Organisation of the proteins of the chromaffin granule membrane

The organisation of the protein components of bovine chromaffin granules has been investigated by labelling or digesting intact granules or broken membranes with the following reagents: lactoperoxidase/Na¹²⁵I as a reagent for tyrosine residues, $\text{N-}\text{(iodoacetylaminoethyl)-5-naphthylamine-1-sulphonic}$ acid as a reagent for cysteine residues, pronase, and galactose oxidase/KB³H₄. Following treatment, membranes were purified and washed and proteins were examined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. Rather more than 60 bands were resolved, of which about 40 were relatively intense and reproducible. The bands were classified according to their molecular weights and sensitivity to reagents. Penetration of the membranes by the reagents was assessed by examination of intragranular proteins.The majority of chromaffin granule membrane polypeptides became labelled when intact granules were treated with impermeant reagents. Eleven were probably protected in the intact granules, reactive sites becoming exposed only on membrane lysis. By contrast, carbohydrate moieties of glycoproteins appear to be exposed only on the matrix side of the membrane. Two proteins were shown to span the membrane, although this is probably an underestimate.     

Is cytoplasmic Ca2+ in lymphocytes elevated in cystic fibrosis

An increased cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i) has been implicated in the pathogenesis of cystic fibrosis. We compared the [Ca²⁺]_i levels of normal and cystic fibrosis peripheral blood lymphocytes and Epstein-Barr virus-transformed lymphoblasts using quin 2, an internally trapped indicator. The [Ca²⁺]_i levels of normal and cystic fibrosis cells were not significantly different. The ionophore-releasable intracellular Ca²⁺ stores were also comparable in both types of individual.     

Purification and characterisation of leukotriene A4 hydrolase from rat neutrophils

Leukotriene A₄ hydrolase was rapidly and extensively purified from rat neutrophils using anion exchange and gel filtration high-pressure liquid chromatography. The enzyme which converts the allylic epoxide leukotriene A₄ to the 5,12-dihydroxyeicosatetraenoic acid leukotriene B₄ was localized in the cytosolic fraction and exhibited an optimum activity at pH 7.8 and apparent K_m for leukotriene A₄ between 2 · 10^?5 and 3 · 10^?5 M. The purified leukotriene A₄ hydrolase was shown to have a molecular weight of 68 000 on sodium dodecylsulfate polyacrylamide gel electrophoresis and of 50 000 by gel filtration. The molecular weight and monomeric native form of this enzyme are unique characteristics which distinguish leukotriene A₄ hydrolase from previously purified epoxide hydrolases.     

The intracellular localisation of proteoglycans and their accumulation in chondrocytes treated with monensin

Pig laryngeal chondrocytes incubated in the presence of monensin showed inhibition of [³⁵S]sulphate incorporation and decreased secretion of proteoglycan into the culture medium, but no large decrease in protein synthesis. This lead to the intracellular accumulation of proteoglycan protein core, which was detected in immunoprecipitates of cell extracts. Using the same antiserum protein core was localised by electron microscopy with protein A-coated gold. In control chondrocytes, it was detected only in elements of the Golgi and in secretory vesicles, but following monensin treatment labelling was more intense in the Golgi and extended into the distended cisternae of the rough endoplasmic reticulum. The results suggest that monensin blocks proteoglycan protein core translocation between different elements of the Golgi and that this occurs prior to the major site of chondroitin sulphate synthesis on proteoglycan.     

Calmodulin and the target size of the (Ca2+ + Mg2+)-ATPase of human red-cell ghosts

An average target size of 251 kDa has been obtained for the (Ca²⁺ + Mg²⁺)-ATPase of calmodulin-depleted erythrocyte ghosts by radiation inactivation with 16 MeV electrons. This is close to twice the size of the purified calcium-pump polypeptide. When calmodulin was included during the ATPase assay, a component of about 1 MDa appeared in addition to the activated dimer.     

Generation of phosphodiesters during fast-to-slow muscle transformation A 31P-NMR study

During rabbit fast-to-slow twitch muscle transformation, in response to electrical stimulation, the compound glycerophosphocholine can be detected in these muscles by ³¹P-NMR. This compound is not detectable in contralateral control muscles but is present in slow twitch soleus.     

