Effect of thiamine, riboflavin and ascorbic acid on tetracycline, erythromycin and levomycetin activity]

The effect of antibiotics was estimated by inhibition of the protein increase in the broth culture of Staph, aureus during incubation at a temperature of 37 degrees for 18 hours. In some experiments preincubation of the antibiotic solutions with the vitamins for 2 hours at light and in dark was used. The antibiotic concentrations in gamma per 1 ml were equal to those of the vitamins. In the experiments with tetracycline and 2-hour preincubation at light the antibiotic in a concentration of 0.1gamma ml inhibited for certain the protein increase by 58.9%, in combination with thiamin it inhibited the protein by 60 per cent and in combination with ascorbic acid by 59%. Riboflavin lowered the activity of tetracycline to a value not differing for certain from the control one. In the experiments with preincubation in dark tetracycline inhibited the protein increase by 55.2%, in combination with thiamin it inhibited the protein increase by 50.5%, in combination with riboflavin by 53% and in combination with ascorbic acid by 57.2%. Erythromycin in a concentration of 0.03gamma/ml when preincubated at light inhibited the protein increase by 48.8% and in combinations with thiamin, riboflavin or ascorbic acid by 23, 27, 47.2% respectively. When preincubated in the darkness erythromycin alone inhibited the protein increase by 47.8% and in combinations with thiamin, riboflavin or ascorbic acid by 32.5, 51.1 or 49.8% respectively. The above vitamins has no effect on levomycetin activity.     

Human beta-glucuronidase pinocytosis and binding to the immobilized phosphomannosyl receptor. Effects of treatment of the enzyme with alpha-N-acetylglucosaminyl phosphodiesterase

beta-D-Glucuronidase (EC 3.2.1.31) was purified to homogeneity from human spleen, and enzyme fractions from CM-Sephadex were examined for uptake by fibroblasts and retention by a column of immobilized phosphomannosyl receptor. Uptake and binding were enhanced by treatment of the enzyme with alpha-N-acetylglucosaminyl phosphodiesterase, greatly reduced by prior treatment with alkaline phosphatase, and restored by subsequent treatment with alpha-N-acetylglucosaminyl phosphodiesterase. Immobilized phosphomannosyl receptor was used to separate high and low uptake enzyme forms. About 25% of the total beta-glucuronidase was retained by the receptor column and eluted with mannose 6-phosphate. The rate of uptake of retained enzyme was 2.5-3.0-fold greater than that of the enzyme applied to the receptor column. The fraction retained by the column was reduced to 5-10% by prior treatment of the enzyme with alkaline phosphatase. This phosphatase-resistant, receptor-retained fraction was taken up at only 24% the rate of non-phosphatase-treated, receptor-retained enzyme. However, its uptake was increased 7-fold by treatment with alpha-N-acetylglucosaminyl phosphodiesterase. The enhanced rate of pinocytosis conferred by treatment of the enzyme with alpha-N-acetylglucosaminyl phosphodiesterase was destroyed by a subsequent treatment with alkaline phosphatase. These studies demonstrate that although most of the "high uptake" enzyme in beta-glucuronidase from human spleen binds to receptors through phosphomonoesters of mannose, a significant fraction can interact with immobilized phosphomannosyl receptor and be taken up by fibroblasts through interactions involving mannose 6-phosphate in diester linkage with N-acetyl-D-glucosamine.     

Detergent-induced distinctions between fluoride- and vanadate-stimulated adenylate cyclases and their responses to guanine nucleotides

