Multifunctional enzyme, bisphosphoglyceromutase/2,3-bisphosphoglycerate phosphatase/phosphoglyceromutase, from human erythrocytes. Evidence for a common active site.

Bisphosphoglyceromutase and 2,3-bisphosphoglycerate phosphatase activities responsible for 2,3-bisphosphoglycerate metabolsim in human red cells are displayed by the same enzyme protein which has phosphoglyceromutase activity [Sasaki, R., et al. (1975) Eur J. Biochem. 50, 581-593]. This enzyme was subjected to chemical modification by trinitrobenzenesulfonate. The three enzyme activities were inactivated by trinitrobenzenesulfonate at the same rate. The sulfhydryl content of the enzyme was unchanged during trinitrophenylation, indicating that derivatization was through the amino group. Trinitrophenylation of about one amino group per mole of the enzyme resulted in complete loss of the three activities. Both 2,3-bisphosphoglycerate and 1,3-bisphosphoglycerate inhibited trinitrophenylation and effectively protected the enzyme from inactivation. Although monophosphoglycerates did not show any protective effect at concentrations which should be adequate based upon their kinetic constants, they were protective at higher concentrations. Inactivation by trinitrophenylation was an apparent first-order reaction. The dissociation constant of the enzyme - 2,3-bisphosphoglycerate complex was determined by analyzing the first-order reaction on the assumption that the protective effect of 2,3-bisphosphoglycerate was due to competition with trinitrobenzenesulfonate. The dissociation constant was in good agreement with kinetic constants of 2,3-bisphosphoglycerate in the enzyme reactions, which indicated that 2,3-bisphosphoglycerate did indeed exert its protective effect through competition with trinitrobenzenesulfonate for an amino group of the enzyme. The protective effect of monophosphoglycerates could be rationalized with kinetic evidence that 2-phosphoglycerate at high concentrations interacts with the 2,3-bisphosphoglycerate binding site. These results indicate that the enzyme exhibits the three enzyme activities at a common active site at which one amino group essential for binding of bisphosphoglycerates is located. Based on the multifunctional properties of this enzyme, a possible mechanism was discussed for regulation of 2,3-bisphosphoglycerate metabolism in human red cells.     

Evidence for the presence of volume-sensitive KCl transport in 'young' human red cells

'Young' human red cells are shown to possess a specific K+ pathway which is dependent on Cl- and sensitive to cell volume. This system was latent in 'mature' cells but was revealed by high hydrostatic pressure. This suggests the pathway is functionally active in 'young' cells but becomes masked with cell maturation.     

Isolation and partial characterization of a proteoglycan from the red alga Laurencia spectabilis

A proteoglycan was isolated from the red alga Laurencia spectabilis Postels & Ruprecht (Ceramiales) by extraction in a dilute buffer—NaCl solution followed by gel and anion exchange chromatography. The molecule was composed of 92% carbohydrate and 8% protein. Galactose and uronic acids were the major monosaccharides. Ester sulfate was not detected. A small quantity of hydroxyproline was present in the protein component. The proteoglycan accounted for a very small portion (less than 1% by fr. wt) of the alga.     

Partial characterization of the inactive mutant form of human red cell bisphosphoglyceromutase and comparison with an alkylated form

The trifunctional enzyme bisphosphoglyceromutase (or diphosphoglycerate mutase) (EC 2.7.5.4) was purified from human red cells and injected into two chickens. Specific anti-bisphosphoglyceromutase antibodies were produced that displayed a single precipitation line on Ouchterlony plates and on immunoelectrophoresis. No cross-reaction of these antibodies was detected with phosphoglyceromutase, the common glycolytic enzyme. Immunoneutralization of bisphosphoglyceromutase and of its two other activities, i.e., bisphosphoglycerate phosphatase and phosphoglyceromutase, was observed for a purified preparation. The anti-bisphosphoglyceromutase antibody reacts with the inactive enzyme present in the hemolysate of a mutant human subject. It also binds bisphosphoglyceromutase inactivated by N-ethylmaleimide, a strong alkylating agent of SH groups. Active bisphosphoglyceromutase is stable at 55°C, whereas the inactive forms of the mutant and of the alkylated hemolysates are thermolabile. These forms can be protected against thermal precipitation by 4 mM 2,3-diphosphoglycerate and 4 mM 3-phosphoglycerate. These findings afford evidence that the binding of the substrates on the bisphosphoglyceromutase molecule is not prevented by alkylation nor by the mutation of the hereditary inactive enzyme.     