Identification of products of protein phosphorylation in T37-transformed cells and comparison with normal cells

Proteins from crown gall tissue labelled in vivo with [³²P]orthophosphate were analysed by SDS-polyacrylamide gel electrophoresis. The major phosphorylated proteins were of 50.6 and 48.3 kDa, with minor bands at 80.1, 73.9, 68, 40.4, 30, 21.5, 20.2 and 15.2 kDa. Partial hydrolysates of total ³²P-labelled proteins were analysed in a number of ways. A two-dimensional separation on paper by electrophoresis in pyridine/acetic acid at pH 3.5 followed by chromatography in isobutyric acid/0.5 M ammonia revealed radioactive spots coincident with phosphoserine and phosphothreonine markers and only partially coincident with the phosphotyrosine marker. Two-dimensional electrophoresis at pH 1.9 followed by pH 3.5, however, unequivocally showed the presence of phosphotyrosine after elution of the phosphotyrosine marker. Phosphoserine, phosphothreonine and phosphotyrosine were present in the ratio 89.4:8.5:2.1. This is a much higher level of phosphotyrosine than normally found in animal cells. The three phosphoamino acids were confirmed by chromatography with authentic samples in four solvent systems on cellulose or silica TLC, and by dansylation followed by silica TLC. The radioactive compound running almost coincident with phosphotyrosine on two-way electrophoresis, pH 3.5, followed by chromatography in isobutyric acid/0.5 M ammonia was identified tentatively as uridine 5′-monophosphate on the basis of electrophoretic and chromatographic behaviour. Further experiments to compare normal (growing and non-growing) tobacco callus and T37-transformed cells did not give markedly different ratios of the three phosphoamino acids, although the rapidly-growing normal tobacco (i.e., plus cytokinin) appeared to have a greater abundance of the two minor phosphoamino acids (approx. 2-times). The lack of effect of transformation is in contrast to animal cells where transformation results in a 10-fold increase in the virally affected cells.     

Resting potential of the mouse neuroblastoma cells: II. Significant contribution of the surface potential to the resting potential of the cells under physiological conditions

In the previous paper, we showed that the K⁺ channels of the mouse neuroblastoma cell (clone N-18) are closed at low concentration of external K⁺ ([K⁺]₀) including the physiological concentration for the cells. In the present study, the origin of the resting membrane potential of N-18 cells has been examined. (1) The resting membrane potential of N-18 cells was depolarized by increasing concentration of the polyvalent cations (La³⁺, Fe³⁺, Co²⁺, Ca²⁺, Sr²⁺, Mg²⁺) and by decreasing the pH of the medium. The input membrane resistance was slightly increased during the depolarization. The depolarization was not explained in terms of the diffusion of the cations across the membrane, since the trivalent cations of greater ionic size were effective at much lower concentrations than the divalent cations. The results obtained from the measurements of ⁸⁶Rb efflux suggested that the depolarization cannot be explained in terms of blocking of the K⁺ channels by the cations. (2) An increase in Ca²⁺ concentration from 0.3 to 1.8 mM induced depolarization of about 10 mV at low [K⁺]₀ where the K⁺ channels are closed, but did not induce any depolarization at high [K⁺]₀ where the channels are open. (3) In order to estimate the changes in the zeta-potential, the electrophoretic mobility of N-18 cells was measured under various conditions. There was a close correlation between the changes in the zeta-potential and those in the membrane potential in response to the polyvalent cations and proton. On the other hand, an increase in K⁺-concentration in the medium, which induced a large depolarization in the cells, did not affect the zeta-potential. (4) The results obtained were explained by an electrical circuit model for the membranes of N-18 cells. In this model, an electrical circuit for the membrane part carrying no selective ionic channels, in which changes in the surface potential directly affect the transmembrane potential, is connected in parallel to the usual circuit model representing selective ionic channel systems. It was concluded that the surface potential contributes significantly to the resting membrane potential of N-18 cells at low [K⁺]₀ where the K⁺ channels are closed.     

