Bis(sulfo-N-succinimidyl) [15N,2H16]doxyl-2-spiro-4'-pimelate, a stable isotope-substituted, membrane-impermeant bifunctional spin label for studies of the dynamics of membrane proteins: application to the anion-exchange channel in intact human erythrocytes

We have synthesized and characterized an isotopically substituted homologue of the membrane-impermeant bifunctional spin label bis(sulfo-N-succinimidyl) doxyl-2-spiro-4'-pimelate (BSSDP) [Beth et al. (1986) Biochemistry 25, 3824-3832] in which the nitroxide N is substituted with 15N and all of the protons in the doxylpimelate moiety are replaced by deuterons ([15N,2H16]BSSDP). Like its normal isotope homologue, [15N,2H16]BSSDP reacts with the anion-exchange channel in intact human erythrocytes at a site that spans the single extracytoplasmic chymotryptic cleavage site and that overlaps the stilbenedisulfonate site. The narrower line widths in the EPR spectrum of [15N,2H16]BSDP-labeled anion channels allow calculation of a minimum separation of 16 A between spin labels bound at the functionally important stilbenedisulfonate sites on adjacent subunits of an anion channel dimer. The 15N and 2H isotopic substitutions also provide substantial improvement in signal to noise of motionally sensitive regions of the ST-EPR spectrum of [15N,2H16]BSSDP-labeled anion channels in intact erythrocytes. [15N,2H16]BSSDP-labeled anion channels in intact erythrocytes were cross-linked to covalent dimers in the extracytoplasmic domain with the membrane-impermeant cross-linking reagent bis(sulfo-N-succinimidyl) suberate [Staros (1982) Biochemistry 21, 3950-3955], and the saturation-transfer EPR spectrum of these cells was compared with that of cells treated with [15N,2H16]BSSDP but not subsequently cross-linked. The spectra were essentially identical, supporting the hypothesis that anion channel subunits form stable dimers in the membranes of intact erythrocytes.     

Cross-linking and chymotryptic digestion of the extractoplasmic domain of the anion exchange channel in intact human erythrocytes

We have applied our new high yield, membrane-impermeant, protein cross-linking reagents (J.V. Staros, 1982. Biochemistry 21:3950-3955) together with chymotryptic digestion of the surface of intact erythrocytes (T.L. Steck, B. Ramos, and E. Strapazon, 1976. Biochemistry 15:1154-1161) in an investigation of the topology of the extracytoplasmic domain of the anion exchange channel of intact human erythrocytes. In intact erythrocytes, these cross-linking reagents have been shown to cross-link subunits of the anion exchange channel to dimers in the extracytoplasmic domain of the protein. Chymotryptic treatment of intact erythrocytes has been shown to cleave subunits of the anion exchange channel into two fragments of distinct Mr. Sequential treatment of intact erythrocytes with either of two membrane-impermeant cross-linkers, followed by digestion with chymotrypsin, yields chymotryptic fragments of the anion exchange channel cross-linked to one another. The cross-linked products observed appear to arise by cross-linking of unlike chymotryptic fragments, whether the cross-links are intersubunit or intrasubunit. These results are consistent with a model of the anion exchange channel in which the subunits form a head-to-head dimer with a twofold center of symmetry perpendicular to the plane of the membrane.     

Identification of the eosinyl-5-maleimide reaction site on the human erythrocyte anion-exchange protein: overlap with the reaction sites of other chemical probes.

