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1.
The nature of protein phosphatases that are active against the phosphorylated proteins of glycogen metabolism was investigated in rabbit skeletal muscle and liver. Six 32P-labelled substrates corresponding to the major phosphorylation sites on glycogen phosphorylase, phosphorylase kinase, glycogen synthase and inhibitor-1 were used in these studies. The results showed that the four protein phosphatases defined in the preceding paper, namely protein phosphatases-1, 2A, 2B and 2C [Ingebritsen, T. S. and Cohen, P. (1983) Eur. J. Biochem. 132, 255-261] were the only significant enzymes acting on these substrates. The four enzymes can be conveniently separated and identified by a combination of ion-exchange chromatography and gel filtration and by the use of specific inhibitors. Three species of protein phosphatase-2A were resolved on DEAE-cellulose, termed protein phosphatases-2Ao (0.12 M NaCl), 2A1 (0.2 M NaCl) and 2A2 (0.28 M NaCl) that had apparent molecular weights of 210000, 210000 and 150000 respectively. Protein phosphatase-2Ao was a completely inactive enzyme whose activity was only expressed after dissociation to a 34000-Mr(app) catalytic subunit by freezing and thawing in 0.2 M 2-mercaptoethanol. This treatment also dissociated protein phosphatases 2A1 and 2A2 to more active 34000-Mr(app) catalytic subunits. The catalytic subunits derived from protein phosphatases-2Ao, 2A1 and 2A2 possessed identical substrate specificities, preferentially dephosphorylated the alpha-subunit of phosphorylase kinase, were unaffected by inhibitor-1 and inhibitor-2 and were inhibited by similar concentrations of ATP. The properties of protein phosphatases-2A1 and 2A2 were very similar to those of the catalytic subunits, except that they were less sensitive to inhibition by ATP. Protein phosphatase-2B was eluted from DEAE-cellulose in the same fraction as protein phosphatase-2Ao. These activities were resolved by gel filtration, the Mr(app) of protein phosphatase-2B being 98000. Protein phosphatase-2B was completely inhibited by 100 microM trifluoperazine, which did not affect the activity of protein phosphatase-2Ao or any other protein phosphatase. Freezing and thawing in 0.2 M 2-mercaptoethanol resulted in partial inactivation of protein phosphatase-2B. Protein phosphatase-2C was eluted from DEAE-cellulose at the leading edge of the peak of protein phosphatase-2A1. These activities were completely resolved by gel filtration, since the Mr(app) of protein phosphatase-2C was 46000. Two forms of protein phosphatase-1 can be identified by chromatography on DEAE-cellulose, namely protein phosphatase-1 itself and the Mg X ATP-dependent protein phosphatase. Both these species were eluted at 0.16 M NaCl just ahead of protein phosphatases-2C and 2A1. These enzymes did not interfere with measurements of type-2 protein phosphatases, since it was possible to block their activity with inhibitor-2... 相似文献
2.
1. The distribution of ribosomal protein S1 in subcellular fractions of E. coli was determined by radioimmunoassay. It was found that about 70%, 20% and 10% of protein S1 were present in the high salt (1.0 M NH4Cl)-washed ribosomes, the ribosomal wash and the S100 fraction, respectively. 2. Protein S1 was purified from unwashed ribosomes by an improved procedure which included: (i) extraction of protein S1 from unwashed ribosomes with 1.2 M LiCl and 1.0 M NH4Cl, (ii) ammonium sulfate fractionation, (iii) two successive column chromatographies on DEAE-Sephadex, and (iv) hydroxylapatite column chromatography. Purified protein S1 was homogeneous in polyacrylamide gel electrophoresis under native and denatured conditions. 3. The molecular weights determined by sedimentation equilibrium and by SDS-polyacrylamide gel electrophoresis were 83,000 and 70,000 respectively. The sedimentation coefficient was estimated as 3.0S by glycerol gradient centrifugation. The stokes radius determined by Sephadex G-200 gel filtration was 45 A. From these data, the frictional ratio of protein S1 was calculated to be 1.65, assuming the molecular weight and partial specific volume to be 70,000 and 0.736, respectively. Protein S1 had an elongated shape with an axial ratio of approximately 8.5. 4. Protein S1 contained 2 residues of half-cystine and about 10 residues of tryptophan. From CD measurements, the contents of alpha-helix and beta-structure were estimated to be 32 and 27%, respectively. 5. As reported by Kolb et al. (1977) (Proc. Natl. Acad. Sci. U.S. 74, 2379-2383), and Draper et al. (1977) (Proc. Natl. Acad. Sci. U.S. 74, 4786-4790), the intrinsic fluorescence of protein S1 was markedly quenched on interaction with poly(U). The maximal quenching was observed when 30 mol of poly(U) (as UMP residues) was added to one mol of the protein. 相似文献
3.