The influence of detergents on fluoride- and vanadate-stimulated adenylate cyclases was investigated with enzyme from liver and adipocyte plasma membranes. Stimulation of the adipocyte cyclase by Na3VO4 was maximal (sixfold) at 3 mM, was not additive with fluoride stimulation, and was readily reversed by washing of the membranes. Vanadate stimulation of the hepatic cyclase was specifically blocked by catechol, which had no effect on basal activity or on fluoride- or glucagon-stimulated activities. The hepatic enzyme, stimulated by fluoride ion, guanyl-5'-yl-(beta,gamma-imino)diphosphate (GPP(NH)P), or GPP(NH)P and glucagon, was inhibited by vanadate with 50% inhibition seen with 2 to 6 mM vanadate. The fluoride-activated adipocyte adenylate cyclase was inhibited by guanosine 5'-O-(3-thio-triphosphate) (GTP gamma S) more potently than by GPP(NH)P, with 50% inhibition being seen with 10 nM GTP gamma S or 100 nM GPP(NH)P. These nucleotides also inhibited the vanadate-stimulated enzyme, but with one-third the potency seen with the fluoride-activated cyclase. Dispersion of the adipocyte cyclase by Lubrol-PX into a 30,000g supernatant fraction caused no change in activation of the enzyme by fluoride, but reduced vanadate-stimulated activity 80%. By comparison, this treatment enhanced stimulation by GPP(NH)P twofold and by GTP gamma S threefold. More importantly, perhaps, the treatment with detergent blocked inhibition of the basal enzyme by GTP, blocked inhibition of fluoride- and vanadate-stimulated cyclases by GTP, GPP(NH)P, or GTP gamma S, and rendered vanadate-stimulated activity sensitive to enhancement by guanine nucleotides. The data indicate differences in the actions of vanadate and fluoride, made evident by the influence of guanine nucleotides and detergent treatment. The observations would be consistent with the idea that the effects of vandate may be due to the formation of GDP X V on the enzyme. The data strongly suggest that treatment of adenylate cyclase with Lubrol-PX causes a functional blockade in the guanine nucleotide-dependent inhibitory regulation (mediated by Ni), thereby allowing activation by the stimulatory guanine nucleotide-dependent regulatory component (Ns).     

Immunohistochemical Artifact for Nitrotyrosine in Eosinophils or Eosinophil Containing Tissue

Immunohistochemical artifacts for nitrotyrosine were investigated in eosinophils with regard to fixatives. Immunoreactivity for nitrotyrosine was revealed in separated eosinophils and in gastric mucosa fixed with periodate, lysine-paraformaldehyde (PLP). The increase in immunoreactivity by PLP was due to periodate itself, a component of PLP. Nitrotyrosine formed by peroxidase using NO 2 &#109 and H 2 O 2 or by peroxynitrite was not completely inhibited by 100 mM dithionite but the immunoreactivity for nitrotyrosine antibodies by PLP was completely inhibited by 5.7 mM dithionite. Although untreated eosinophils or ovalbumin (OVA) did not show protein tyrosine nitration in a standard Western blot, the treatment of the blotted membrane with PLP increased the reactivities of proteins from eosinophils with anti-nitrotyrosine antibodies. The increase in immunoreactivity of OVA with anti-nitrotyrosine antibodies by PLP did not change with pre-treatment with dithionite but was abolished by treatment with dithionite after PLP fixation. In HPLC assays, periodate did not generate nitrotyrosine from l -tyrosine and aminotyrosine. These results suggest that the treatment of eosinophils or eosinophil-containing tissues with PLP fixative augments the immunoreactivity of nitrotyrosine antibodies with eosinophils due to the formation of epitopes similar to nitrotyrosine by an oxidation reaction of periodate, which evokes an artifact in nitrotyrosine immunohistochemistry.     

Elementary steps in the reaction mechanism of chicken liver fatty acid synthase: beta-ketoacyl reductase and enoyl reductase

The following reactions catalyzed by chicken liver fatty acid synthase have been studied with the stopped-flow method in 0.1 M potassium phosphate (pH 7.0) and 1 mM ethylenediaminetetraacetic acid at 25 degrees C by monitoring the change in NADPH fluorescence: the transfer of acetoacetyl from acetoacetyl coenzyme A to the enzyme, reduction of the enzyme-bound acetoacetyl by NADPH (beta-ketoacyl reductase), and reduction of enzyme-bound D-hydroxybutyryl/crotonyl by NADPH (enoyl reductase). The first two reactions were studied by mixing enzyme-NADPH with acetoacetyl-CoA under conditions where the kinetics can be analyzed as two consecutive pseudo-first-order processes: a mechanism consistent with the aceto-acetyl-CoA dependence of the pseudo-first-order rate constant associated with formation of the aceto-acetyl-enzyme is a relatively rapid binding of substrate to the enzyme, with a dissociation constant of 650 microM, followed by formation of covalently bound acetoacetyl, with a rate constant of 10.2 s-1. The aceto-acetyl-enzyme is reduced by enzyme-bound NADPH with a rate constant of 20 s-1, and the NADPH binding is characterized by a dissociation constant of 5.3 microM. Reduction of the D-hydroxybutyryl-/crotonyl-enzyme was studied by mixing NADPH with enzyme that was equilibrated with D-hydroxybutyryl-CoA or crotonyl-CoA; the rate constant for reduction of an equilibrium mixture of D-hydroxybutyryl- and crotonyl-enzyme is 36.6 s-1. Steady-state kinetic studies of the reduction of acetoacetyl-CoA and crotonyl-CoA by NADPH also have been carried out.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunohistochemical artifact for nitrotyrosine in eosinophils or eosinophil containing tissue

