found to be associated with membranes. Of the membrane-associated viral RNA, 70 % was recovered as 26-S RNA. Similar results were obtained with fibroblasts infected by the temperature-sensitive Sindbis mutant ts2, which is defective in the co-translational processing of the viral gene products at the nonpermissive temperature. Sucrose gradient analysis of membrane-bound polyribosomes solubilized by detergent indicated that as much as 50 % of the membrane-associated viral 26-S RNA is not integrated into potyribosomes and that most of the ribosomes are present as monomers or ribosomal subunits. Treatment with puromycin of living cells or of isolated membrane fractions under a variety of ionic conditions revealed that the viral RNA-membrane linkage is insensitive to puromycin but sensitive to high concentrations of monovalent ions. The bulk of the membrane-bound ribosomes were detached by high salt and recovered as ribosomal subunits on sucrose gradients. These results are consistent with the idea that in chick embryo fibroblasts infected with Sindbis virus only a small percentage of the ribosomes are engaged in protein synthesis, and that the Sindbis messenger RNA may attach to endoplasmic reticulum membranes through a ribosome-independent, salt-sensitive link. [ABSTRACT FROM AUTHOR]

Cathepsin B from rat liver was purified to apparent homogeneity by cell-fractionation, freezing and thawing, acetone treatment, gel filtration, DEAE-Sephadex and CM-Sephadex column chromatography, and was crystallized. The purified enzyme formed spindle-shaped crystals and its homogeneity was proved by disc gel electrophoresis in the presence of sodium dodecyl sulfate and by ultracentrifugical analysis. Its s20,w value was 2.8 S and its relative molecular mass was calculated to be 22500 (±900) by sedimentation equilibrium analysis. Crystalline cathepsin B was shown to consist of four isozymes with isoelectric points between pH 4.9 and 5.3, the main isozyme having an isoelectric point of pH 5.0. The enzyme was irreversibly inactivated by exposure to weak alkali. The pH optimum was 6.0 with α-N-benzoyl-DL-arginine-4-nitroanilide as substrate. Amino acid analysis showed that the enzyme contained hexosamine, glucosamine and galactosamine. Cathepsin B inactivated aldolase, glucokinase, apo-ornithine aminotransferase, and apo-cystathionase, but the rates of inactivation of glucokinase, apo-ornithine aminotransferase, and apocystathionase were lower than that of aldolase. Studies by polyacrylamide gel electrophoresis in the presence and absence of sodium dodecyl sulfate showed that cathepsin B degraded apo-ornithine aminotransferase to two polypeptide chains differing in relative molecular mass and electrophoretic mobility. [ABSTRACT FROM AUTHOR]

The intracellular localization of exogenously supplied human platelet β-glucuronidase in cultured skin fibroblasts derived from a β-glucuronidase-deficient patient was studied. Four cellular fractions were obtained by differential speed centrifugation. Following two days of incubation, the exogenously supplied enzyme exhibited a distribution pattern identical to that of endogenous β-hexosaminidase. Disruption of membranes by freezing and thawing caused a 35% increase of the enzyme activity, thus indicating a latent activity following the internalization. This indicated localization in the lysosomal fractions. Longer incubation periods led to an intracellular shift of the engulfed enzyme from the lighter lysosomal fraction to heavier particles. Once located in the heavier fraction, the enzyme was relatively stable, and participated in the catabolism of 35S-labeled mucopolysaccharides which had accumulated in the lysosomes of these fibroblasts. A marked reduction in the accumulated mucopolysaccharides of the lysosomal fraction was observed following addition of the enzyme, This was accompanied by the formation of smaller sized molecules. [ABSTRACT FROM AUTHOR]

Techniques allowing the recovery of large free and membrane-bound polysomes in high yield are reported. Subcellular fractions were prepared from rat liver homogenates as described in the preceding paper. Purified microsomal membranes (obtained from the post-lysosomal supernatant) were adjusted to 50 mM Mg(CH3COO)2 and treated with 2% Triton X-100 and 0.3% sodium deoxycholate in the presence of yeast RNA and cell sap, and polysomes were purified by overnight centrifugation through low-ionic-strength discontinuous sucrose gradients containing 2 mg/ml of cell sap proteins. Polysomes were isolated from the mitochondria/endoplasmic reticulum complex (fraction C) by treatment with 2 % Triton X-100 and 0.5 % sodium deoxycholate in the presence of 50 mM Tris-HC1, pH 7.6, 0.1 M KC1, 0.15 M NH4Cl and 50 mM Mg(CH3COO)2 and purified through sucrose layers of decreasing ionic strength containing 2 mg/ml of cell sap proteins. Analyses of polysomes in isokinetic sucrose gradients showed that the free polysome fraction and both membrane-bound polysome fractions had 14-15 ribosomes per mRNA at the maximum of absorbance. Experiments from which these methods were derived are described. [ABSTRACT FROM AUTHOR]