When highly purified myelin from rat sciatic nerve was incubated with [gamma-32P]ATP, protein components of the membrane were phosphorylated indicating the presence of both the substrate (receptor protein) and an endogenous kinase in the membrane. Polyacrylamide gel electrophoresis of the phosphorylated membrane proteins followed by scintillation counting of gel slices and autoradiography showed that the polypeptides of molecular weights 28000, 23000 and 19000 were phosphorylated, and 32P from [gamma-32P]ATP having been incorporated into serine residues of the substrate proteins. Phosphorylation of purified myelin was Mg2+-dependent, was optimal at pH 6.5 and was not stimulated by adenosine 3',5'-monophosphate. We found that proteins other than those in myelin, such as phosvitin, casein, protamine and histones, can also act as a substrate for the membrane associated kinase. Muscle protein kinase inhibitor had no effect on the endogenous phosphorylation of myelin proteins or on the phosphorylation of phosvitin by peripheral nerve myelin protein kinase. However, the phosphorylation of histone by peripheral nerve myelin protein kinase was inhibited by the protein kinase inhibitor. After washing the membrane with 150 mM KCl the protein kinase that utilizes histone as substrate was found in the supernatant. In contrast, the endogenous phosphorylation of membrane proteins or the phosphorylation of phosvitin by the membrane associated kinase was not affected by washing. From these findings we conclude that at least two protein kinase systems exist in purified peripheral nerve myelin. One system is not inhibited by muscle kinase inhibitor, is tightly bound to the membrane and utilizes as its receptor proteins either exogenous phosvitin or endogenous membrane proteins. The second system is inhibited by muscle kinase inhibitor, is removable from the membrane and utilizes histones as its receptor proteins. 相似文献
4.
Cardiac alpha-crystallin. I. Isolation and identification 总被引:3,自引:0,他引:3
A water soluble protein, a major component of the cytosolic fraction of rat heart cells, was purified using either reverse phase HPLC or antibodies affinity chromatography procedures and characterized. The protein has an apparent Mr of 24 k, as judged by SDS-gel electrophoresis. Under non-denaturing conditions, however, the protein occurs as a homomultimer (Mr between 400 and 650 k) of the monomeric 24 kDa species and could be selectively enriched by fractionation of the cytosolic fraction on 10 to 40% sucrose gradients. Polyclonal antibodies, raised against the denatured 24 kDa protein, were used to investigate its tissue distribution. Besides the heart, where it is very abundant, the 24 kDa protein is expressed also in other red muscles and in kidneys, but was not detectable in stomach, thymus, liver, and brain. The amino acid composition of the protein and the partial amino acid sequence of various proteolytic fragments was determined. A search for homologies of the primary structure of known proteins has shown that the 24 kDa protein is strikingly similar, if not identical to alpha-B-crystallin. In fact, the two proteins were found to be indistinguishable also by immunological criteria. This study demonstrates that the lens protein alpha B-crystallin is a major cytosolic component of heart cells. 相似文献
5.