Immunohistochemical artifacts for nitrotyrosine were investigated in eosinophils with regard to fixatives. Immunoreactivity for nitrotyrosine was revealed in separated eosinophils and in gastric mucosa fixed with periodate, lysine-paraformaldehyde (PLP). The increase in immunoreactivity by PLP was due to periodate itself, a component of PLP. Nitrotyrosine formed by peroxidase using NO 2 - and H 2 O 2 or by peroxynitrite was not completely inhibited by 100 mM dithionite but the immunoreactivity for nitrotyrosine antibodies by PLP was completely inhibited by 5.7 mM dithionite. Although untreated eosinophils or ovalbumin (OVA) did not show protein tyrosine nitration in a standard Western blot, the treatment of the blotted membrane with PLP increased the reactivities of proteins from eosinophils with anti-nitrotyrosine antibodies. The increase in immunoreactivity of OVA with anti-nitrotyrosine antibodies by PLP did not change with pre-treatment with dithionite but was abolished by treatment with dithionite after PLP fixation. In HPLC assays, periodate did not generate nitrotyrosine from l -tyrosine and aminotyrosine. These results suggest that the treatment of eosinophils or eosinophil-containing tissues with PLP fixative augments the immunoreactivity of nitrotyrosine antibodies with eosinophils due to the formation of epitopes similar to nitrotyrosine by an oxidation reaction of periodate, which evokes an artifact in nitrotyrosine immunohistochemistry.     

Plant-assisted degradation of phenanthrene as assessed by solid-phase microextraction (SPME)

The soil bacterium Sphingomonas yanoikuyae was isolated from a petroleum-contaminated soil and grown on mineral salts agar overlaid with the polycyclic aromatic hydrocarbon phenanthrene. The effect of white mustard, Sinapis alba, on phenanthrene degradation by S. yanoikuyae in artificially contaminated Redi-earth-sand was examined. Solid-phase-microextraction (SPME) gas chromatography-flame ionization detection (GC-FID) was used to quantify the concentration of phenanthrene in the gas phase of Magenta jars containing S. alba and S. yanoikuyae, each alone and with no additions. Gas chromatography-mass spectrometry (GC-MS) of Soxhlet extracts was used to determine the concentration of phenanthrene remaining in Redi-earth-sand. The gas phase concentration of phenanthrene in nonsterile Redi-earth-sand decreased by 99.7% in treatments with S. alba plus S. yanoikuyae, by 98.6% with S. alba, by 96.7% with S. yanoikuyae, and by 95.8% with no additions. Under gnotobiotic conditions, the gas phase concentration of phenanthrene in Redi-earth-sand decreased by 94% in treatments with S. alba plus S. yanoikuyae, by 77% with S. yanoikuyae, by 26% with S. alba, and 0% with no additions. The concentration of phenanthrene in Redi-earth-sand under gnotobiotic conditions decreased in treatments with S. alba plus S. yanoikuyae by 88%, by 67% with S. yanoikuyae, by 13% with S. alba, and 0% with no additions as measured in Soxhlet extracts. These results suggest that SPME-GC can be used to rapidly assess the potential of plants and microorganisms to reduce the level of unaged polyaromatic hydrocarbons such as phenanthrene in soil. This method provided results that were consistent with the more costly Soxhlet extraction method and was less time consuming.     

Scientific Report on the Belgian Expedition to the Great Barrier Reef in 1967. Nematodes IX Four new species of Quadricoma Filipjev (Nematoda, Desmoscolecida)

The following species are described: Q. papillata sp.n., characterized by its head-shape with broad truncated anterior end and in males by the possession of 2 single short, stout ventral genital papillae situated anterior to the middle of the body; Q. lizardiensis sp.n., closely related to Q. papillata but distinguished from the latter by its head-shape with a pronounced distally widened anterior border and the absence of genital papillae in the male; Q. freudenhammeri sp.n., characterized by its head-shape with an anteriorly tapering naked front part with thickened, sclerotized cuticle and a rounded posterior part covered with thin cuticle and by the stoma with cheilorhabdia; Q. noffsingerae sp.n., characterized by its head-shape with widened truncated anterior border surrounding 6 sometimes protruding lips, each with a tube-like papilla and by the position of the ocelli situated along the posterior half of the long cylindrical oesophagus.     