Evidence for bumetanide-sensitive, Na(+)-dependent, partial Na-K-Cl co-transport in red blood cells of a primitive fish

Tracer uptake studies identified the major routes for K+ transport in hagfish red cells, resolving them into ouabain-sensitive, loop diuretic-sensitive, and residual components. The K1/2 values for ouabain, bumetanide, and furosemide were 10(-5), 6 x 10(-7), and 5 x 10(-6) M, respectively. The properties of the Na-K-Cl co-transporter were investigated further by varying K+, Na+, and Cl- concentrations. The measured K1/2 values were similar to those for human red cells. Finally, the stoichiometry of Na:K:Cl uptake was determined, giving 1:1 for K+:Cl-; in contrast, no significant Na+ flux could be measured, although Na+ content must be present for measurable bumetanide-dependent K+ or Cl- flux to occur. The Na-K-Cl transport therefore shows Na(+)-dependent KCl co-transport or partial flux of the system.     

Purification of bisphosphoglyceromutase, 2,3-bisphosphoglycerate phosphatase and phosphoglyceromutase from human erthrocytes. Three enzyme activities in one protein.

Bisphosphoglyceromutase, 2,3-bisphosphoglycerate phosphatase and phosphoglyceromutase have been purified from human red cells. Three enzymes were co-purified throughout all purification steps. Three fractions (peaks I, II and III) which were chromatographically separable and had three activities in different ratios were obtained. Peak III which contained the main bisphosphoglyceromutase and 2,3-bisphosphoglycerate phosphatase activities was purified to homogeneity by electrophoretic and ultracentrifugal analyses. The homogeneous preparation had the phosphoglyceromutase activity. The three activities were lost at the same rate during thermal inactivation. Thus, bisphosphoglyceromutase and 2,3-bisphosphoglycerate phosphatase activities, which are responsible for 2,3-bisphosphoglycerate metabolism in red cells, are displayed by the same enzyme protein which has phosphoglyceromutase activity. Peaks I and II were rich in the phosphoglyceromutase activity. Both peaks showed bisphosphoglyceromutase and 2,3-bisphosphoglycerate phosphatase activities, although these two activities were much smaller than those of peak III. Some of the enzymic properties of peak III are described. Comparative studies on three peaks showed that the phosphoglyceromutase of peak III differed from that of peaks I and II in the kinetic property and thermostability.     

Haemolytic factor from the crop of Rhodnius prolixus: Evidence and partial characterization

A haemolytic factor, which lysed sheep red cells in an isotonic buffer, was found in the crop of all larval stages and adult Rhodnius prolixus. Little or no haemolytic factor occurred in unfed insects but haemolytic activity increased for 2–4 days after feeding. From the 4th day on, the activity declined gradually. Fifth-instar larvae fed on whole blood, erythrocytes and haemoglobin produced large quantities of haemolytic factor, while those fed on plasma and erythrocyte stroma did not. The haemolytic factor was purified approximately 1200-fold by a two-step procedure: (1) Bio-Gel P-6 Gel-Filtration and (2) SP-Sephadex chromatography. Purified haemolytic factor was heatstable (100°C, 10 min), dialysable, inactivated by trypsin treatment, and could be recovered in the supernatant after addition of ethanol. It was concluded that the haemolytic factor is a peptide displaying a basic character.     