Microtubule-membrane interactions in cilia. I. Isolation and characterization of ciliary membranes from Tetrahymena pyriformis 总被引:7,自引:6,他引:1
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《The Journal of cell biology》1980,84(2):364-380
Tetrahymena ciliary membranes were prepared by four different techniques, and their protein composition was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), electron microscopy, and two-dimensional thin-layer peptide mapping. Extraction of the isolated cilia by nonionic detergent solubilized the ciliary membranes but left the axonemal microtubules and dyneine arms intact, as determined by quantitative electron microscopy. The proteins solubilized by detergent included a major 55,000-dalton protein, 1-3 high molecular weight proteins that comigrated, on SDS-PAGE, with the axonemal dynein, as well as several other proteins of 45,000-50,000 daltons. Each of the major proteins contained a small amount of carbohydrate, as determined by PAS-staining; no PAS-positive material was detected in the detergent-extracted axonemes. The major 55,000- dalton protein has proteins quite similar to those of tubulin, based on SDS-PAGE using three different buffer systems as well as two- dimensional maps of tryptic peptides from the isolated 55,000-dalton protein. To determine whether this tubulin-like protein was associated with the membrane or whether it was an axonemal or matrix protein released by detergent treatment, three different methods to isolate ciliary membrane vesicles were developed. The protein composition of each of these differetn vesicle preparations was the same as that of the detergent-solubilized material. These results suggest that a major ciliary membrane protein has properties similar to those of tubulin. 相似文献
6.
E A Beck L Tranqui-Pouit A Chapel B A Perret M Furlan G Hudry-Clergeon M Suscillon 《Biochimica et biophysica acta》1979,578(1):155-163
Human factor VIII-related protein precipitates with specific heterologous anti-bodies directed against purified factor VIII and supports ristocetin-induced aggregation of washed platelets. We purified human factor VIII from cryoprecipitate by subsequent gel filtration on crosslinked large-pore agarose. Factor VIII-related protein appeared as a large aggregate following electrophoresis on 3% polyacrylamide gels in the presence of sodium dodecyl sulfate (SDS). The same material was separated into multiple bands (molecular weight in excess of several millions) following electrophoresis on SDS-1% agarose gels. After complete disulfide reduction of factor VIII-related protein and electrophoresis on SDS-5% polyacrylamide gels a single subunit chain (Mr approximately equal to 200 000) was revealed. Analysis of this protein, in its non-reduced state, by negative contrast electron microscopy showed filaments of markedly variable size. The calculated molecular weight of such filaments ranged from about 0.6.10(6) to 20.10(6). We conclude that size heterogeneity is an essential feature of human factor VIII-related protein. 相似文献
7.
J J Jackson A Hagopian D J Carlo G A Limjuco E H Eylar 《The Journal of biological chemistry》1975,250(15):6141-6150
A unique highly soluble aspermatogenic protein (AP1) was isolated from guinea pig testes and was shown by immunofluorescence to occupy the outer surface of the sperm acrosome. This protein is a potent inducer of allergic orchitis and aspermatogenesis; as little as 0.2 mug induced orchitis in 60 percent of guinea pig tested. The AP1 protein, relatively small and neutral, is stable under acid conditions, but at pH 8.6 shows a variety of forms due either to aggregation or polymorphism. The purified AP1 protein appeared homogeneous by polyacrylamide gel electrophoresis at pH 2.7 and in sodium dodecyl sulfate and by immunoelectrophoresis using rabbit antisera to either the purified protein or the testes extract. It also showed a single band on immunodiffusion over a wide concentration range. The purification procedure consisted of delipidation with chloroform/methanol (2/1); acid extraction at pH 3.0; precipitation with 85 percent saturated ammonium sulfate; trichloroacetic acid extraction and gel filtration on Bio-Gel A-1.5; gel filtration on Bio-Gel P-10; chromatography on CM52 cellulose; and preparative gel electrophoresis at pH 2.7. Approximately 20 mg of purified AP1 protein were obtained from 5000 g of wet guinea pig testes. The AP1 protein induced an autoimmune disease characterized by infiltration of mononuclear cells around and within the seminiferous tubules (orchitis), followed by extensive damage and destruction of the germinal cells (aspermatogenesis). The course of the disease induced by this protein (0.5 to 1 mug) was essentially identical with that seen with whole testicular tissue or other purified fractions. 相似文献
8.