Characterization of the Participation of Sodium Channels on the Rise in Na+ Induced by 4-Aminopyridine (4-AP) in Synaptosomes

The participation of voltage-sensitive Na+ channels (VSSC) on the changes on internal (i) Na+, K+, Ca2+, and on DA, Glu, and GABA release caused by different concentrations of 4-AP was investigated in striatum synaptosomes. TTX, which abolished the increase in Na(i) (as determined with SBFI), induced by 0.1 mM 4-AP only inhibited by 30% the rise in Na(i) induced by 1 mM 4-AP. One millimolar 4-AP markedly decreased the fluorescence of the K+ indicator dye PBFI but 0.1 mM 4-AP did not. Like 1 mM 4-AP, ouabain decreased PBFI fluorescence and increased a considerable fraction of Na(i) in a TTX-insensitive manner. In contrast with the different TTX sensitivity of the rise in Na(i) induced by 0.1 and 1 mM 4-AP, the rise in Ca(i) (as determined with fura-2) induced by the two concentrations of 4-AP was markedly inhibited by TTX, as well as by omega-agatoxin in combination with omega-conotoxin GVIA, indicating that only the TTX-sensitive fraction of the rise in Na(i) induced by 4-AP is linked with the activation of presynaptic Ca2+ channels. It is concluded that the TTX-sensitive fraction of neurotransmitter release evoked by 4-AP is released by exocytosis, and the TTX insensitive fraction involves reversal of the neurotransmitters transporters. This contrasts with the exocytosis evoked by high K+ that is unchanged by TTX and with the neurotransmitter-transporter-mediated release evoked by veratridine, which is highly TTX sensitive and does not require activation of Ca2+ channels.     

Sialic acids in gastropods.

The occurrence of N-acetylneuraminic acid and N-glycolylneuraminic acid residues in preparations of the slug Arion lusitanicus (Gastropoda) was determined by sodium dodecyl sulphate electrophoresis of the proteins followed by lectin blots stained with the sialic acid specific lectin from Maackia amurensis, by the sensitivity of this binding to sialidase from Clostridium perfringens, by specific fluorescent labelling of sialic acids with 1,2-diamino-4,5-methylenedioxybenzene, by the determination of the sensitivity to sialate-pyruvate-lyase, by co-migration with standards on high performance anion exchange chromatography with pulsed amperometric detection and by identification of the typical masses in the fragmentation patterns of the trimethylsilyl derivatives after gas chromatography. It is the first time sialic acids are identified in gastropods.     

Phospholipase C-mediated signalling is not required for histamine-induced catecholamine secretion from bovine chromaffin cells

A possible role for signalling through phospholipase C in histamine-induced catecholamine secretion from bovine adrenal chromaffin cells has been investigated. Secretion evoked by histamine over 10 min was not prevented by inhibiting inositol-1,4,5-trisphosphate receptors with 2-APB, by blocking ryanodine receptors with a combination of ryanodine and caffeine, or by depleting intracellular Ca(2+) stores by pretreatment with thapsigargin. Inhibition of protein kinase C with Ro31-8220 also failed to reduce secretion. Inhibition of phospholipase C with ET-18-OCH(3) reduced both histamine- and K(+) -induced inositol phosphate responses by 70-80% without reducing their secretory responses. Stimulating phospholipase C with Pasteurella multocida toxin did not evoke secretion or enhance the secretory response to histamine. The secretory response to histamine was little affected by tetrodotoxin or by substituting extracellular Na(+) with N -methyl-d-glucamine(+) or choline(+), or by substituting external Cl(-) with nitrate(-). Blocking various K(+) channels with apamin, charybdotoxin, Ba(2+), tetraethylammonium, 4-aminopyridine, tertiapin or glibenclamide failed to reduce the ability of histamine to evoke secretion. These results indicate that histamine evokes secretion by a mechanism that does not require inositol-1,4,5-trisphosphate-mediated mobilization of stored Ca(2+), diacylglycerol-mediated activation of protein kinase C, or activation of phospholipase C. The results are consistent with histamine acting by depolarizing chromaffin cells through a phospholipase C-independent mechanism.     