Substantial improvements of cellular fractionation in ionic conditions allowing preservation of polysome structure and polysome-membrane interactions are reported. They consist primarily in minimizing the lysosomal content of fractions containing endoplasmic reticulum by isolating a lysosome-rich fraction with little loss (6 %) of RNA. Endoplasmic reticulum membranes were recovered in high yield, mainly in association with mitochondria, the remainder being found in the postlysosomal supernatant. The latter also contained practically all the free polysomes, as judged by metrizamide gradient analyses. The distributions of various constituents (RNA, DNA, protein and marker enzymes) among cell fractions is presented. [ABSTRACT FROM AUTHOR]

Subfractionation of sarcoplasmic reticulum from fast-twitch and slow-twitch rabbit skeletal muscles was performed on a sucrose density gradient. Vesicle fractions were characterized by: measurement of (Ca2-,M2+)-dependent (extra) ATPase. Mg2+-dependent (basal) ATPase, Ca2+ uptake characteristics, polypeptide patterns in sodium dodecylsulphate polyacrylamide gel electrophoreses, phosphoprotein formation and electronmicroscopy of negatively stained samples. In fast-twitch muscle, low and high density vesicles were separated. The latter showed high activity of (Ca2+,Mg2+)-dependent ATPase, negligible activity of Mg2--dependent ATPase, high initial rate and high capacity of Ca2- uptake, high amount of phosphorylated 115000-Mr polypeptide, and appeared morphologically as thin-walled vesicles covered with particles of 4 nm in diameter. Low density vesicles had little (Ca2-,M2+)-dependent ATPase but high Mg2+-dependent ATPase. Although the initial rate of Ca2- uptake was markedly lower, the total capacity of uptake was comparable with that of high density vesicles. Phosphorylated 115000-Mr polypeptide was detectable at low concentrations. Instead, 57000 and 47000-Mr polypeptides were characterized as forming stable phosphoproteins in the presence of ATP and Mg2+. Negatively stained, these vesicles appeared to have smooth surfaces. It is suggested that low density vesicles represent a Ca2+ sequestering system different from that of high density vesicles and that Mg2+-dependent (basal) ATPase as well as the 57000 and 47000-Mr polypeptides are part of the Ca2+ transport system within the low density vesicles. According to the results from slow-twitch muscle, Ca2+ sequestration by the sarcoplasmic reticulum functions in this muscle type only through the tow density vesicles. [ABSTRACT FROM AUTHOR]

1. Cathepsin L was purified from rat liver lysosomes by cell fractionation, osmotic disruption of the lysosomes in the lysosomal mitochondrial pellet, gel filtration of the lysosomal extract and chromatography on CM-Sephadex. 2. Cathepsin L is a thiol proteinase and exists in several multiple forms visible on the disc electropherogram By polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate, its molecular weight was found to be 23 000-24 000. The isoelectric points of the multiple forms of cathepsin L extended from pH 5.8-6.1 ascertained by analytical isoelectric focusing. 3. Using various protein substrates, cathepsin L was found to be the most active endopeptidase from rat liver lysosomes acting at pH 6-7. In contrast to cathepsin B1, its capability of hydrolyzing N-substituted derivatives of arginine is low and it does not split esters. 4. Greatest activity is obtained close to pH 5.0 with 70-90% of maximal activity at pH 4.0 and pH 6.0 and 30-40% at pH 7.0. 5. The enzyme is strongly inhibited by leupeptin and the chloromethyl ketone of tosyl-lysine. Leupeptin acts as a pseudo-irreversible inhibitor. 6. The enzyme is stable for several months at slightly acid pH values in the presence of thiol compounds in a deep-frozen state. [ABSTRACT FROM AUTHOR]

Human polymorphonuclear leucocytes were obtained from the synovial fluids of patients with inflamed knee joints suffering either from Reiter's syndrome or from, rheumatoid arthritis. The polymorphonuclear leucocytes were collected by gentle centrifugation followed by disruption and their subcellular fractionation by centrifugation in 0.34 M sucrose to provide a granule fraction and a post-granule supernatant fraction. 0.5M KCl extraction of the granule fraction yielded neutral protease activity, similar to trypsin, when assayed against fluorescein-labelled polymeric collagen fibrils.. The post-granule supernatant fraction contained an inhibitor' towards the neutral protease and trypsin. The inhibition of the neutral protease was found to be time-dependent, this inhibition being released. after 1.5-2 h. In contrast, the inhibition of trypsin was irreversible and this property was used to devise an assay procedure for the inhibitor. [ABSTRACT FROM AUTHOR]