K Panneerselvam A S Balasubramanian 《Indian journal of biochemistry & biophysics》1991,28(5-6):395-400
The lysosomal enzyme binding receptor protein isolated from monkey brain by phosphomannan-Sepharose affinity chromatography was phosphorylated by [gamma-32P] ATP by protein kinases tightly associated with the receptor protein. A greater than 200 kDa protein was phosphorylated on both serine and tyrosine residues and a approximately 45 kDa protein was phosphorylated on only serine residues as evidenced by SDS-gel electrophoresis, autoradiography and phosphoamino acid analysis [(Panneerselvam, Ramamoorthy & Balasubramanian (1987) Biochem Biophys Res Commun, 147, 927-935)]. 125I-labelled lysosomal enzymes could be cross-linked to the receptor protein in the presence of disuccinimidyl suberate. Phosphorylation of the receptor on both serine and tyrosine residues was inhibited by quercetin, polylysine and polymyxin B. Catalytic subunit of cyclic AMP-dependent protein kinase preferentially phosphorylated the approximately 45 kDa protein. In the presence of Triton X-100, phosphorylation of a few additional protein bands on non-tyrosine residues was observed. There was a marked reduction in the efficiency of binding lysosomal enzymes by the phosphorylated receptor protein in comparison to the unphosphorylated receptor protein. 相似文献
9.
Normal and Rous sarcoma virus transformed chicken embryo fibroblasts cultured on petri dishes were labeled with 125I by two enzymic methods and one chemical method. The enzymic labeling systems employed chloroperoxidase and lactoperoxidase. Hydrogen peroxide was generated by glucose oxidase and glucose. The chemical method used chloramine T as the oxidant for iodide ion. After solubilization of cells, SDS disc gel electrophoresis and gamma counting, it was found that only one cell protein was predominantly labeled in all three reactions. This protein, a major cell surface protein, has been previously identified and termed Zeta protein. Zeta protein disappears from transformed cells and is susceptible to trypsin digestion. 相似文献
10.
The UDP-apiose/UDP-xylose synthase from cell suspension cultures of parsley has been purified 1400-fold by an improved method. The ratio of apiose to xylose formed from UDP-D-glucuronic acid (UDP-GlcUA) remained constant throughout the purification procedure. Dodecylsulfate-gel electrophoresis and sedimentation equilibrium measurements showed that this enzyme preparation is composed of two proteins with molecular weights of 65000 and 86000. The two proteins which are present in a molar ratio of about 1:0.7 to 1:0.9 could not be separated by ammonium sulfate fractionation, chromatography on DEAE-cellulose at different pH-values, and on omega-aminoalkyl-Sepharose, and by gel filtration on Acrylex P-100. Each protein is composed of two apparently identical subunits. The presence of only two different subunits was confirmed by end group analysis in which glycine was found as N-terminal amino acid for the larger and lysine for the smaller protein. Crosslinking with dimethylsuberimidate gave dimers of the identical subunits but no hybrids. Separation of the two proteins was achieved on DEAE-cellulose in the presence of urea. After dialysis only the 86000-Mr protein showed enzyme activity with no significant change in the apiose/xylose ratio. However, in the absence of the 65000-Mr protein enzyme stability was decreased drastically. By equilibrium dialysis it was found that 0.5 mol UDP-GlcUA are bound per mole of 86000-Mr protein. NAD+ alone was not bound, but in the presence of UDP it was also bound in a ratio of 0.5 mol/mol catalytic protein. Experiments in which sodium borohydride was added to the enzyme incubation gave no indication that the 4-keto intermediate is bound as a Schiff base to the enzyme. Also no evidence for epimerization at C-3 of the 4-ulose intermediate prior to ring contraction to apiose was found. 相似文献
11.
beta-Bungarotoxin, a specific presynaptic blocking agent, was prepared in two stages from the crude venom of Bungarus multicinctus by ion-exchange chromatography on the weakly acidic ion exchanger, CM-Sephadex, and on the strongly acidic ion exchanger, sulphopropyl-Sephadex. By these procedures it was purified to a single protein, which was shown by reduction to contain two polypeptide chains with mol.wts. of less than 15000. During purification of beta-bungarotoxin three other proteins were isolated. Two of these proteins have similar molecular weights, subunit structure and physiological properties to the major protein component. This latter is referred to as beta-bungarotoxin, since it has the same physiological properties as those described for unpurified beta-bungarotoxin by other workers. The first protein has very different physiological effects and biochemical properties from beta-bungarotoxin. This protein has a single class of polypeptide chains with an apparent molecular weight that is lower than the main beta-bungarotoxin protein, and appears to block synaptic transmission by a predominantly postsynaptic effect. It has been suggested [Oberg & Kelly (1976) J. Neurobiol. 7, 129-141] that the action of beta-bungarotoxin depends on its phospholipase A activity; however, in this preparation of the toxin less than 50 muunits of phospholipase A activity were detected (1 unit of activity is the amount of enzyme forming 1 mumol of L-alpha-phosphatidylcholine/min per mg of protein). 相似文献
12.