Sediment-related growth limitation of Elodea nuttallii as indicated by a fertilization experiment

1. A fertilization experiment was performed to identify the limiting nutrient for the growth of submerged vegetation in ditches of a peat-grassland system in the Netherlands, in which restoration measures involved ceasing fertilization, exporting nutrients by removal of above-ground plant mass and large-scale introduction of calcium-rich, nutrient-poor artesian water.
2. Growth of Elodea was significantly enhanced by enrichment with nitrogen alone, and by fertilization with nitrogen in combination with phosphorus, and by nitrogen in combination with phosphorus and potassium.
3. Plant tissue nutrient concentrations increased significantly, for nitrogen by enrichment with nitrogen alone, and with nitrogen in combination with phosphorus and potassium; for phosphorus by enrichment with phosphorus alone and with phosphorus in combination with nitrogen and potassium; tissue concentrations of potassium were not enhanced by any treatment.
4. The elemental ratios of treated plants indicated that nitrogen, rather than phosphorus, was limiting in all treatments, except in those involving nitrogen and NK enrichment (when phosphorus was limiting).
5. The efficiency with which plants used nutrients declined with increased supply of nitrogen and phosphorus, but was unchanged when potassium was increased. Efficiencies were similar to those of other aquatic macrophytes.     

Sediment-related growth limitation of Elodea nuttallii as indicated by a fertilization experiment

1. A fertilization experiment was performed to identify the limiting nutrient for the growth of submerged vegetation in ditches of a peat-grassland system in the Netherlands, in which restoration measures involved ceasing fertilization, exporting nutrients by removal of above-ground plant mass and large-scale introduction of calcium-rich, nutrient-poor artesian water.
2. Growth of Elodea was significantly enhanced by enrichment with nitrogen alone, and by fertilization with nitrogen in combination with phosphorus, and by nitrogen in combination with phosphorus and potassium.
3. Plant tissue nutrient concentrations increased significantly, for nitrogen by enrichment with nitrogen alone, and with nitrogen in combination with phosphorus and potassium; for phosphorus by enrichment with phosphorus alone and with phosphorus in combination with nitrogen and potassium; tissue concentrations of potassium were not enhanced by any treatment.
4. The elemental ratios of treated plants indicated that nitrogen, rather than phosphorus, was limiting in all treatments, except in those involving nitrogen and NK enrichment (when phosphorus was limiting).
5. The efficiency with which plants used nutrients declined with increased supply of nitrogen and phosphorus, but was unchanged when potassium was increased. Efficiencies were similar to those of other aquatic macrophytes.     

Isolation and Purification of Chondroitin 6-Sulfate Protein From Umbilical Cord

Intact chondroitin 6-sulfate protein can be extracted from umbilical cord with dilute saline. Hyaluronic acid which is also extracted, is removed by precipitation with cetylpyridinium chloride followed by washing of the precipitate with aqueous sodium chloride. Subsequent purification is effected by passage through cation and anion exchange resins. Elution from the latter with salt solutions of increasing concentration yields chondroitin 6-sulfate proteoglycan in two fractions. The product is isolated from each of the fractions as the calcium salt by fractional precipitation with ethanol. The protein moiety can be cleaved from the mucopolysaccharide either by proteolytic digestion or treatment with alkali. The results obtaired on reaction with alkali and with sodium borohydride indicate that the polysaccharide is covalently linked to the protein through a serine unit.     

Inhibition of hydrogen peroxide release from activated macrophages by prior ingestion of erythrocytes or haemoglobin

Production of hydrogen peroxide by mouse peritoneal macrophages activated with Corynebacterium parvum was induced by incubating the cells with opsonised zymosan. H2O2 release was reduced by 47% when macrophages were preincubated with opsonised sheep erythrocytes. A significant decrease also occurred when the cells were preincubated with heat-denatured haemoglobin, but not when preincubated with opsonised erythrocyte ghosts, even though the latter were taken up by the macrophages. The ability of macrophages in an infected lesion to destroy microorganisms may therefore be impaired by ingestion of extravasated erythrocytes.     

Inhibition of hydrogen peroxide release from activated macrophages by prior ingestion of erythrocytes or haemoglobin

Abstract Production of hydrogen peroxide by mouse peritoneal macrophages activated with Corynebacterium parvum was induced by incubating the cells with opsonised zymosan. H₂O₂ release was reduced by 47% when macrophages were preincubated with opsonised sheep erythrocytes. A significant decrease also occurred when the cells were preincubated with heat-denatured haemoglobin, but not when preincubated with opsonised erythrocyte ghosts, even though the latter were taken up by the macrophages. The ability of macrophages in an infected lesion to destroy microorganisms may therefore be impaired by ingestion of extravasated erythrocytes.     