The anion-exchange protein (band 3) reaction site in human erythrocytes for the fluorescent/phosphorescent probe eosinyl-5-maleimide (EMA) has been identified. Proteolytic dissection of band 3 in situ indicated that EMA reacts with the membrane-spanning Mr 17K peptide produced by chymotrypsin cleavage of band 3 in intact erythrocytes followed by removal of the cytoplasmic domain by mild trypsin digestion of ghost membranes. Sequencing of the major eosin-labeled peptide obtained from HPLC purification of an extensive chymotrypsin digest of purified Mr 17K peptide allowed assignment of the covalent reaction site for EMA to lysine-430 of the human erythrocyte protein [Tanner et al. (1988) Biochem. J. 256, 703-712]. Hydropathy plots based upon the primary structure of the protein [Lux et al. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 9089-9093] suggest that this residue is in an extracellularly accessible loop connecting membrane-spanning segments 1 and 2 of native band 3 in the erythrocyte membrane. Inhibition of sequential labeling of intact erythrocytes by pairs of chemical probes including EMA, the anion transport inhibitor 4,4'-diisothiocyanodihydrostilbene-2,2'-disulfonate (H2-DIDS), and the reactively bifunctional spin-label bis(sulfo-N-succinimidyl) doxyl-2-spiro-5'-azelate (BSSDA) has also been investigated. Each of these reagents affinity labels band 3 when added separately to a suspension of intact human erythrocytes by formation of one or more stable covalent bonds. Prelabeling of intact erythrocytes with EMA reduced subsequent labeling of band 3 by H2-DIDS by approximately 95% and by BSSDA by 90%. Similarly, prelabeling with H2-DIDS reduced subsequent labeling of band 3 by EMA by over 90%, and BSSDA prelabeling reduced EMA labeling by approximately 95%. Therefore, though having widely divergent chemical structures and protein modification reactivities, each of these negatively charged reagents may be competing for reaction with spatially overlapping sites on band 3 which are accessible from the extracellular space.     

Adenosine cyclic 3',5'-monophosphate uptake and regulation of membrane protein kinase in intact human erythrocytes

The uptake of adenosine cyclic 3',5'-monophosphate (cAMP) and stimulation of membrane-associated protein kinase in mature human erythrocytes were investigated. cAMP transport across the membrane was temperature dependent, and cAMP binding to the isolated membrane had less temperature dependence. More than 99% of the [3H]-cAMP taken up by erythrocytes was nonmembrane bound. Maximal stimulation of membrane protein kinase and maximal occupancy of membrane cAMP binding sites by extracellular cAMP cccurred at 30 degrees C within 30 min after initiation of the incubation of erythrocytes with cAMP. The concentration of extracellular cAMP that gave half-maximal stimulation of membrane protein kinase was 5.4 X 10-4 M, a value consistent with the concentrations of cAMP (5.2 X 10-4 M) found to occupy half-maximally the membrane cAMP binding sites in erythrocytes. Extracellular cAMP and to a lesser extent guanosine cyclic 3',5'-monophosphate and inosine cyclic 3',5'-monophosphate stimulated membrane protein kinase in erythrocytes. The cAMP uptake by human erythrocytes as well as cAMP binding to membranes in the erythrocyte was blocked by an inhibitor [4,4'-bis(isothiocyano)stilbene-2,2-disulfonate] of the anion channel. These studies indicate that cAMP can be transported across membranes into human erythrocytes and can bind to membranes to activate membrane protein kinase. It appears that there is a shared transport channel for cAMP and anion transport.     

Inhibition of protein synthesis by the cordycepin analog of (2'-5')ppp(Ap)nA, (2'-5')ppp(3'dAp)n3'dA, in intact mammalian cells

The introduction of the cordycepin analog of (2'-5')An, (2'-5')ppp(3'dAp)n3'dA [referred to as (2'-5')p33'dAn], into mouse L929 cells and cultured human fibroblasts resulted in a dose-dependent inhibition of protein synthesis which was comparable to the inhibition observed by (2'-5')ppp(Ap)nA [referred to as (2'-5')p3An]. The inhibition of protein synthesis by (2'-5')p33'dAn was much more persistent than that of the naturally occurring (2'-5')p3An following prolonged incubation of cells. Furthermore, the (2'-5')p3An was cytotoxic to mammalian cells in culture, whereas the (2'-5')p33'dAn was not.     