F J Walker 《The Journal of biological chemistry》1984,259(16):10335-10339
Thrombin treatment of the vitamin K-dependent protein S resulted in the loss of the activated protein C cofactor activity associated with protein S. The addition of phospholipid vesicles inhibited the inactivation. Thrombin treatment did not alter the molecular weight of the native protein. However, upon reduction, a peptide of approximately 3000 daltons was released from the treated protein. The interaction between calcium and protein S was reduced by thrombin treatment. When the calcium interaction was determined by the quenching of the intrinsic fluorescence of protein S, thrombin treatment appeared to inhibit the interaction between calcium and the protein. When the calcium interaction was observed by measuring the effect on the electrophoretic mobility of the protein, thrombin treatment reduced the interaction between calcium and protein S. However, the effect of thrombin treatment on the interaction between calcium and protein S was less than observed by the fluorescent method. This observation suggests that fluorescence quenching may be a result of a structural change induced by calcium binding. Thrombin treatment of protein S appears to uncouple the calcium binding from the structural change. In addition, the interaction between protein S and phospholipid vesicles was reduced by thrombin treatment. These results suggest that the thrombin conversion of protein S into a two-chain protein causes the loss of a calcium-induced change in protein structure, loss of the lipid-binding properties, and the loss of cofactor activity. 相似文献
13.
Regulation of microsomal stearoyl-coenzyme A desaturase. Purification of a non-substrate-binding protein that stimulates activity.
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Crude cytosolic fraction from rat liver was examined for proteins that may be involved in regulation of microsomal stearoyl-CoA desaturase activity. Gel filtration revealed the presence of several components that either stimulate or inhibit this enzyme. In addition, other components bind the acyl-CoA substrate, thus affecting observed activities in vitro. A protein that stimulates stearoyl-CoA desaturase but does not bind substrate was purified approx. 1100-fold. The purified protein had no visible absorption spectrum and an approximate mol.wt. of 26500. Maximal stimulation of desaturase activity occurred with less than 2 micrometer purified protein. The protein was heat-labile and exhibited neither catalase nor glutathione peroxidase activity. Addition of the cytosolic protein produced no effect on the desaturase reaction stoicheiometry; the proportions O2 consumed/NADH oxidized/stearoyl-CoA desaturated remained 1:1:1. Because the Km' for stearoyl-CoA was also unchanged by addition of the cytosolic protein, no change in substrate affinity was suggested. Furthermore addition of the cytosolic protein also produced no effect on desaturase inhibition by oleoyl-CoA, which suggested that the protein does not simply relieve apparent product inhibition. These results indicate that, in analogy to other cytosolic proteins that stimulate microsomal oxidase activities, the protein may have a regulatory function, perhaps related to activity modulation via organization of the multienzymic desaturase in the membrane. 相似文献
14.
C R Alving D H Conrad J P Gockerman M B Gibbs G H Wirtz 《Biochimica et biophysica acta》1975,394(2):157-165
Incorporation of vitamin A aldehyde (retinal) into liposomes had an inhibitory effect on the amount of human complement protein bound in the presence of specific antiserum. The total membrane-bound protein was directly measured on liposomes which were washed after incubation in antiserum and fresh human serum (complement). At every concentration of complement, decreased protein binding was found with liposomes which contained retinal. Binding of the third component of complement (C3) was also measured directly on washed liposomes and was found to be decreased in the presence of retinal. The diminution in protein binding due to retinal was not caused by differences in the amount of antibody bound and this was shown by two experiments. First, specific antibody protein binding to liposomes was directly measured and was essentially unaffected by retinal. Second, liposomes were prepared from lipid extracts of sheep erythrocytes. These liposomes were used as as immunoadsorbants to remove antisheep erythrocyte antibodies. The immunoadsorbant capacity was the same in both the presence and the absence of retinal. A further conclusion from these experiments was that retinal did not change the number of liposomal glycolipid antigen molecules available for antibody binding and thus presumably did not change the total number of lipid molecules present on the outer surface of the liposomes. Retinal did have an effect on the geometric structure of the liposomes. Size distribution measurements were performed in the diameter range of 1-6.35 mum by using an electronic particle size analyzer (Coulter Counter). Liposomes containing retinal were shifted toward smaller sizes and had less total surface area and volume. It was suggested that retinal-containing liposomes may have had a tighter packing of the molecules in the phospholipid bilayer. This effect of retinal on liposomal structure may have been responsible for the observed decreased binding of C3 and total complement protein. 相似文献
15.