Na+-dependence of 45Ca2+ uptake in adult rat isolated cardiac cells

We developed a technique that yields isolated adult rat myocytes, 70% of which are elongated and morphologically similar to intact tissue. Electrophysiological studies showed most of these cells were quiescent, Ca²⁺-tolerant and exhibited normal action potentials accompanied by contractions. We analyzed ⁴⁵Ca²⁺ uptake data in terms of instantaneous, fast and slow compartments. 69% of total exchangeable Ca²⁺ was found in the slow compartment; the rest was almost equally divided between the instantaneous and fast compartments. Replacement of extracellular Na⁺ by Li⁺ or Tris increased ⁴⁵Ca²⁺ uptake by the fast compartment; high [K⁺]_o increased this uptake further. These increases appeared to be related also to internal concentrations of Na⁺. This conclusion was supported by experiments with digitonin-treated cells. Our results indicate that the way Na⁺-dependent ⁴⁵Ca²⁺ uptake is affected by [Na⁺]_o, [Na⁺]_i and [K⁺]_o is compatible with the Na⁺-Ca²⁺ exchange mechanism. Our preparation should prove useful in studies of regulation of Ca²⁺ transport in cardiac muscles.     

Receptor-mediated endocytosis of fibroblast β-glucuronidase by peritoneal macrophages

β-Glucuronidase secreted by mouse 3T3 fibroblasts in vitro was taken up into mouse peritoneal macrophages and into human fibroblasts by a process which was rapid and saturable. High concentrations of mannose-containing compounds inhibited uptake into macrophages but had no effect on uptake into fibroblasts. Mannose-6-phosphate inhibited uptake into both types of cell, reducing uptake into macrophages by 34% and abolishing uptake into fibroblasts completely at a concentration of 5 mM. Fructose-1-phosphate was almost equally as effective at inhibiting uptake into fibroblasts but had no effect on macrophages. Pre-treatment of β-glucuronidase with alkaline phosphatase totally prevented its uptake into fibroblasts but had no effect on its uptake into macrophages. These results indicate that fibroblasts can secrete a lysosomal enzyme in a form recognised as a high uptake ligand not only by other fibroblasts but also by peritoneal macrophages and that endocytosis appears to be mediated by different receptors present on each type of cell. This has important implications for the potential treatment of mucopolysaccharidoses by fibroblast transplants.     

Effects of divalent metal ions on the calcium pump and membrane phosphorylation in human red cells

In inside-out red cell membrane vesicles ATP-dependent calcium transport is activated by the divalent metal ions Mg²⁺, Mn²⁺, Co²⁺, Ni²⁺ and Fe²⁺. This activation is based on the formation of Me²⁺-ATP complexes which can serve as energy-donor substrates for the calcium pump, and probably, satisfy the requirement for free Me²⁺ in this transport process. Higher Me²⁺ concentrations inhibit calcium transport with various efficiencies. Mn²⁺ directly competes with Ca²⁺ at the transport site, while other divalent metal ions investigated have no such effect. The formation of the hydroxylamine-sensitive phosphorylated intermediate (EP) of the red cell membrane calcium pump from [γ-³²P]ATP is induced by Ca²⁺ while rapid dephosphorylation requires the presence of Mg²⁺. At higher concentrations Mn²⁺ and Ni²⁺ inhibit predominantly the formation of EP, while Co²⁺ and Fe²⁺ block dephosphorylation. The possible sites and nature of the divalent metal interactions with the red cell calcium pump are discussed. Hydroxylamine-insensitive membrane phosphorylation in inside-out vesicles from [γ-³²P]ATP is significantly stimulated by Mn²⁺ and Co²⁺, as compared to that produced by Mg²⁺, Fe²⁺ and Ni²⁺. Part of this labelling is found in phospholipids, especially in phosphatidylinositol. The results presented for the metal dependency of protein and lipid phosphorylation in red cell membranes may help in the characterization of ATP consumptions directly related to the calcium pump and those involved in various regulatory processes.     