Discovery,synthesis and characterization of a series of (1-alkyl-3-methyl-1H-pyrazol-5-yl)-2-(5-aryl-2H-tetrazol-2-yl)acetamides as novel GIRK1/2 potassium channel activators

The present study describes the discovery and characterization of a series of 5-aryl-2H-tetrazol-3-ylacetamides as G protein-gated inwardly-rectifying potassium (GIRK) channels activators. Working from an initial hit discovered during a high-throughput screening campaign, we identified a tetrazole scaffold that shifts away from the previously reported urea-based scaffolds while remaining effective GIRK1/2 channel activators. In addition, we evaluated the compounds in Tier 1 DMPK assays and have identified a (3-methyl-1H-pyrazol-1-yl)tetrahydrothiophene-1,1-dioxide head group that imparts interesting and unexpected microsomal stability compared to previously-reported pyrazole head groups.     

Bis(acridinylthiourea)platinum(II) complexes: synthesis,DNA affinity,and biological activity in glioblastoma cells

The preparation of two novel bis(acridine)platinum(II) complexes is reported. The 4+ charged conjugates associate strongly with double-stranded native DNA (K(i)>10(6)), possibly through bisintercalation. A cell viability assay was used to demonstrate that both compounds are capable of mediating cytotoxicity at micromolar concentrations in SNB19 brain tumor cells.     

Short and efficient synthesis of (2S,3R,4R,5R) and (2S,3R,4R,5S)-tetrahydroxyazepanes via the Henry reaction

The Henry reaction with the easily available alpha-d-xylo-pentodialdose afforded a diastereomeric mixture of nitroaldoses with the alpha-d-gluco- and beta-l-ido-configuration, respectively, in good yield. When n-BuLi was used as the base, the reaction afforded the alpha-d-gluco-nitroaldose as the only product. The reduction of the nitro group in the alpha-d-gluco- and beta-l-ido-nitroaldoses, removal of the protecting groups and intramolecular reductive cyclo-amination afforded the corresponding (2S,3R,4R,5R) and (2S,3R,4R,5S) tetrahydroxyazepanes.     

Purification and characterization of an (ADP-ribose)n glycohydrolase from human erythrocytes

An (ADP-ribose)n glycohydrolase from human erythrocytes was purified approximately 13,000-fold and characterized. On sodium dodecyl sulfate/polyacrylamide gel the purified enzyme appeared homogeneous and had an estimated relative molecular mass (Mr) of 59,000. Amino acid analysis showed that the enzyme had a relatively high content of acidic amino acid residues and low content of basic amino acid residues. Isoelectrofocusing showed that the enzyme was an acidic protein with pI value of 5.9. The mode of hydrolysis of (ADP-ribose)n by this enzyme was exoglycosidic, yielding ADP-ribose as the final product. The Km value for (ADP-ribose)n (average chain length, n = 15) was 5.8 microM and the maximal velocity of its hydrolysis was 21 mumol.min-1.mg protein-1. The optimum pH for enzyme activity was 7.4 KCl was more inhibitory than NaCl. The enzyme activity was inhibited by ADP-ribose and cAMP but not the dibutyryl-derivative (Bt2-cAMP), cGMP or AMP. These physical and catalytic properties are similar to those of cytosolic (ADP-ribose)n glycohydrolase II, but not to those of nuclear (ADP-ribose)n glycohydrolase I purified from guinea pig liver [Tanuma, S., Kawashima, K. & Endo, H. (1986) J. Biol. Chem. 261, 965-969]. Thus, human erythrocytes contain (ADP-ribose)n glycohydrolase II. The kinetics of degradation of poly(ADP-ribose) bound to histone H1 by purified erythrocyte (ADP-ribose)n glycohydrolase was essentially the same as that of the corresponding free poly(ADP-ribose). In contrast, the glycohydrolase showed appreciable activity of free oligo(ADP-ribose), much less activity on the corresponding oligo(ADP-ribose) bound to histone H1. The enzyme had more activity on oligo(ADP-ribose) bound to mitochondrial and cytosolic free mRNA ribonucleoprotein particle (mRNP) proteins than on oligo(ADP-ribose) bound to histone H1. It did not degrade mono(ADP-ribosyl)-stimulatory guanine-nucleotide-binding protein (Gs) and -inhibitory guanine-nucleotide-binding protein (Gi) prepared with cholera and pertussis toxins, respectively. These results suggest that cytosolic (ADP-ribose)n glycohydrolase II may be involved in extranuclear de(ADP-ribosyl)n-ation, but not in membrane de-mono(ADP-ribosyl)ation.     