The effect of inhibitors of protein synthesis on the killing of tumor cells by in vitro activated macrophages was determined. Cytotoxicity was inhibited by concentrations of puromycin, pactamycin, and actinomycin D that almost completely inhibited protein synthesis by guinea pig macrophages, but not by concentrations of drug that inhibited protein synthesis by only ± 50%. Cytotoxicity was inhibited when the effector macrophages were pretreated with the metabolic inhibitors, but not when the drugs were added 30 to 60 min after the initiation of the reaction. Pretreatment with puromycin or pactamycin also markedly inhibited the binding of tumor cells by mediator activated macrophages. These results are consistent with the hypothesis that one possible mechanism by which inhibitors of protein synthesis inhibit macrophage mediated cytotoxicity is by inhibiting close contact between effector and target cells. The finding that pretreatment of activated macrophages with trypsin also inhibits tumor cell killing suggests that protein synthesis may be necessary to maintain an adequate number of “recognition structures” on the macrophage membrane, structures that mediate the initial contact between the activated macrophage and the target tumor. 相似文献
16.
D J Millward P J Garlick R J Stewart D O Nnanyelugo J C Waterlow 《The Biochemical journal》1975,150(2):235-243
Because of turnover, protein synthesis and breakdown can each be involved in the regulation of the growth of tissue protein. To investigate the regulation of skeletal-muscle-protein growth we measured rates of protein synthesis and breakdown in growing rats during development on a good diet, during development on a marginally low-protein diet and during rehabilitation on a good diet after a period of severe protein deficiency. Rates of protein synthesis were measured in vivo with a constant intravenous infusion of [14C]tyrosine. The growth rate of muscle protein was measured and the rate of breakdown calculated as breakdown rate=synthesis rate-growth rate. These measurements showed that during development on a good diet there was a fall with age in the rate of protein synthesis resulting from a fall in capacity (RNA concentration) and activity (synthesis rate per unit of RNA). There was a fall with age in the breakdown rate so that the rate was highest in the weaning rats, with a half-life of 3 days. There was a direct correlation between the fractional growth and breakdown rates. During rehabilitation on the good diet, rapid growth was also accompanied by high rates of protein breakdown. During growth on the inadequate diet protein synthesis rates were lesss than in controls, but growth occurred because of decreased rates of protein breakdown. This compression was not complete, however, since ultimate muscle size was only one-half that of controls. It is suggested that increased rates of protein breakdown are a necessary accompaniment to muscle growth and may result from the way in which myofibrils proliferate. 相似文献
17.
Ag independent adhesion between lymphocytes and target cells is mediated in part by the interaction between lymphocyte function associated Ag-1 (LFA-1) and its coreceptor intercellular adhesion molecule-1 (ICAM-1). Within minutes, PMA treatment of JY cells, which express both LFA-1 and ICAM-1, induced capping of LFA-1 and augmentation of intercellular adhesion lasting for several hours. However, over the course of 15 to 30 min, both of these events were blocked by elevation of intracellular cAMP concentration ([cAMP]i) presumably via activation of protein kinase A. This short term inhibition of protein kinase C-induced adhesion was in contrast to the long term augmentation of adhesion caused by increased [cAMP]i as demonstrated in the companion article. Intercellular adhesion, due to LFA-1/ICAM-1 interactions, could also be induced by LPS treatment of JY cells. At submaximal concentrations, the extent of aggregation induced by LPS had two maxima, one at 30 to 60 min and the other with a plateau at 5 to 8 h. LPS is known to activate protein kinase C and we show that LPS treatment induced increased [cAMP]i. Using inhibitors of protein kinases C and A, possible mediators of the two components of adhesion induced by LPS could be identified. The early component was abrogated by inhibition of protein kinase C although the later component was unaffected. In contrast, an inhibitor of protein kinase A had no affect on the early component and attenuated, but did not entirely eliminate, the late component. These results suggest a model of sequential induction, inhibition, and reinduction of LFA-1/ICAM-1-mediated lymphocyte adhesion that is regulated by temporally ordered actions and interactions of protein kinases C and A. 相似文献
18.
