Activation of pigeon erythrocyte adenylate cyclase by cholera toxin partial purification of an essential macromolecular factor from horse erythrocyte cytosol

A cytosolic, macromolecular factor required for the cholera toxin-dependent activation of pigeon erythrocyte adenylate cyclase and cholera toxin-dependent ADP-ribosylation of a membrane-bound 43 000 dalton polypeptide has been purified 1100-fold from horse erythrocyte cytosol using organic solvent precipitation and heat treatment. This factor, 13 000 daltons, does not absorb to anionic or cationic exchange resins, is sensitive to trypsin or 10% trichloroacetic acid and is not extractable by diethyl ether. Activation of adenylate cyclase by cholera toxin requires the simultaneous presence of ATP (including possible trace GTP), NAD⁺, dithiothreitol, cholera toxin, membranes and the cytosolic macromolecular factor. Reversal of cholera toxin activation of adenylate cyclase, and of the toxin-dependent ADP-ribosylation, requires the presence of the cytosolic factor. The ability of the purified cytosolic factor to influence the hormonal sensitivity of liver membrane adenylate cyclase may provide clues to its physiological functions.     

Mechanisms of peroxidase oxidation of o-dianisidine, 3,3′,5,5′-tetramethylbenzidine, and o-phenylenediamine in the presence of sodium dodecyl sulfate

Peroxidase oxidation of o-dianisidine, 3,3′,5,5′-tetramethylbenzidine, and o-phenylenediamine in the presence of sodium dodecyl sulfate (SDS), an anionic surfactant, was spectrophotometrically studied. It was found that 0.1–100 mM SDS concentrations stabilize intermediates formed in the peroxidase oxidation of these substrates. The cause of the stabilization is an electrostatic interaction between positively charged intermediates and negatively charged surfactant.     

Monoclonal antibodies against beta-galactoside-binding lectin of chick embryo recognizes conformational change of the antigen

Monoclonal antibodies against chick embryonic beta-galactoside-binding lectin were obtained. One of the monoclonal antibodies was ineffective in Western blotting and seemed to be unable to bind the SDS-denatured lectin. When the native lectin was dotted on a nitrocellulose filter and subjected to denaturation by treatment with SDS, urea or heat, binding of this antibody no longer occurred, though other monoclonal antibodies bound normally. This antibody seems to have been raised against an epitope which is destroyed upon denaturation.     

Human lymphocyte tubulin: Purification and characterization in normal and leukemic cells

Tubulin has been purified from human blood and tonsil lymphocytes. Using gel filtration, the molecular weight of human lymphocyte tubulin was estimated to be 119 000. The proteins was shown to consist of two subunits, with molecular weights of 61 000 and 58 000 comparable to the α and β polypeptides of human brain tubulin. A partial identity reaction was observed between lymphocyte tubulin and human tubulin when tested by double immunodiffusion against a rabbit anti-human brain tubulin antibody. In the presence of GTP, the purified protein polymerized to form microtubules. Tubulin was localized to the cell's juxtacentriolar region by immunofluorescence and electron microscopy. When assayed by a colchicine-binding assay corrected for time decay, the binding affinity was 1.50 ± 0.86 · 10⁶M^?1 and a level in normal lymphocytes of 1.21 · 10^?2 ± 0.79 g/g of soluble protein was determined. Since chronic lymphocytic leukemia lymphocytes have an anomalous capping behavior as well as an unusual susceptibility to colchicine toxicity, the properties and levels of tubulin were determined in these cells. Similar values were obtained for the level, decay rate, molecular weight, and K_a for colchicine as for normal lymphocytes. Chronic lymphocytic leukemia lymphocyte tubulin polymerized in a normal fashion. It thus appears that a decrease in the quantity or function of tubulin does not account for these anomalies in the chronic lymphocytic leukemia lymphocyte.     

Isolation, partial chemical and biological characterization of thymone B

A new approach to the study of the molecular arrangements of proteins in membranes is described. Irradiation with visible light of native erythrocytes or washed erythrocyte membranes suspended in buffers containing a) riboflavin, fluorescein or fluorescein coupled to dextran and b) ³H-labelled tryptophan resulted in incorporation of radioactivity into the membrane proteins. Polyacrylamide gel electrophoresis of solubilized membranes followed by radioactivity measurements of the separated membrane proteins revealed that in native erythrocytes the protein components known to be located at the exterior cell surface, Band 3 and the major sialoglycoproteins became specifically labelled, whereas in washed lysed cells all of the major membrane proteins were labelled.     

Purification and characterization of chick intestine brush border membrane Effects of 1α(OH) vitamin D3 treatment

A new technique has been developed for the isolation of membrane vesicles from the vitamin D-deficient and vitamin D-treated chick intestinal brush border membrane. The technique involves removal of nuclei from a low speed pellet by discontinuous sucrose gradient centrifugation. The resulting intact brush borders are then homogenized in 0.5 M Tris and the membrane fragments purified on a glycerol gradient. This preparation represents a 20-fold purification of the brush border marker sucrase. After 1α-hydroxyvitamin D₃ treatment there is a significant increase in membrane phospholipid phosphorous, an alteration in the fatty acid composition of the phosphatidylcholine fraction of membrane phospholipid, and a decrease in sucrase specific activity.     

