Analogues of NADP+ as inhibitors and coenzymes for NADP+ malic enzyme from maize leaves

Structural analogues of the NADP⁺ were studied as potential coenzymes and inhibitors for NADP⁺ dependent malic enzyme from Zea mays L. leaves. Results showed that 1, N⁶-etheno-nicotinamide adenine dinucleotide phosphate ( NADP⁺), 3-acetylpyridine-adenine dinucleotide phosphate (APADP⁺), nicotinamide-hypoxanthine dinucleotide phosphate (NHDP⁺) and -nicotinamide adenine dinucleotide 2: 3-cyclic monophosphate (23NADPc⁺) act as alternate coenzymes for the enzyme and that there is little variation in the values of the Michaelis constants and only a threefold variation in V_max for the five nucleotides. On the other hand, thionicotinamide-adenine dinucleotide phosphate (SNADP⁺), 3-aminopyridine-adenine dinucleotide phosphate (AADP⁺), adenosine 2-monophosphate (2AMP) and adenosine 2: 3-cyclic monophosphate (23AMPc) were competitive inhibitors with respect to NADP⁺, while -nicotinamide adenine dinucleotide 3-phosphate (3NADP⁺), NAD⁺, adenosine 3-monophosphate (3AMP), adenosine 2: 5-cyclic monophosphate (25AMPc), 5AMP, 5ADP, 5ATP and adenosine act as non-competitive inhibitors. These results, together with results of semiempirical self-consistent field-molecular orbitals calculations, suggest that the 2-phosphate group is crucial for the nucleotide binding to the enzyme, whereas the charge density on the C₄ atom of the pyridine ring is the major factor that governs the coenzyme activity.Abbreviations NADP⁺ 1, N⁶-etheno-nicotinamide adenine dinucleotide phosphate - NHDP⁺ nicotinamide-hypoxanthine dinucleotide phosphate - APADP⁺ 3-acetylpyridine-adenine dinucleotide phosphate - SNADP⁺ thionicotinamide-adenine dinucleotide phosphate - AADP⁺ 3-aminopyridine-adenine dinucleotide phosphate - 23NADPc⁺ -nicotinamide adenine dinucleotide 2: 3-cyclic monophosphate - 3NADP⁺ -nicotinamide adenine dinucleotide 3-phosphate - 2AMP adenosine 2-monophosphate - 3AMP adenosine 3-monophosphate - 23AMPc adenosine 2: 3 monophosphate cyclic - A adenosine - RuBP ribulose 1,5-bisphosphate - SCF-MO Self-Consistent Field-Molecular Orbitals (method)     

A fluorometric assay of ceramide glycanase with 4-methylumbelliferyl β-d-lactoside derivatives

4-Methylumbelliferyl 6-O-benzyl--d-lactoside (6Bn-MU-Lac) and some related compounds were synthesizedvia different selective reactions including phase-transfer glycosylation. Their suitability as substrates for a fluorometric assay of ceramide glycanase (CGase) was evaluated. Among others, the 6Bn-MU-Lac, which is resistant to exogalactosidase, was found to be a suitable substrate for routine assay of the CGase activity. For American leech CGase, theK _m value is 0.232 mM at pH 5. Abbreviations: CGase, ceramide glycanase; Gal, galactose; Glc, Glucose; Lac, lactose; MU, 4-methylumbelliferone; MU-Lac, 4-methylumbelliferyl -d-lactoside; bBn-Lac, 6-O-benzyl-lactose; 6Bn-MU-Lac, 4-methylumbelliferyl 6-Obenzyl--d-lactoside; 46Bd-MU-Lac, 4-methylumbelliferyl 4,6-O-benzylidene--d-lactoside; MU-Cel, 4-methylumbellifery -d-cellobioside; 46Bd-MU-Cel, 4-methylumbelliferyl 4,6-O-benzylidene--d-cellobioside; TLC, thin layer chromatography;¹H-NMR, proton nuclear magnetic resonance; GSL, glycosphingolipids; CSA, 10-camphorsulfonic acid. See Scheme 1 for chemical structures.     

