Biochemical characterization of I-Ak proteins from mutant antigen-presenting B-cell--B-lymphoma hybridomas

Chemically induced mutants of an I-Ak,d expressing antigen-presenting B-cell--B-lymphoma hybridoma have recently been generated by immunoselection in vitro and were found to possess alterations in some of their serologically and functionally defined I-Ak region dependent functions. In order to identify at the structural level the origin of the differences in serological and functional properties of these mutants, I-Ak molecules from several of these mutant hybridomas were compared biochemically to wild-type I-Ak polypeptides by two-dimensional gel electrophoresis and high-pressure liquid chromatographic tryptic peptide analyses. Two-dimensional gel electrophoresis indicated that no major structural alterations, resulting in changes in mol. wt or charge, had occurred in the Ak alpha or Ak beta polypeptides from the mutant cells. Likewise, Ak alpha peptide maps of the mutants were indistinguishable from the normal Ak alpha peptide maps. However, two of the three mutants studied did exhibit one additional peptide in their Ak beta peptide maps. These results suggest that the major deficiencies in T-cell-activating functions of these mutants are a result of a limited alteration in the Ak beta polypeptide primary structure.     

Effect of short physical exercise on the levels of zinc and carbonic anhydrase isoenzyme activities in human erythrocytes

Summary Studies of the influences of physical exercise of short duration (bicycle ergometer, 200 W for 30 min) on the activities of carbonic anhydrase isoenzymes (CA-B and CA-C types) and zinc concentration in erythrocytes were made on 5 untrained healthy male volunteers. The subjects were rested for 30 min after the exercise. There were significant decreases in the levels of zinc, CA-B, total carbonic anhydrase activity and CA-B-dependent activity immediately after exercise, but after 30 min of rest they all returned to their pre-exercise levels. No significant change in CA-C level or CA-C-dependent activity was found after exercise. Immediately after exercise, total carbonic anhydrase activity and CA-B-dependent activity following the addition of Zn²⁺ showed significant increases, compared with their respective activities without Zn²⁺ addition. However, no such effects were observed just before exercise or after rest; the addition of Zn²⁺ had no effect on CA-C-dependent activity at any time. A significant correlation was found between the changes in concentration of zinc and CA-B-dependent activity after exercise (r=0.711). The findings of the present study suggest that active CA-B enzymes are converted in part to inactive enzymes during acute physical exercise, possibly by decreased zinc binding. Moreover, the change in CA-B-dependent activity correlated well with the changes in pH and HCO₃ concentrations in venous blood (r=0.853 and r=0.718, respectively). One may speculate that an adaptive decrease in CA-B-dependent activity in erythrocytes occurs with increased acidification in blood during heavy physical exercise of short duration.The present study was presented to the Fifth International Symposium on the Biochemistry of Exercise, Boston, June 1–5, 1982     

Stimulus-dependent modulation of smooth muscle intracellular calcium and force by altered intracellular pH

Measurements of simultaneous force and intracellular Ca²⁺ concentration ([Ca²⁺]_i) in rat uterine smooth muscle have been made to elucidate the mechanisms involved when force produced spontaneously, by high-K⁺ depolarization or carbachol is altered by a change of intracellular pH (pH_i). Rises in force and [Ca²⁺]_i were closely correlated for all forms of contraction, with the Ca²⁺ transient peaking before force. In spontaneously active preparations, alkalinization significantly increased, and acidification decreased, force and [Ca²⁺]_i. Inhibition of the sarcoplasmic reticulum ATPase (cyclopiazonic acid) did not affect these changes, whereas removal of external Ca²⁺ abolished both responses, suggesting that the effect of pH_i is on Ca²⁺ entry. Alkalinization caused a prolongation of the action potential complex, associated with a potentiation of contractile activity. Acidification produced hyperpolarization and abolition of action potentials and spontaneous activity, but did not prevent brief applications of carbachol or high-K⁺ from producing depolarization and increasing force, suggesting no impairment of the mechanism of generation of the action potential. For depolarized preparations, acidification increased tonic force and [Ca²⁺]_i; the increase in the calcium signal persisted in zero-external calcium. In the presence of carbachol, acidification transiently increased force and [Ca²⁺]_i, followed by a reduction in both. It is concluded that changes in pH_i act at more than one step in excitation-contraction coupling and that changes in [Ca²⁺]_i can account for most of the changes in uterine force. Received: 1 April 1996 /Accepted: 8 May 1996     