Volume restoration in osmotically swollen lymphocytes does not involve changes in free Ca2+ concentration

An increase in cytoplasmic free [Ca²⁺], [Ca²⁺]_i, has been suggested as the trigger for the permeability changes that bring about cell volume restoration following exposure to anisotonic media. This idea was directly tested in human peripheral lymphocytes undergoing regulatory volume decrease following a hypotonic dilution of the suspension. [Ca²⁺]_i was measured with the intracellularly trapped fluorescent indicator, quin2, and showed no measurable change on hypotonic swelling or during the subsequent volume decrease. Moreover, even though the incorporated quin2 adds significant Ca-buffering to the cytoplasm, regulatory volume decrease occurred normally in the quin2-loaded cells. It appears that alterations in [Ca²⁺]_i are not involved in these processes of volume regulation. An intracellularly trapped derivative of fluorescein, bis(carboxyethyl)carboxyfluorescein, was used to monitor cytoplasmic pH, which also showed no change during regulatory volume decrease.     

Intracellular transport and degradation of 125I-labelled denatured serum albumin in isolated nonparenchymal rat liver cells

The intracellular movement, following uptake of ¹²⁵I-labelled denatured serum albumin into nonparenchymal liver cells, was followed by means of subcellular fractionation. Isolated nonparenchymal rat liver cells were prepared by means of differential centrifugation. The cells were homogenized in a sonifier and the cytoplasmic extract subjected to isopycnic centrifugation in a sucrose gradient. The intracellular movement of the labelled albumin was followed by comparing the distribution profile of radioactivity in the sucrose gradient with those of marker enzymes for plasma membrane and lysosomes. The distribution profiles for radioactivity after the cells had been exposed to the labelled denatured albumin for different time periods indicated that the radioactivity was first associated with subcellular fractions of lower modal densities than the lysosomes. With time of incubation the radioactivity moved towards higher densities. After prolonged incubations in the absence of extracellular labelled denatured albumin the radioactivity peak coincided with that of the lysosomal marker β-acetylglucosaminidase. When the cells were treated with the lysosomal inhibitor leupeptin, degradation of the labelled albumin was decreased, resulting in a massive intracellular accumulation of radioactivity. The radioactivity peak coincided with the peak of activity for the lysosomal marker β-acetylglucosaminidase, suggesting lysosomal degradation.     

Cellular mode of serotonin action on CI− transport in the rabbit corneal epithelium

The present work examines serotonin-induced changes in cell potential difference and barrier resistances in the corneal epithelium in vitro using voltage-measuring microelectrodes and related techniques. Component resistances were determined using voltage and resistance profiles of the epithelium before and during the serotonin response. Serotonin, added to the stromal side of the cornea in the presence of nialamide, markedly reduced transcorneal and apical membrane resistances, while basal barrier resistance increased slightly and shunt resistance was unchanged. The marked drop in apical membrane resistance after serotonin treatment reflects an increase in apical membrane chloride permeability, inasmuch as the serotonin-stimulated short-circuit current is indistinguishable from the increase in net chloride flux. Prolonged (more than 1 h) exposure of corneas to serotonin markedly depolarized the epithelial cells and reduced the voltage divider ratio from 12.3 ± 2.1 to 1.5 ± 0.5, while not significantly affecting the stimulated short-circuit current. These later effects suggest changes in epithelial ion distribution during long periods of stimulation by serotonin.     

Identification and partial characterization of plasma membrane polypeptides of Trypanosoma brucei