Red-cell amino acid transport. Evidence for the presence of system ASC in mature human red blood cells.

The properties of Na+-dependent L-alanine transport in human erythrocytes were investigated using K+ as the Na+ substitute. Initial rates of Na+-dependent L-alanine uptake (0.2 mM extracellular amino acid) for erythrocytes from 22 donors ranged from 40 to 180 mumol/litre of cells per h at 37 degrees C. Amino acid uptake over the concentration range 0.1-8 mM was consistent with a single saturable component of Na+-dependent L-alanine transport. Apparent Km and Vmax. values at 37 and 5 degrees C measured in erythrocytes from the same donor were 0.27 and 0.085 mM respectively, and 270 and 8.5 mumol/litre of cells per h respectively. The transporter responsible for this uptake was identified as system ASC on the basis of cross-inhibition studies with a series of 42 amino acids and amino acid analogues. Apparent Ki values for glycine, L-alpha-amino-n-butyrate, L-serine and L-leucine as inhibitors of Na+-dependent L-alanine uptake at 37 degrees C were 4.2, 0.12, 0.16 and 0.70 mM respectively. Reticulocytes from a patient with inherited pyruvate kinase deficiency were found to have a 10-fold elevated activity of Na+-dependent L-alanine uptake compared with erythrocytes from normal donors. Separation of erythrocytes according to cell density (cell age) established that even the oldest mature erythrocytes retained significant Na+-dependent L-alanine transport activity. Amino acid transport was, however, a more sensitive indicator of cell age than acetylcholinesterase activity. Erythrocytes were found to accumulate L-alanine against its concentration gradient (distribution ratio approx. 1.5 after 4 h incubation), an effect that was abolished in Na+-free media. Na+-dependent L-alanine uptake was shown to be associated with L-alanine-dependent Na+ influx, the measured coupling ratio being 1:1.     

Isolation and partial characterization of biologically active Fc receptor of chicken red cells

It has previously been demonstrated that chicken red cells have a receptor with the capacity to bind aggregated IgG, IgM 7 S or antigen-complex IgG. This receptor was isolated from Nonidet P-40 soluble extracts of chicken red cells by immunoadsorption with either immobilized aggregated IgG or monomeric IgM (IgM 7 S) and further gel filtration through a Sephacryl S-300 column. The Fc binding material was characterized as a glycoprotein with a molecular weight of 30,000 which retained its Fc receptor activity after the isolation procedure. This was demonstrated by its capacity to inhibit the binding of 125I-IgM 7 S or 125I-labelled aggregated IgG to chicken red cells. After Bacillus cereus phospholipase C treatment the Fc receptor activity remained unchanged, but the molecular weight (15,000) did not, suggesting that the phospholipids cleaved by this treatment were not essential for the interactions of the receptor with specific ligands. However, this Fc-binding component was shown to have a molecular weight of 13,000 and a diminished Fc receptor activity after reduction with dithiothreitol, suggesting the presence of at least one disulphide bridge, necessary to maintain the total ligand-binding activity.     

Evidence for the presence of androgen receptors in human Leydig cells

Localization of androgen receptors (ARs) in the human testis Leydig cells was examined with an AR assay and Northern blot analysis. Leydig cells, highly purified on a Percoll gradient, were used for the experiments. AR concentration in the total cell extract containing both the cytosol and nuclear fractions in Leydig cells was measured using [3H]methyltrienolone. ARs in Leydig cells showed a high affinity for [3H]methyltrienolone and the Kd and Bmax of the receptors were 1.24 nM and 11.7 fmol/mg protein, respectively. Northern blot analysis, using a 32P-labeled full-length human AR complementary DNA (cDNA) detected a 9.5-kb hybridizing band in the total RNA extracted from Leydig cells. These data can be interpreted as evidence of the existence of ARs in human Leydig cells.     