Cell shrinkage stimulates bradykinin-induced cell membrane potential oscillations in NIH 3T3 fibroblasts expressing the ras-oncogene

In NIH 3T3 fibroblasts expressing the Ha-ras oncogene (+ras) bradykinin leads to sustained oscillations of cell membrane potential due to oscillations of intracellular Ca²⁺ with subsequent activation of Ca²⁺-sensitive K⁺ channels. In cells not expressing the oncogene (-ras), bradykinin leads only to a single transient hyperpolarization of the cell membrane. The present study has been performed to elucidate the possible interaction of cell volume, intracellular pH and bradykinin-induced oscillations of the cell membrane potential. Bradykinin leads to cell shrinkage and intracellular alkalinization of both +ras cells and –ras cells. Inhibition of Na⁺/H⁺ exchanger by HOE 694 abolishes the bradykinin-induced alkalinization but does not significantly interfere with the bradykinin-induced oscillations of cell membrane potential. In contrast, prevention of bradykinin-induced cell shrinkage by simultaneous reduction of extracellular osmolarity blunts the oscillations. Thus, cell shrinkage stimulates bradykinin-induced oscillations of cell membrane potential. On the other hand, cell shrinkage alone does not elicit oscillations unless, in addition, Ca²⁺ entry is stimulated by ionomycin.     

Polarized expression of Na+/H+ exchange activity in LLC-PK1/PKE20 cells: II. Hormonal regulation

LLC-PK₁/PKE₂₀ cells (a continuous epithelial cell line) has two different Na/H exchange activities: Na/H-1 located in the basolateral membrane and Na/H-2 located in the apical membrane [Casavola et al. (1989) Biochem Biophys Res Commun 165:833–837; Haggerty et al. (1988) Proc Natl Acad Sci USA 86:6797–6801]. In the present report we have studied hormone regulation of these exchange activities by measuring Na-dependent recovery of pH_i from an acid load (by using microspectrofluorometry and 2,7-bis(carboxyethyl)-5,6-carboxyfluorescein) in response to activation of regulatory cascades by either pharmacological agents or by vasopressin or calcitonin. Agents leading to activation of protein kinase A (cAMP-dependent), such as forskolin (10 M), 8-Br-cAMP (0.25 mM), and isobutylmethylxanthine (0.5 mM), inhibited Na/H-2 and Na/H-1 by an average of 49%. Stimulation of protein kinase C by a phorbol ester (phorbol 12-myristate 13-acetate, TPA, 100 nM) inhibited Na/H-2 (by an average of 48%) and stimulated Na/H-1 (by an average of 38%); these effects of TPA were also observed in the presence of forskolin (100 M). Addition of either vasopressin (2 M) or calcitonin (0.3 M) onto both sides of the monolayer decreased the activity of Na/H-2 by an average of 26.3% and 27.7% respectively, and stimulated the activity of Na/H-1 by an average of 17.4% and 38.7% respectively; exposure of cells to either hormone stimulated production of cAMP and inositol trisphosphate, respectively. Separate hormone additions to either the apical or basolateral cell surface led to effects similar to those produced by simultaneous hormone additions onto both cell surfaces, although the relative response of Na/H exchangers to either agonist is variable. In summary, these results suggest that in LLC-PK ₁/PKE₂₀ cells, vasopressin and calcitonin can act via receptor systems coupled either to adenylate cyclase or to phospholipase C. Activation of these receptor systems can lead to inhibition of Na/H-2 and stimulation of Na/H-1.     

Mechanisms of the inhibition of Shaker potassium channels by protons

Potassium channels are regulated by protons in various ways and, in most cases, acidification results in potassium current reduction. To elucidate the mechanisms of proton-channel interactions we investigated N-terminally truncated Shaker potassium channels (K_v1 channels) expressed in Xenopus oocytes, varying pH at the intracellular and the extracellular face of the membrane. Intracellular acidification resulted in rapid and reversible channel block. The block was half-maximal at pH 6.48, thus even physiological excursions of intracellular pH will have an impact on K⁺ current. The block displayed only very weak voltage dependence and C-type inactivation and activation were not affected. Extracellular acidification (up to pH 4) did not block the channel, indicating that protons are effectively excluded from the selectivity filter. Channel current, however, was reduced greatly due to marked acceleration of C-type inactivation at low pH. In contrast, inactivation was not affected in the T449V mutant channel, in which C-type inactivation is impaired. The pH effect on inactivation of the wild-type channel had an apparent pK of 4.7, suggesting that protonation of extracellular acidic residues in Kv channels makes them subject to pH regulation.     