A plasma membrane-enriched vesicle fraction has been prepared from Trypanosoma brucei by sonication and differential centrifugation on sucrose gradients. This fraction is enriched 5-fold in the plasma membrane marker enzymes adenyl cyclase (EC 4.6.1.1) and ouabain-inhibitable, ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}$)-dependent adenosine triphosphatase (EC 3.6.1.3). It is also enriched up to 14-fold in iodinated surface proteins, and up to 4-fold in [³H]mannose-labeled glycoproteins, of which the major variable surface coat glycoprotein is the main constituent. Proteins of the plasma membrane fraction and other subcellular fractions have been identified by electrophoretic analysis in sodium dodecyl sulfate-polyacrylamide gradient slab gels. Several high molecular weight surface glycopeptides have been selectively investigated and partially characterized by a combination of metabolic labeling with [³H]mannose, lactoperoxidase-catalyzed surface iodination, and affinity chromatography on Con A-Sepharose. In addition to the major variable surface coat glycoprotein (estimated $\text{M}{}_{\mathrm{<mtext>r</mtext>}}\text{= 58 000}$), there are several minor surface glycopeptides ($\text{M}{}_{\mathrm{<mtext>r</mtext>}}\text{= 76 000, 86 000}\text{and}\text{92 000–100 000}$) which are apparent extrinsic membrane components, and two surface glycopeptides ($\text{M}{}_{\mathrm{<mtext>r</mtext>}}\text{= 42 000}\text{and}\text{130 000}$) which are intrinsic membrane components.     

Evidence that high mobility group protein 17 is not phosphorylated in human colon carcinoma cells

The high mobility group proteins 14 and 17 were reported previously to be phosphorylated in murine and human tumor cell lines. Recently, it was suggested that subgroups of HMG-14, HMG-14a and 14b, but not HMG-17, were phosphorylated in situ in HeLa cells. In order to definitively determine whether HMG-17 is indeed phosphorylated or whether the protein previously identified as [³²P]HMG-17 was a subgroup of HMG-14, we have used the technique of electroblotting in conjunction with an immunochemical procedure utilizing anti-HMG-17 IgG. Our results indicate that HMG-17 was not phosphorylated in human colon carcinoma cell line HT-29 incubated for 18 h with ³²P_i, but that HMG-14a and HMG-14b were phosphorylated. In contrast, HMG-14a, -14b and -17 were phosphorylated in vitro in isolated nuclei incubated with [γ-³²P]ATP.     

Unusual redox behaviour of cytochrome b-561 from bovine chromaffin granule membranes

(1) Redox titrations of cytochrome b-561 have been performed with the purified cytochrome and with intact and detergent-solubilized chromaffin-granule membranes. (2) The midpoint redox potential of the cytochrome is 100–130 mV; this depends upon the composition of the buffer, but is independent of pH in the range 5.5–7.5; partial proteolysis of the cytochrome raises the midpoint potential to 160 mV. (3) The Nernst plots of titration data have slopes of 75–115 mV, and are in some cases sigmoid in shape. This may be explained by negative cooperativity during redox transitions in oligomeric cytochrome b-561. (4) Measurements of the haem and cytochrome content of chromaffin granule membrane suggest a haem content of 1 mol/mol protein. (5) Chemical crosslinking of cytochrome b-561 suggests that it may exist as an oligomer of 4–6 polypeptide chains within the chromaffin granule membrane. Aggregation of purified cytochrome b-561 was shown by gel filtration studies and by immunological methods in SDS-polyacrylamide gels. Studies of the molecular weight of the aggregates suggest that the monomer has a molecular weight close to 22 000, but migrates anomalously slowly during electrophoresis.     

PhoE protein pores in the outer membrane of Escherichia coli K-12 not only have a preference for Pi and Pi-containing solutes but are general anion-preferring channels

Previous studies on the question of whether the PhoE protein pore has a preference for P_i and P_i-containing solutes only or whether it constitutes a general anion-preferring channel, have not given an unequivocal answer either because the presence of the phosphate binding protein was not ascertained or because only arsenate was tested as a non P_i-containing control solute. Permeability properties of PhoE, OmpF and OmpC protein pores for negatively charged solutes were measured in vivo in the presence of phosphate-binding protein. It appeared that the PhoE protein pore is the most efficient channel for the three tested solutes phosphate, succinate and sulphate. Conditions were established to measure the frequency of ethyl methane sulphonate induced mutations as a function of the presence of pore proteins. These results indicate that PhoE protein also forms the most efficient channel for ethyl methane sulphonate. We conclude that the preference of the PhoE protein pore is not restricted to P_i and P_i-containing solutes but also concerns several other negatively charged solutes.