Analogs of (2'-5')oligo(A). Endonuclease activation and inhibition of protein synthesis in intact cells

Synthetic analogs of (2'-5')oligo(A) were assayed for endonuclease activation in cell extracts and for inhibition of protein synthesis in intact cells. The analogs are triadenylates: (i) methylated in the terminal 3'-OH; (ii) methylated at all three 3'-OH groups; (iii) with different numbers of phosphate groups at the 5' terminus or with a methylene group between the beta- and gamma-phosphate. Only 5'-phosphorylated monomethylated analogs activate an endonuclease in cell extracts and are powerful inhibitors of protein synthesis in intact cells. The analogs with only one 5'-terminal phosphate may require addition of another phosphate for activity since the kinase inhibitor 2-aminopurine prevents endonuclease activation by this compound but not by the di- and triphosphate-terminated triadenylates. These results suggest that two terminal phosphates and one or two free 3'-OH are required for endonuclease activation and inhibition of protein synthesis. The monomethylated analogs are more active than (2'-5')pppA3 because of their resistance to degradation by cellular enzymes. Accordingly, the monomethylated analogs cause a prolonged inhibition of protein synthesis in human fibroblasts treated with nanomolar concentrations of these compounds.     

Isolation and characterization of the human CLC-5 chloride channel gene promoter

The human CLC-5 chloride channel is expressed mainly in the kidney and its mutations cause Dent's disease (a familial renal tubular syndrome with hypercalciuria, tubular proteinuria, rickets, nephrocalcinosis, and eventual renal failure). To gain insight into the regulatory mechanism of CLC-5 expression, a genomic clone that contains the 5'-flanking region of the human CLC-5 gene was isolated and characterized. Two types of 5'-ends of cDNA were isolated by 5'-rapid amplification of cDNA ends, and one of them, approximately 2.1 kbp upstream of ATG-containing exon II, was first identified in human. The major promoter activity was detected in the 5'-flanking region of this newly identified exon Ia. The sequence of the proximal 5'-flanking region contained an activator protein (AP)-1-like site and cAMP-responsive element, but it lacked a TATA box, a GC-rich element, and an SP-1 site. Deletion analysis of the 5'-flanking region showed that the fragments containing the AP-1-like element (TGACTCC) positioned at -38 exhibited high promoter activities in CLC-5 expressing LLC-PK1 cells, but that further deletions not containing this AP-1-like element resulted in a great loss of luciferase activities. Gel-retardation analysis demonstrated the existence of a specific protein binding to this AP-1-like element in LLC-PK1 cells, which seemed to differ from an authentic AP-1. This study clarified the key element of the human CLCN5 promoter, and the mutation in this region could be the cause of Dent's disease.     

Recombinant human CIS2 (SOCS2) protein: subcloning,expression, purification,and characterization

The 1x myc-tagged cDNA encoding for human CIS2 protein was subcloned into a pET-29a+ vector in order to express and produce a recombinant S-peptide tagged and 1x myc-tagged protein in Escherichia coli BL21(DE3). The constitutively expressed protein was isolated from inclusion bodies by a simple solubilization-renaturation procedure and purified by anion-exchange chromatography on Q-Sepharose. The recombinant form was found to be pure and monomeric as judged by both SDS-PAGE and gel-filtration chromatography and its biological activity was proven by its ability to bind to the tyrosine-phosphorylated cytosolic fragment of human growth hormone receptor fused to glutathione-S-transferase. Recombinant CIS2 was compared by biochemical, immunological, and molecular methods to the CIS2 protein expressed in eukaryotic cells. This report describes the first substantial production of biologically active recombinant human CIS2.     