Purification and characterization of mouse glucose 6-phosphate dehydrogenase

Summary Glucose-6-phosphate dehydrogenase was purified to homogeneity from testes and kidneys of the inbred strain of mice (DBA/2J) by a simple two-step affinity column procedure. This involved the sequential application of 8-(6-aminohexyl)-amino-AMP-and -2, 5-ADP-Sepharose columns and biospecific elution with NADP⁺ in both steps. The molecular and biochemical properties of the purified enzyme were studied in detail. These include the molecular weight determination, amino acid composition, steady-state kinetics, inactivation by high temperature, urea and iodoacetate, and immunology. The purified enzyme from mouse kidneys or testes was shown to be a tetramer with a molecular weight of 220,000. The enzyme is highly specific for glucose-6-phosphate, exhibits almost no activity with NAD⁺ as a coenzyme and is little inhibited by AMP or ATP. Michaelis constants for glucose-6-phosphate and NADP⁺ were determined to be 50 m and 10 m respectively. NADPH is a competitive inhibitor of NADP⁺ and has a K_i of 18 µm. Rabbit antisera against glucose-6-phosphate dehydrogenase were raised. The antisera also cross-react with the same enzyme from human and guinea pig.     

Amino acid esters of 9-(2′,3′-dihydroxypropyl-1′)-adenine are the specific inhibitors of protein synthesis on ribosomes

Peptide acceptor properties of phenylalanine and glycine esters of 9-(2,3-dihydroxypropyl-1)-adenine and 1-(2,3-dihydroxypropyl-1)-4-thiouracyl were investigated. All these esters appeared to be powerful inhibitors of polyphenylalanine synthesis in E. coli MRE-600 ribosomes charged with poly U. Like puromycin, esters of adenine derivatives accepted the AcPhe residue from Ac-[¹⁴C] Phe-tRNA in a ribosomal system charged with poly U. However, peptidyl esters of 9-(2,3-dihydroxypropyl-1)-adenine remained bound with ribosomes. The structure of the peptide esters synthesized was ascertained after dissociation of ribosomes into subparticles by direct comparison with the synthetic specimens.Abbreviations AcPhe acetyl-l-phenylalanine - HP-Ade 9-(2,3-dihydroxypropyl-1)-adenine - Phe-HP-Ade and Gly-HP-Ade l-phenylalanine and glycine esters of HP-Ade - Phe-HP-TUra l-phenylalanine ester 1-(2,3-dihydroxypropyl-1)-4-thiouracyl - AcPhePhe-HP-Ade and AcPheGly-HP-Ade acetyl-l-diphenylalanine and acetyl-l-phenylalanylglycine esters of HP-Ade respectively - AcPhe-puromycin acetyl-l-phenylalanyl-puromycin     

Adenosine-5′-phosphosulfate (APS) as sulfate donor for assimilatory sulfate reduction in Rhodospirillum rubrum

Crude extracts of Rhodospirillum rubrum catalyzed the formation of acid-volatile radioactivity from (³⁵S) sulfate, (³⁵S) adenosine-5-phosphosulfate, and (³⁵S) 3-phosphoadenosine-5-phosphosulfate. An enzyme fraction similar to APS-sulfotransferases from plant sources was purified 228-fold from Rhodospirillum rubrum. It is suggested here that this enzyme is specific for adenosine-5-phosphosulfate, because the purified enzyme fraction metabolized adenosine-5-phosphosulfate, however, only at a rate of 1/10 of that with adenosine-5-phosphosulfate. Further, the reaction with 3-phosphoadenosine-5-phosphosulfate was inhibited with 3-phosphoadenosine-5-phosphate whereas this nucleotide had no effect on the reaction with adenosine-5-phosphosulfate. For this activity with adenosine-5-phosphosulfate the name APS-sulfotransferase is suggested. This APS-sulfotransferase needs thiols for activity; good rates were obtained with either dithioerythritol or reduced glutathione; other thiols like cysteine, 2-3-dimercaptopropanol or mercaptoethanol are less effective. The electron donor methylviologen did not catalyze this reaction. The pH-optimum was about 9.0; the apparent K _m for adenosine-5-phosphosulfate was determined to be 0.05 mM with this so far purified enzyme fraction. Enzyme activity was increased with K₂SO₄ and Na₂SO₄ and was inhibited by 5-AMP. These properties are similar to assimilatory APS-sulfotransferases from spinach and Chlorella.Abbreviations APS adenosine-5-phosphosulfate - PAPS 3-phosphoadenosine-5-phosphosulfate - 5-AMP adenosine-5-monophosphate - 3-AMP adenosine-3-monophosphate - 3-5-ADP 3-phosphoadenosine-5-phosphate (PAP) - DTE dithiorythritol - GSH reduced glutathione - BAL 2-3-dimercaptopropanol     