Electrophoretic profiles of cyanobacterial membrane polypeptides showing heme-dependent peroxidase activity

The photosynthetic membranes of Anacystis nidulans R2 were examined electrophoretically following solubilization with lithium dodecyl sulfate. Electrophoresis yielded six prominent chlorophyll-containing bands. In addition, five polypeptides were observed which possessed heme-dependent peroxidase activity, monitored by incubating gels with 3,3′,5,5′-tetramethylbenzidine plus hydrogen peroxide. One such polypeptide, at 105 kdaltons, was removed by repeated washing of the membranes. Four remaining peroxidase-active polypeptides were observed at 7.2, 13.5, 18.5 and 33 kdaltons. Further examination of these four polypeptides yielded the following results. (1) The mobility of the 33 kdalton polypeptide was altered from 29 to 33 kdaltons upon heating (70°C) during membrane solubilization. (2) All four polypeptides showed stable heme-protein associations in the presence of 8 M urea; however, in the presence of urea, alterations in protein mobility were observed for each poly-peptide and only two (at 13.5 and 33 kdaltons) showed peroxidase activity following heating (70°C) during membrane solubilization. (3) The presence of thiols during membrane solubilization at 0°C was required to observe peroxidase activity at 7.2 kdaltons. These results, when compared to known properties of isolated cytochromes, suggest that the four polypeptides characterized here correspond to the subunits of photosynthetic cytochromes. Electrophoretic assessment of maize mutants lacking cytochrome f and b₆ activity supports this suggestion.     

P-glycoprotein as multidrug transporter: a critical review of current multidrug resistant cell lines

MDR has been studied extensively in mammalian cell lines. According to usual practice, the MDR phenotype is characterized by the following features: cross resistance to multiple chemotherapeutic agents (lipophilic cations), defective intracellular drug accumulation and retention, overexpression of P-gp (often accompanied by gene amplification), and reversal of the phenotype by addition of calcium channel blockers. An hypothesis for the function of P-gp has been proposed in which P-gp acts as a carrier protein that actively extrudes MDR compounds out of the cells. However, basic questions, such as what defines the specificity of the pump and how is energy for active efflux transduced, remain to be answered. Furthermore, assuming that P-gp acts as a drug transporter, one will expect a relationship between P-gp expression and accumulation defects in MDR cell lines. A review of papers reporting 97 cell lines selected for resistance to the classical MDR compounds has revealed that a connection exists in most of the reported cell lines. However, several exceptions can be pointed out. Furthermore, only a limited number of well characterized series of sublines with different degrees of resistance to a single agent have been reported. In many of these, a correlation between P-gp expresson and transport properties can not be established. Co-amplification of genes adjacent to the mdr1 gene, mutations [122], splicing of mdr1 RNA [123], modulation of P-gp by phosphorylation [124] or glycosylation [127], or experimental conditions [26,78] could account for some of the complexity of the phenotype and the absence of correlation in some of the cell lines. However, both cell lines with overexpression of P-gp without increased efflux [i.e., 67,75] and cell lines without P-gp expression and accumulation defects/increased efflux [i.e., 25,107] have been reported. Thus, current results from MDR cell lines contradict - but do not exclude - that P-gp acts as multidrug transporter. Other models for the mechanism of resistance have been proposed: (1) An energy-dependent permeability barrier working with greater efficacy in resistant cells. This hypothesis is supported by studies of influx which, although few, all except one demonstrate decreased influx in resistant cells; (2) Resistant cells have a greater endosomal volume, and a greater exocytotic activity accounts for the efflux. Furthermore, large amounts of P-gp in the plasma membrane altering the ultrastructure and generalized changes, such as increases or decreases in membrane fluidity, alterations in lipid composition, changes in transmembrane pH gradient and membrane potential have been described in MDR cell lines and could account for some of the findings.     

Ammoniacal silver staining of proteins: mechanism of glutaraldehyde enhancement

When the conditions for detecting proteins by ammoniacal silver staining (B. R. Oakley, D. R. Kirsch, and N. R. Morris (1980) Anal. Biochem. 105, 361-363.) following gel electrophoresis were varied, it was noted that glutaraldehyde pretreatment was necessary for maximal staining, which could not be explained simply as the result of "fixation." Further studies indicated that glutaraldehyde enhancement of protein staining with this silver reagent was probably due to oxidation of the aldehyde groups by silver ions, resulting in metallic silver depositions within the gel which act as nucleation sites for additional metallic silver localization in the protein bands upon the addition of formaldehyde developer. This proposed mechanism is consistent with the Tollen's reaction, as well as some aspects of the photographic process. Consistent with this notion, silver-staining intensities are directly related to mole percentage lysine of various standard proteins.