Water-soluble 3-O-(2-methoxyethyl)cellulose: synthesis and characterization

The synthesis of novel 3-O-(2-methoxyethyl)cellulose via 2,6-di-O-thexyldimethylsilyl ethers was successfully carried out. Treatments of 3-O-(2-methoxyethyl)-2,6-di-O-thexyldimethylsilylcellulose with tetrabutylammonium fluoride trihydrate led to a complete removal of the protecting groups. Structure characterization carried out by means of 1D and 2D NMR spectroscopy proves a high regioselectivity. The novel cellulose ether is soluble in dimethyl sulfoxide, N,N-dimethylacetamide, N-methylpyrrolidone, and water. Size-exclusion chromatography revealed a distinct aggregation behavior in water.     

Bis( -cysteinato)gold(I): Chemical characterization and identification in renal cortical cytoplasma

-Cysteinatogold(I) was prepared by the reaction of -cysteine with KAuBr₄ in acidic media and its solubility determined from pH 4 to 10. The solubility at pH 7.4 and 37° C is 1 μM. In the presence of excess cysteine, the solubility increases because of formation of bis( -cysteinato)gold(I). The equilibrium constant for formation of the bis complex is 2.1 ± 0.4 × 10⁻³, which at pH 7.4 corresponds to an apparant formation constant of 4.4 × 10⁴. The formation of the bis adduct was confirmed by chromatographic separation of the products of the reaction between [³⁵S]- -cysteine and Na₂AuTM. This complex elutes with K_av = 1.15 which allows it to be distinguished from other gold thiolates that might form in vivo. The bis(cysteinato)gold(I) complex is shown to be present in kidney cytosol isolated from rats given Na₂AuTM in vivo. When additional cysteine is added to the cytosol in vitro, the peak at 1.15 is increased, but if glutathione is added, the low molecular weight gold elutes at K_av = 1.00, which is taken as evidence for the existence of bis(cysteinato)gold(I) in the cytosol preparation. The amount of gold present as bis(cysteinato)gold(I) after 4 different dose schedules has been measured and found to increase with the total cytosol gold concentration. -Cysteinatogold(I) does not dissolve in the presence of bovine serum albumin to form an adduct.     

Partial purification and characterization of cytosolic Tyr-protein kinase(s) from human erythrocytes

Tyrosine-protein kinase, phosphorylating tyrosine residues of transmembrane band 3 protein, has been partially purified from human erythrocyte cytosol by DEAE-Sepharose chromatography followed by heparin-Sepharose chromatography. Such a Tyr-protein kinase (36 kDa), as distinct from the Ser/Thre-protein kinases (casein kinase S and TS), appears to display a broader site specificity than does the previously described human erythrocyte P-Tyr-protein phosphatase, dephosphorylating band 3 protein. That is, it is able to phosphorylate not only the highly acidic copolymer poly(Glu-Tyr) but also angiotensin II, lacking an acidic amino acid sequence around the target Tyr residue. Moreover, the phosphorylation of these two substrates exhibits a different pH dependence and a different response to NaCl and 2,3-bisphosphoglycerate. These results suggest that in intact erythrocytes the cytosolic Tyr-protein kinase might phosphorylate band 3 not only on Tyr-8, surrounded by several acidic side-chains (as demonstrated preferentially to occur in isolated ghosts), but also on other Tyr residues surrounded by other amino acid sequences.     

Identification and characterization of a novel linkage isomerization in the reaction of trans-diamminedichloroplatinum(II) with 5'-d(TCTACGCGTTCT)