Stereoselective formation of a 2′(3′)-aminoacyl ester of a nucleotide

Summary Reaction ofDl-serine and adenosine-5-phosphorimidazolide in the presence of adenosine-5-(O-methylphosphate) and imidazole resulted in the stereoselective synthesis of the aminoacyl nucleotide ester 2(3)-O-seryl-adenosine-5-(O-methylphosphate). The enantiomeric excess ofd-serine incorporated into 2(3)-O-seryl-adenosine-5-(O-methylphosphate) was about 9%. Adenylyl-(5N)-serine and an unknown product also incorporated an excess ofd-serine; however, serylserine showed an excess ofl-serine. The relationship of these results to the origin of the biological pairing ofl-amino acids and nucleotides containingd-ribose is discussed.     

δ andk opiate receptors in primary astroglial cultures part II: Receptor sets in cultures from various brain regions and interactions with ß-receptor activated cyclic AMP

In a previous paper, and opiate receptors were shown to be co-localized on the same cell in enriched primary cultures of astroglia from neonatal rat cerebral cortex. Activation of the receptors inhibited adenylate cyclase. In this work, the presence of opiate receptors was investigated in astroglial primary cultures from neonatal rat striatum and brain stem. Cyclic adenosine 3, 5-monophosphate accumulation was quantified in the presence of different opioid receptor ligands after stimulation of the cyclic adenosine 3,5-monophosphate system with forskolin. Morphine was used as a receptor agonist. [d-Ala², D-Leu⁵]-enkephalin or[d-Pen²,d-Pen⁵]-enkephalin were used as receptor agonists and dynorphin 1–13 or U-50,488H were used as receptor agonists. Specific antagonists for the respective receptors were used. After striatum or brain stem cultures had been incubated in 10^–9–10^–5M of each [d-Ala²,d-Leu⁵]-enkephalin, [d-Pen², D-Pen⁵]-enkephalin and Dynorphin 1–13 or U-50,488H, dose related inhibitions of the 10^–5M rorskolin stimulated cyclic adenosine 3,5-monophosphate accumulation were observed. The changes were reversed to the forskolin-induced control level in the presence of the respective antagonists. 10^–9–10^–5M morphine did not significantly change the forskolin-induced accumulation of cyclic adenosine 3,5-monophosphate in the cultures studied. Furthermore, cultures from cerebral cortex, striatum or brain stem were incubated with isoproterenol alone or together with morphine or [d-Ala²,d-Leu⁵]-enkephalin. Isoproterenol stimulated cyclic adenosine 3,5-monophosphate accumulation more prominently in the cerebral cortex and striatum cultures than in the brain stem cultures. Morphine did not influence isoproterenol-induced cyclic adenosine 3,5-monophosphate accumulation, while [d-Ala²,d-Leu⁵]-enkephalin inhibited the accumulation. The results indicate that astroglial cells in primary cultures from striatum, brain stem and cerebral cortex express andk opioid receptors linked to the adenylate cyclase/cyclic adenosine 3,5-monophosphate system. No receptors were detected, however, in the present model. Aspects of the relation between the expression of opioid peptides and opioid receptors are discussed, while speculations are also made on the functional aspects of opioid receptors on astroglia.     