A model mechanism was developed for the binding of a rigid multisite protein with a randomly coiled multivalent ligand. Probabilities of the formation of chain loops between sites located at given distances at the protein were calculated by an extension of the concept of ring closure in coiled chain molecules. Expressions were derived for the dependence of overall equilibrium quantities, such as the binding constant between the protein and the ligand, on intrinsic parameters such as intrinsic binding constants, number of sites at the protein and their distances and on the chain length of the polymeric ligand. A pronounced chain length dependence of the overall binding constant was predicted even at chain lengths much longer than the size of the protein. Such a dependence was previously observed for the enzyme prolyl hydroxylase which acts on polymeric substrates like (ProProGly)n. This so far unexplained feature is quantitatively described by the model mechanism which is believed to be applicable to many other interactions of biological importance. 相似文献
19.
Summary A quantitative ultrastructural study of the protein bodies of the lupin cotyledonary cells was performed to determine the protein content per cell. Two kinds of protein body were observed by transmission and scanning electron microscopy whatever the cellular type within the cotyledon. Some of these were conventional spherical structures, entirely filled with a dense protein matrix, others exhibited one large or several small light inclusions within the dense matrix. Even when a few light areas contained globoids, the majority remained of unknown nature, but could not be considered proteinaceous since they never reacted with specific protein dyes. The reserve protein content per cell was determined by image analysis on two seeds (L1 and L2) selected because they had a markedly different total protein content. The volume occupied by the dense matrix of the intracellular protein bodies was considered representative of the reserve protein quantity. The protein content per cell increased from the periphery to the centre of the cotyledon in the two seeds studied. The protein content per cell of the L2 seed was generally found to be greater than the L1 seed, in particular in the abaxial zone where it was markedly higher. The difference in total protein content of the two seeds was demonstrated to be primarily due to a differential alveolation of the protein bodies. These results will be used to study the relationship between the protein content of the cotyledonary cells and their nuclear DNA content. 相似文献
20.
1. Protein extracts obtained from Salmonella minnesota Re mutant cells by treatment with EDTA/NaC1 solution contain a protein which exhibits high affinity to bacterial lipopolysaccharides. The isolation and partial characterization of this lipopolysaccharide-binding protein is described. 2. The protein was purified from EDTA extracts by a two-step procedure consisting of ion-exchange chromatography on CM-Sephadex and preparative polyacrylamide gel electrophoresis at pH 9.5. The yield of the total purification procedure was around 16%. 3. The resulting protein preparation was homogeneous on the basis of disc gel electrophoresis, dodecylsulfate gel electrophoresis, isoelectric focusing in polyacrylamide gel and immunoelectrophoresis. 4. The isoelectric point of the protein was found to be 10.3 at 4 degrees C. Its molecular weight determined by dodecylsulfate gel electrophoresis is 15000. Its amino acid composition is characterized by the absence of histidine and proline, a low content in tyrosine and high amounts of alanine, lysine, aspartic and glutamic acid residues, or their respective amides. 5. The lipopolysaccharide-protein association was shown to be mainly due to ionic interactions of the basic protein with negatively charged groups (probably phosphate and pyrophosphate groups) of the lipid A moiety. 6. Purified lipopolysaccharide-binding protein is immunogenic in rabbits, thus enabling the preparation of specific antiserum. 7. The protein is located at the surface of Salmonella minnesota Re mutant cells as revealed by antiserum absorption with total bacteria. Ferritin-labelling studies further demonstrated that it is evenly spread over the entire cell surface. 8. Comparative antiserum absorption studies using smooth and rough strains of Salmonella minnesota, Salmonella typhimurium, Escherichia coli, Klebsiella and Shigella revealed the presence of lipopolysaccharide-binding protein (or a serologically cross-reacting antigen) in most of the strains tested. From these results the protein can be considered as a common antigen of Enterobacteriaceae. 相似文献