A study of the cell surface of tumour, foetal and lymph-node cells by cell electrophoresis after antibody and enzymic treatment

1. Rabbit anti-(rat foetal liver) serum, absorbed with adult rat liver cells, decreased the electrophoretic mobility of foetal liver cells by 51% and rat hepatoma cells by 45%, indicating the presence of a foetal-type antigen on the hepatoma cell membrane. 2. The chemical nature of the surface antigen was investigated. Incubation with neuraminidase had no effect on adult liver cells but decreased the electrophoretic mobility of foetal liver cells by 51% and of hepatoma cells by 34%; the effect of antiserum was decreased to one-fifth. 3. Sialic acid, or the supernatant from neuraminidase-treated cells, partially blocked the decrease in electrophoretic mobility induced by antiserum. 4. The pH-electrophoretic mobility curves of hepatoma cells treated with antisera were consistent with a sialic acidcontaining antigen on the surface of the tumour cells. 5. Treatment with ribonuclease did not decrease the electrophoretic mobility of adult-liver cells, but decreased that of the foetal liver cells by 17% and hepatoma cells by 29%. 6. In parallel studies made with mouse BP8 ascites-tumour cells ribonuclease decreased the electrophoretic mobility by 39%, that of normal mouse lymph-node cells by 4.8% and allergized mouse lymph-node cells by 13.3%. 7. Trypsin treatment also decreased the electrophoretic mobility of hepatoma cells by 22%.     

Mycoplasma pneumoniae induces interleukin-8 production via the epidermal growth factor receptor pathway

In mycoplasmal pneumonia, the bronchi are histopathologically filled with polymorphonuclear leukocytes. The EGFR pathway is involved in IL-8 production. We investigated the contribution of the EGFR pathway to IL-8 production by bronchial epithelial cells (A549) stimulated with Mp-Ag. The IL-8 production by A549 cells stimulated with Mp-Ag was decreased by the addition of an EGFR kinase inhibitor or transfection with small interfering RNA against EGFR. The levels of epiregulin mRNA in A549 cells were increased by stimulation with Mp-Ag. In conclusion, the EFGR pathway participates in IL-8 production by bronchial epithelial cells stimulated with Mp-Ag.     

Cysteine desulfhydrase activity in cucurbit plants: Stimulation by preincubation with l- or d-cysteine

Cysteine desulfhydrase activity in leaf discs of cucurbit plants is enhanced 2–4-fold by preincubation with l or d-cysteine. Preincubation with structural analogs of cysteine also stimulated the activity of the enzyme, but to a smaller extent. Maximal increase in cysteine desulfhydrase activity was observed by preincubation with 5 mM or higher concentrations of cysteine. Although not caused by activation, stimulation of the enzyme activity was half-maximal within less than 15 min. Whereas the increase in cysteine desulfhydrase activity by preincubation of leaf discs with cysteine was light independent, pretreatment of the entire plant with light or dark determined the leaf discs' potential for stimulation of the enzyme. Exposure to darkness for 4 hr reduced this potential by 60%. It is concluded that the potential for stimulation of cysteine desulfhydrase activity by preincubation with cysteine is regulated by a compound not synthesized, but metabolized, in the leaf tissue. This regulatory compound may be supplied to the leaves by long-distance transport.     

Phosphorylation of cellular proteins in Rous sarcoma virus-infected cells: analysis by use of anti-phosphotyrosine antibodies.

The protein substrates for the tyrosine protein kinases in cells transformed by avian sarcoma viruses were analyzed by gel electrophoresis in combination with immunoblotting or immunoprecipitation by antibodies against phosphotyrosine. We found that greater than 90% of phosphotyrosine-containing cellular proteins can be immunoprecipitated by these antibodies. The level of phosphotyrosine-containing cellular proteins detectable by this method markedly increased upon transformation with Rous sarcoma virus, and more than 20 distinct bands of such proteins were found in lysates of Rous sarcoma virus-transformed cells. Most of these phosphotyrosine-containing proteins had not been identified by other methods, and their presence appeared to correlate with morphological transformation in cells infected with various Rous sarcoma virus mutants and Y73, PRCII, and Fujinami sarcoma viruses. However, considerably different patterns were obtained with cells infected with nontransforming Rous sarcoma virus mutants that encode nonmyristylated src kinases, indicating that most substrates that correlate with transformation can only be recognized by p60v-src associated with the plasma membrane.