Pyridoxal 5′-phosphate binds to a lysine residue in the adenosine 3′-phosphate 5′-phosphosulfate recognition site of glycolipid sulfotransferase from human renal cancer cells

In the course of characterization of glycolipid sulfotransferase from human renal cancer cells, the manner of inhibition of sulfotransferase activity with pyridoxal 5-phosphate was investigated. Incubation of a partially purified sulfotransferase preparation with pyridoxal 5-phosphate followed by reduction with NaBH₄ resulted in an irreversible inactivation of the enzyme. When adenosine 3-phosphate 5-phosphosulfate was co-incubated with pyridoxal 5-phosphate, the enzyme was protected against this inactivation. Furthermore, pyridoxal 5-phosphate was found to behave as a competitive inhibitor with respect to adenosine 3-phosphate 5-phosphosulfate with aK _i value of 287 µm. These results suggest that pyridoxal 5-phosphate modified a lysine residue in the adenosine 3-phosphate 5-phosphosulfate-recognizing site of the sulfotransferase.     

Nonidentity of the cDNA sequence of human breast cancer cell malic enzyme to that from the normal human cell

A cDNA coding for human breast cancer cell cytosolic NADP⁺-dependent malic enzyme was obtained. This cDNA is composed of a length of 2084 base pairs, with 1698 base pairs coding for 565 amino acid residues and a length of 386 base pairs representing a 3-noncoding region. Comparing this nucleotide sequence with that from the normal human tissue [Loeber, G., Dworkin, M. B., Infante, A., and Ahorn, H. (1994),FEBS Lett. 344, 181–186] reveals that three nucleotides in the open reading frame and the length of 3-noncoding region of the cDNA are different. One of the changes results in a substitution of serine at position 438 for proline, which, however, may not cause significant changes in the predicted secondary structure. A partial cDNA lacking the first 84 nucleotides in the open reading frame was successfully cloned and expressed functionally inEscherichia coli cells. ItsK _m value forl-malate (1.21±0.11 mM) is four times higher than that for the natural human breast cancer cell malic enzyme (0.29±0.04 mM) but similar to that for the full-length recombinant enzyme (1.06±0.07 mM). TheK _m values for Mn²⁺ and NADP⁺ (0.26±0.03 and 0.97±0.4M, respectively) are similar to those for the natural enzyme (0.12±0.02 and 1.9±0.3M, respectively) or the recombinant wild-type enzyme (0.56±0.04 and 0.44±0.02M, respectively). A recombinant pigeon liver malic enzyme without the first 13 amino acid residues was used for comparison. TheK _m values forl-malate and Mn²⁺ of the truncated enzyme (11.2±0.9 mM and 61.2±4.6M, respectively) are over 40 times larger than those for the natural pigeon liver malic enzyme (0.21±0.02 mM and 1.06±0.08M, respectively) or the recombinant wild-type enzyme (0.25±0.01 mM and 1.48±0.05M, respectively). We suggest that the N-terminus of malic enzyme may be required for the substrate binding during the catalytic cycle.     

IP3-and cAMP-induced responses in isolated olfactory receptor neurons from the channel catfish

Summary Olfactory receptor neurons enzymatically dissociated from channel catfish olfactory epithelium were depolarized transiently following dialysis of IP₃ or cAMP (added to the patch pipette) into the cytoplasm. Voltage and current responses to IP₃ were blocked by ruthenium red, a blocker of an IP₃-gated Ca²⁺-release channel in sarcoplasmic reticulum. In contrast, the responses to cAMP were not blocked by extracellularly applied ruthenium red, nor by l-cis-diltiazem or amiloride and two of its derivatives. The current elicited by cytoplasmic IP₃ in neurons under voltage clamp displayed a voltage dependence different from that of the cAMP response which showed marked outward rectification. A sustained depolarization was caused by increased cytoplasmic IP₃ or cAMP when the buffering capacity for Ca²⁺ of the pipette solution was increased, when extracellular Ca²⁺ was removed or after addition of 20–200 nm charibdotoxin to the bathing solution, indicating that the repolarization was caused by an increase in [Ca _i ] that opened Ca²⁺-activated K⁺ channels. The results suggest that different conductances modulated by either IP₃ or cAMP are involved in mediating olfactory transduction in catfish olfactory receptor neurons and that Ca²⁺-activated K⁺ channels contribute to the termination of the IP₃ and cAMP responses.Abbreviations ATP adenosine 5-triphosphate - BAPTA (bis-(o-aminophenoxy)-ethane-N-N-N-N)-tetraacetic acid - cAMP adenosine cyclic 3,5-monophosphate - cGMP guanosine cyclic 3,5-monophosphate - CTX charybdotoxin - DCB 3,4-dichlorobenzamil - EDTA ethylenediaminetetraacetic acid - EGTA ethylenglycol-bis-(b-aminoethyl)-N-N-N-N-tetraacetic acid - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - IP₃ inositol-1,4,5-triphosphate - NMDG N-methyl-d-glucamine We would like to thank the Tanabe Seiyaku Co., Ltd., for their gift of l-cis-diltiazem. This work was supported by National Institutes of Health grants DC00566 and BRSG S07RR05825.     