1. The degree of homology between ribosomal RNA isolated from microplasmodia and amoebae of the slime mould Physarum polycephalum has been determined by competitive hybridisation of the RNA from the two sources to homologous DNA in solution. The extent of competition was measured both by hybridisation to saturation and by following the kinetics of hybrid formation. In each case competition was found to be 100%, indicating that the ribosomal RNAs from the two, quite different vegetative forms of the organism exhibit a high degree of homology and are probably transcribed from the same genes. 2. The relationship between the amount of nuclear DNA that codes for ribosomal RNA (rDNA) and ploidy has been investigated in three strains of P. polycephalum which exhibit a 1 : 2: 5 variation in the amount of DNA per nucleus. Ribosomal RNA saturation values were determined by hybridisation to DNA isolated from prophase nuclei of plasmodia. The proportion of rDNA was found to be constant at 0.16–0.18% of the total genome in the three strains. [ABSTRACT FROM AUTHOR]

Ficoll-gradient fractionation of a metabolic lysate from yeast spheroplasts was used to prepare vacuole, mitochondria and cytosol fractions. Tryptophan synthase activity was exclusively found in the soluble fraction. Proteinases A, B and C as well as the tryptophan-synthase-inactivating activity were mainly found in the vacuole fraction. The heat-stable activities inhibiting proteinase and tryptophan-synthase-inactivating enzyme appeared in the soluble fraction. The subceltular separation of tryptophan synthase and the tryptophan-synthase-inactivating activities explains why in crude extracts from stationary yeast cells, high tryptophan synthase activities are observed in spite of high tryptophan-synthase-inactivating activity. The proteinases of a freshly prepared vacuole fraction from yeast spheroptasts are unable to inactivate externally added tryptophan synthase unless the organelles are disrupted by sonication or osmotic shock. [ABSTRACT FROM AUTHOR]

Following incubation of Escherichia coli with puromycin hydrochloride increased proteolysis was detected both in vivo and in vitro. Cell fractionation showed that puromycin-stimulated proteolysis occurred primarily in the 150 000 × g supernatant fraction. Incubation with canavanine sulphate also gave rise to stimulated proteolysis in vivo but little proteolysis was detected when cell-free extracts of these cells were prepared. It is suggested that degradation of canavanyl proteins (proteins of normal length but altered composition) involves at, least one step which is not involved in the degradation of at least some puromycin peptides which are presumably significantly shorter than normal gene products. [ABSTRACT FROM AUTHOR]

Various sub-cellular fractions of rat liver exert catalytic effects on the oxidation of [4-14C]-cholesterol to 7α-hydroxy-[4-14C]cholesterol and to other [4-14C]cholesterol oxidation products. Some of these oxidation products created in these studies in vitro appear to be aberrant oxidation products in that there is no evidence that they are intermediates in the physiological oxidation of cholesterol. These aberrant oxidation products have been classified as autoxidation products despite the fact that the autoxidation reactions in these cell fractions appear to require the participation of NADPH, native proteins and perhaps certain nucleotides for the production of these compounds. The enzyme system catalysing the oxidation of [4-14C]cholesterol to 7α-hydroxy-[4-14C]cholesterol could be assayed in a 18000 × g supernatant fraction of rat liver This enzymic reaction was adversely affected by extensive homogenisation of liver tissue and by prolonged incubation periods, both of which tended to increase the autoxidation reactions. The cholesterol-7α-hydroxylase enzyme showed optimal activity m the presence of NAIDPH and oxygen. In the washed liver microsomal fraction, [4-14C]cholesterol was extensively oxidised in the presence of NADPH, ADP and ferrous ions to 7β-hydroxycholesterol, cholesterol -3β, 5α, 6β-triol and 7-ketocholesterol. Further investigation showed that another compound was also formed, having the same mobility as 7-ketocholesterol in the thin layer solvent systems used Reduction and further metabolism of this compound compared with reference standards suggested that it may be a cholesterol oxide, formed under conditions where peroxidation of microsomal lipids could occur. [ABSTRACT FROM AUTHOR]

A fractionation procedure for avian erythrocyte histones was devised to give the six known, reproducible histone fractions from a single nuclear preparation, with limited manipulation and little wastage. The proposed method includes selective, two-step acid extraction, followed by cation-exchange chromatography and exclusion chromatography. The fractions were identified by amino acid analysis and by starch gel electrophoresis. The method is flexible in scale and permits a continuous balance of compositions and activities in metabolic experiments. Factors contributing to the relative extractability of histones from chromatin in acid were considered. The essential role of this step was the separation of erythrocyte-specific, serine-rich histone V, and arginine-rich histones III, IV, which otherwise would be eluted together from Amberlite CG-50 by guanidinium chloride. Two other arginine-rich components, more easily eluted in chromatograms of other tissues, were absent from the usual extracts of mature erythrocytes. However, similar components were produced from histones III, IV by treatment with β-mercaptoethanol. The conditions for good resolution of sub-fractions of moderately lysine-rich histones and of arginine-rich histones by exclusion chromatography on Bio-Gel P-60 and P-30 were examined. Reversible adsorption appeared to be more significant than pore size of the gel; the sulphates of arginine-rich histones were resolved better than the chlorides. [ABSTRACT FROM AUTHOR]