Evidence for the presence of a cell-surface ribonuclease in mechanically prepared rat-liver cells in suspension

Rat-liver parenchymal cells obtained in suspension by a mecahnical method are shown to contain a cell-surface nuclease(s ) that rapidly degrades exogenously added totalEscherichia coli RNA. However, no acid-soluble products are formed; all the degradation products in the incubation medium sediment in the 4–55 RNA region on a sucrose density gradient. A part of the degraded RNA seems to be taken up by the cells; the uptake of the degradation products, presumably derived from rRNAs, is more than that of purified 4–55 RNA. Most of the RNA taken up by the cell sediments in the 4–55 region; only a small proportion is degraded to acid-soluble material within the cell.     

Evidence for the presence of a free C-terminal fragment of cx43 in cultured cells

Migration of the gap junction protein connexin 43 (Cx43) in SDS-PAGE yields 2 to 4 distinct bands, detectable in the 40-47 kDa range. Here, we show that antibodies against the carboxy-terminal domain of Cx43 recognized an additional 20-kDa product. This protein was detected in some culture cell lysates. The presence of the 20-kDa band was not prevented by the use of protease inhibitors (Complete(R) and phenylmethylsulfonyl fluoride (PMSF), 1-5 mM). The band was absent from cells treated with Cx43-specific RNAi, and from those derived from Cx43-deficient mice, indicating that this Cx43-immunoreactive protein is a product of the Cx43 gene. Treatment of CHO cells with cyclosporin A caused a reduction in the amount of full-length Cx43 and a concomitant increase in the amount of the 20-kDa band. Overall, our data show that a fraction of the Cx43-immunoreactive protein pool within a given cell may correspond to a C-terminal fragment of the protein.     

Evidence for the presence of non-receptor protein tyrosine kinases in algal cells

Two orders of green alga (Cladophorales and Charales) were investigated for the presence of protein tyrosine kinase activity. Proteins of 70 and 85 kDa were found to be tyrosine phosphorylated in Cladophora fracta, with an additional phosphorylated band evident at the 120-kDa region in Chara vulgaris, suggestive of the presence of putative tyrosine kinase activity in these algal species. A 70-kDa protein was immunoprecipitated from both species using a polyclonal antibody against non-receptor protein tyrosine kinase Syk. The protein was found to be phosphorylated on tyrosine, which was prevented upon pretreatment of algal cells with piceatannol. The extent of phosphorylation directly correlated with algal growth, suggesting a link between Syk kinase activity and growth signaling. These observations supported the presence of Syk-like kinase in the green algal species, which could have critical role in the algal growth and development.     

Evidence for cadherin-11 cleavage in the synovium and partial characterization of its mechanism

IntroductionEngagement of the homotypic cell-to-cell adhesion molecule cadherin-11 on rheumatoid arthritis (RA) synovial fibroblasts with a chimeric molecule containing the cadherin-11 extracellular binding domain stimulated cytokine, chemokine, and matrix metalloproteinases (MMP) release, implicating cadherin-11 signaling in RA pathogenesis. The objective of this study was to determine if cadherin-11 extracellular domain fragments are found inside the joint and if a physiologic synovial fibroblast cleavage pathway releases those fragments.MethodsCadherin-11 cleavage fragments were detected by western blot in cell media or lysates. Cleavage was interrupted using chemical inhibitors or short-interfering RNA (siRNA) gene silencing. The amount of cadherin-11 fragments in synovial fluid was measured by western blot and ELISA.ResultsSoluble cadherin-11 extracellular fragments were detected in human synovial fluid at significantly higher levels in RA samples compared to osteoarthritis (OA) samples. A cadherin-11 N-terminal extracellular binding domain fragment was shed from synovial fibroblasts after ionomycin stimulation, followed by presenilin 1 (PSN1)-dependent regulated intramembrane proteolysis of the retained membrane-bound C-terminal fragments. In addition to ionomycin-induced calcium flux, tumor necrosis factor (TNF)-α also stimulated cleavage in both two- and three-dimensional fibroblast cultures. Although cadherin-11 extracellular domains were shed by a disintegrin and metalloproteinase (ADAM) 10 in several cell types, a novel ADAM- and metalloproteinase-independent activity mediated shedding in primary human fibroblasts.ConclusionsCadherin-11 undergoes ectodomain shedding followed by regulated intramembrane proteolysis in synovial fibroblasts, triggered by a novel sheddase that generates extracelluar cadherin-11 fragments. Cadherin-11 fragments were enriched in RA synovial fluid, suggesting they may be a marker of synovial burden and may function to modify cadherin-11 interactions between synovial fibroblasts.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-015-0647-9) contains supplementary material, which is available to authorized users.     