Preparation and heteronuclear 2D NMR spectroscopy of a DNA dodecamer containing a thymidine residue with a uniformly 13C-labeled deoxyribose ring

Summary [¹³C₅]-2-Deoxy-d-ribose, synthesized from [¹³C₆]-d-glucose (98% ¹³C), was coupled with thymine to give [1,2,3,4,5-¹³C₅]-thymidine (T) in an 18% overall yield. The thymidine was converted to the 3-phosphoramidite derivative and was then incorporated into a dodecamer 5-d(CGCGAATTCGCG)-3 by solid-phase DNA synthesis. Preparation of 0.24 mole of the labeled dodecamer, which is sufficient for a single NMR sample, consumed only 25 mg of glucose. By virtue of the ¹³C labels, all of the ¹H-¹H vicinal coupling constants in the sugar moieties were accurately determined by HCCH-E.COSY.     

Stereoselective aminoacylation of a dinucleoside monophosphate by the imidazolides ofDl-alanine and N-(tert-butoxycarbonyl)-Dl-alanine

Summary The aminoacylation of diinosine monophosphate (IpI) was studied. When the acylating agent was the imidazolide of N-(tert-butoxycarbonyl)-Dl-alanine, a 40% enantiomeric excess of thel isomer was incorporated at the internal 2 site and the positions of equilibrium for the 23 migration reaction differed for theD andl enantiomers. The reactivity of the nucleoside hydroxyl groups decreased in the order 2(3)>internal 2>5, and the extent of reaction was affected by the concentration of the imidazole buffer (pH 7.1). In contrast, reaction of IpI with the imidazolide of unprotectedDl-alanine led to an excess of theD isomer at the internal 2 site, while reaction with the N-carboxy anhydride ofDl-alanine proceeded without detectable stereoselection. The relevance of these results to the evolution of optical activity and the origin of genetically directed protein synthesis is discussed.     

Cyclic nucleotide phosphodiesterases of the renal cortex

Summary Cyclic nucleotide phosphodiesterase in the basal-lateral segment of plasma membranes from proximal tubule cells of the rabbit renal cortex was studied and compared to that in the brush border segment of the plasma membrane. Both adenosine 3,5-monophosphate and guanosine 3,5-monophosphate were hydrolyzed by the basal-lateral membrane, but activity varied differently with the two substrates in a complex concentration-dependent manner. Activity with adenosine 3,5-monophosphate was greater than, equal to, or less than with guanosine 3,5-monophosphate, at concentrations of 1000, 100, and 10 to 1 m, respectively. Basal-lateral membrane phosphodiesterase activities at 1 and 500 m substrate exhibited differential responses to pH, metals, heat, and a heat stable inhibitor. Stimulation by guanosine 3,5-monophosphate and inosine 3,5-monophosphate of adenosine 3,5-monophosphate hydrolysis was found in basal-lateral but not in brush border membranes. This stimulation was potentiated by ethyleneglycol-bis(-aminoethyl ether)N,N-tetraacetic acid and ethylenediaminetetraacetate, inhibited by Triton X-100, and totally blocked by Zn²⁺. The findings indicate that multiple forms of phosphodiesterase are present in the basal-lateral segment and these differ from the activities in the brush border region of the plasma membrane. The characteristics of (i) allosteric, guanosine 3,5-monophosphate-sensitivity of adensoine 3,5-monophosphate phosphodiesterase, and (ii) relatively high guanosine 3,5-monophosphate phosphodiesterase activity, in basal-lateral membranes, which are also enriched in adenylate and guanylate cyclase, suggest an important physiological role for these phosphodiesterases in the regulation of net production of cyclic nucleotides in the renal cortex.     