Advanced 1H Solid‐State NMR Spectroscopy on Hydrogels, 1

Summary: The nature of the pH dependent collapse of poly(methacrylic acid) (PMAA) hydrogels is investigated using recent ¹H solid‐state NMR methods. In aqueous solution, PMAA changes from an expanded conformation at high pHs to a compact contracted form at low pHs, where hydrogen bonds play a central role. In solid‐state ¹H NMR spectra, recorded under fast magic angle spinning (MAS), dried PMAA samples previously collapsed at low pHs show characteristic signals in the spectral region of the carboxylic acid protons. With the aid of 2D ¹H‐¹H double‐quantum (DQ) MAS NMR spectra, three signals can be distinguished at 8, 10.5 and 12.5 ppm, which are attributed to free carboxylic groups and two different types of hydrogen bonded forms, respectively. The 12.5 ppm signal arises from the hydrogen bond with the shortest H? H distance, corresponding to the form that is most stable with respect to increasing temperature and pH. The weaker hydrogen‐bonded form (with a signal at 10.5 ppm) requires a slightly lower pH, while the free acid signal (at 8 ppm) emerges under the most acidic medium. Moreover, the stabilities of the hydrogen‐bonded carboxylic acid dimers can be inferred from the proton‐proton distances within the dimers, i.e. (275 ± 5) pm and (295 ± 15) pm for the protons at 12.5 and 10.5 ppm, respectively, which are determined by means of DQ MAS sideband patterns. Both the stability of the hydrogen bonds and the acidity of the protons may be related to the stereochemistry and the conformation of the PMAA chains.

     

pH modulates conducting and gating behaviour of single calcium release channels

Intracellular pH changes affect excitation-contraction coupling in skeletal, and cardiac muscles. However the proton implication in modulating the sarcoplasmic reticulum Ca²⁺ release channel activity has never been visualized at single channel level. A large conducting Ca²⁺ release pathway has previously been characterized after incorporation of skeletal and cardiac sarcoplasmic reticulum vesicles into planar lipid bilayers. This channel has been activated by micromolar and millimolar concentrations of Ca²⁺ and ATP, respectively. The pH was independently varied on each side of the channels. Acidification of the cis-chamber (7.4 to 6.6) induced a modification of the gating behaviour, resulting in a decrease of the open probability. This effect was completely reversible. On the other hand, acidification of the trans-chamber (7.4 to 6.8) induced a reduction of the unitary conductance of the sarcoplasmic reticulum Ca²⁺ release channel.     

Monoclonal antibodies against transferrin. Precipitating mixtures and lack of inter-species cross-reactivity

Five stable hybridoma lines were prepared using the myeloma cell line P3-X63-Ag.653 and spleen cells of mice hyperimmunized by pig transferrin. All hybridomas grew well in mouse peritoneal cavity and produced antibodies of the IgG₁ subclass. Antibody preparations obtained from ascitic fluids tested for their capacity of antigen precipitation. No precipitation was obtained with single antibodies and with pairs of antibodies. Three out of 10 possible triads gave clear and sharp precipitation zones and rings in immunodiffusion tests performed in agar gel. All 5 antibodies were shown by quantitative enzyme-immunoassay to be specific for pig transferrin: no cross-reaction was obtained with mouse, human, horse and sheep transferrins.     

人胃黏膜组织蛋白质组二维电泳图像的获得

目的初步建立并优化人类胃黏膜组织蛋白质组分析所需的双向凝胶电泳技术，提高其分辨率及重复性。方法刮取手术胃黏膜组织，对以固相pH梯度为第一向的双向凝胶电泳的关键因素与环节，如样品处理、上样量、电泳参数、凝胶浓度和SDS凝胶电泳染色方法等进行一系列的优化。以固相pH梯度——IPG胶条(pH=3—10)进行第一向等电聚焦，以SDS均一胶(13％)的垂直电泳为第二向。结果成功地得到了胃黏膜组织的双向凝胶电泳图谱。