The oligonucleotide 5'-d(TCTACGCGTTCT) reacts with trans-diamminedichloroplatinum(II) to yield primarily trans-[Pt(NH3)2[d(TCTACGCGTTCT)-N7-G(6),N7-G(8)]], containing the desired trans-[Pt(NH3)2[d(GCG)]] 1,3-cross-link. A key element of the platination reaction is the use of low pH to suppress coordination at A(4). The product was fully characterized by pH-dependent NMR titrations, enzymatic degradation analysis, and 195Pt NMR spectroscopy. Interestingly, the 1,3-cross-linked adduct is unstable at neutral pH, rearranging unexpectedly to form the linkage isomer trans-[Pt(NH3)2[d-(TCTACGCGTTCT)-N3-C(5),N7-G(8)]]. This rearrangement product is more stable than the initially formed isomer and could be characterized by pH-dependent NMR titrations, enzymatic degradation analysis, liquid secondary ion mass spectrometric analysis of an enzymatically digested fragment, 195Pt NMR spectroscopy, and modified Maxam-Gilbert footprinting experiments. By contrast, the 1,3-intrastrand cross-linked isomer rearranges during the course of both pH titration and enzymatic degradation experiments to form the 1,4-adduct. The equilibrium constant for this rearrangement is approximately 3, favoring the 1,4-adduct. Kinetic studies of the linkage isomerization reaction reveal t1/2 values for the first-order disappearance of the 1,3-intrastrand cross-linked isomer ranging from 129 (at 30 degrees C) to 3.6 h (at 62 degrees C), with activation parameters delta H not equal to = 91 +/- 2 kJ/mol and delta S not equal to = -58 +/- 8 J/(mol.K). Mechanistic implications of these kinetic results as well as the general relevance of this linkage isomerization reaction to platinum-DNA chemistry are briefly discussed.     

The interaction of 1-fluoro-2,4-dinitrobenzene with amino-phospholipids in membranes of intact erythrocytes,modified erythrocytes,and erythrocytes ghosts

Summary 1-Fluoro-2,4-dinitrobenzene (FDNB) has been used to study the availability of amino-containing phospholipids in erythrocyte membranes and ghosts in an aqueous, isotonic medium. It was found that the addition of bovine serum albumin (BSA) to the medium protects the cells from cation leak and protects some of the amino-phospholipids from reacting with the probe. In isotonic medium without BSA, 46% of the phosphatidylethanolamine and 12% of the phosphatidylserine of erythrocytes and 73% and 21% of these respective lipids of ghosts react with the probe. In the presence of 70 m BSA, 31% of phosphatidylethanolamine and 6.5% of phosphatidylserine of erythrocytes and 59% and 16% of these respective lipids of ghosts react with the probe. The labeling of these lipids does not change under conditions of varying tonicity, or after treatment of erythrocytes with pronase or lysolecithin. The data suggest that 46% of phosphatidylethanolamine and 12% of phosphatidylserine of the erythrocyte membrane are free in a lipid bilayer; 27% and 9% of these respective lipids are loosely bound to proteins which are lost during the preparation of ghosts and 27% of the phosphatidylethanolamine and 79% of the phosphatidylserine are tightly bound to core proteins which remain part of the erythrocyte membrane even after hemolysis.     

Dynamics and interactions of the anion channel in intact human erythrocytes: an electron paramagnetic resonance spectroscopic study employing a new membrane-impermeant bifunctional spin-label

We have developed a new membrane-impermeant, bifunctional spin-labeling reagent, bis-(sulfo-N-succinimidyl) doxyl-2-spiro-4'-pimelate (BSSDP), and employed it in an electron paramagnetic resonance (EPR) study of the rotational diffusion of the anion-exchange channel (band 3) in intact human erythrocytes. BSSDP reacts in a covalent manner and with high specificity with the extracytoplasmic domain of band 3, forming a complex in which the spin-label is immobilized on the protein. The linear EPR spectrum of BSSDP-labeled intact erythrocytes is characteristic of a highly immobilized, spatially isolated nitroxide probe. The saturation-transfer EPR spectrum of the same sample indicates that the anion channel in intact erythrocytes exhibits rotational dynamics in the 0.1-1 ms correlation time range at 20 degrees C. Rotational dynamics in this motional domain are consistent with a strong interaction of the anion-exchange channel with the erythrocyte cytoskeleton. The saturation-transfer EPR spectrum of ghosts prepared from BSSDP-labeled erythrocytes indicates a significant increase in rotational mobility of the anion channel, suggesting a significant disruption on lysis of interactions between the anion channel and the cytoskeleton.