Leucoplast of the cryptomonad Chilomonas paramecium: Evidence for presence of a true plastid in a colorless flagellate

Chilomonas paramecium is a colorless cryptomonad flagellate which contains a leucoplast. The structural features of the leucoplast parallel those shown in the cryptomonad chloroplast, exemplified by Cryptomonas ovata. An electron microscopic study of this colorless organelle shows it to be divided into two compartments: the outer compartment contains starch granules and 220 Å ribosomes similar to cytoplasmic RNP particles. The inner compartment often contains carotenoid-like masses, 160 Å ribosomes significantly smaller than those in the outer compartment, and DNA-like filaments. Cesium-chloride density gradients of the whole-cell DNAs show three DNase-sensitive satellites in addition to the major fraction. One of them is presumed to be associated with the leucoplast. Evidence for the existence of true plastids in bleached and colorless flagellates is reviewed.     

Evidence for a halotolerant-alkaline laccase in Streptomyces psammoticus: Purification and characterization

An unusual halotolerant-alkaline laccase from Streptomyces psammoticus has been purified to homogeneity through anion exchange and gel filtration chromatography steps with an overall purification fold of 12.1. The final recovery of the enzyme was 22.1%. The molecular mass of the purified laccase was about 43 kDa. The enzyme was active in the alkaline pH range with pH optima at 8.5 and 97% activity retention at pH 9.0. The optimum temperature was 45 °C. The enzyme was stable in the pH range 6.5–9.5 and up to 50 °C for 90 min. The enzyme was tolerant to NaCl concentrations up to 1.2 M. It was inhibited by all the putative laccase inhibitors while the enzyme was activated by metal ions like Fe, Zn, Cu, Na and Mg. Fe enhanced the enzyme activity by twofold (204%). The enzyme showed lowest K_m value with pyrogallol (0.25 mM) followed by ABTS (0.39 mM). The purified enzyme was a typical blue laccase with an absorption peak at 600 nm.     

A new chemical tool for characterization and partial depolymerization of red algal galactans

Complete acid hydrolysis of red algal galactans in the presence of borane - 4-methylmorpholine complex has been shown to prevent the acid degradation of 3,6-anhydrogalactose derivatives by their reduction to the corresponding 3,6-anhydro-galactitols, whereas all the other monosaccharides are liberated essentially in the non-reduced form; the reductive hydrolysis products may be determined quantitatively using gas-liquid chromatography (GLC). The method is recommended for preliminary characterization of the polysaccharide composition of red algal biomass. Partial acid hydrolysis of galactans in the presence of the same reducing agent gives rise to reduced oligosaccharides having terminal 3,6-anhydrogalactitol residues. Based on this reaction, the attribution of unknown galactans to the agar or carrageenan groups is possible by partial reductive hydrolysis of small samples of algal biomass with subsequent identification of agarobiitol or carrabiitol acetates by GLC. Sulfate groups are substantially retained under partial reductive hydrolysis conditions; the isolation by liquid chromatography and elucidation of structures of reduced sulfated oligosaccharides may be of great value for the structural analysis of complex red algal galactans.