Purification and properties of cAMP independent glycogen synthase kinase and phosvitin kinase from human leukocytes

Summary cAMP independent glycogen synthase kinase and phosvitin kinase activity was purified from the 180 000 × g supernatant of human polymorphonuclear leukocytes by ammonium sulphate precipitation and phosphocellulose chromatography. The cAMP independent glycogen synthase kinase eluted from the phosphocellulose at 0.54 m NaCl (peak A) separate from the major phosvitin kinase eluting at 0.68 m NaCl (peak B). The kinase activity of both peaks tended to form aggregates, but in the presence of 0.6 m NaCl, the peak B enzyme had M_r 250 000, 7.2S and the peak A enzyme M_r 38 000, 3.8S. The ratio between synthase kinase and phosvitin kinase activity in peak A was 1:3.2 and in peak B 1:31.4. In addition the kinase activities differed with respect to sensitivity to temperature, ionic strength and CaCl₂. It is suggested that the peak A enzyme represents the cAMP independent glycogen synthase kinase of leukocytes, whereas the peak B enzyme is a phosvitin kinase, which is insignificantly contaminated with some synthase kinase (peak A) and contains a separate, second synthase kinase.Synthase kinase had K _m ^app 4.2 m for muscle glycogen synthease I and K _m ^app 45 m for ATP. GTP was a poor substrate. The activity was not influenced by cyclic nucleotides, Ca²⁺, or glucose-6-P. Synthase I from muscle and leukocytes was phosphorylated to a ratio of independence of less than 0.05.Abbreviations cAMP adenosine cyclic 3:5-monophosphate - DTT dithiothreitol - EGTA ethylene glycol-bis-(-amino-ethylether)-N,N-tetraacetic acid - PMSF phenylmethylsulfonylfluoride - PKI protein kinase inhibitor - RI ratio of independence for glycogen synthase - SDS sodium dodecyl sulphate     

Regioselective alkylation of benzylβ-d-lactoside and its derivatives by stannylation

The preparation of benzyl 2,3,6,2,6-penta-O-benzyl--d-lactoside, which is a key intermediate for chemical synthesis of oligosaccharide components of glycosphingolipids, was achieved by an improved method. The 3-O-p-methoxybenzyl and 3-O-methyl derivatives were prepared from benzyl 2,3,6,2,6-penta-O-benzyl--d-lactoside through stannylation. By using benzyl -d-lactoside as starting material, benzyl 3-O-methyl-, 3-O-benzyl- and 3-O-p-methoxybenzyl--d-lactoside were regioselectively synthesized using the same procedure.     

Purification and characterization of the 5′ → 3′ exonuclease domain-deleted Thermus filiformis DNA polymerase expressed in Escherichia coli

The gene encoding 5 3 exonuclease domain-deleted Tfi DNA polymerase, named 5 3 Exo^– Tfi fragment, from Thermus filiformis was expressed in Escherichia coli under the control of the tac promoter on a high-copy plasmid, pJR. The expressed enzyme was purified 27-fold with a 19% yield and a specific activity of 2621 U mg^–1 protein. The 5 3 exonuclease domain of Tfi DNA polymerase was removed without significant effect on enzyme activity and stability. PCR conditions for the 5 3 Exo^– Tfi fragment were more tolerant to the buffer composition as compared to the full-length enzyme.     

Re-evaluation of the specificity of adenylyl (beta,gamma-methylene)diphosphonate as a substrate for adenylate cyclase.

Summary The conversion of the ATP-analogue adenylyl(,-methylene)diphosphonate (AMPPCP) to cyclic AMP by adenylate cyclase of rat liver membranes was demonstrated using a radioimmunoassay for cyclic AMP. The conversion was only insignificantly lower than with adenylylimidodiphosphate (AMPPNP), another ATP-analogue which is usually used in the histochemical adenylate cyclase assay. The unspecific phosphate production was lower with AMPPCP as compared to AMPPNP. Therefore AMPPCP is considered to be a more suitable substrate for the histochemical assay.Unspecific phosphate deposition in the histochemical assay was due to ATP:pyrophosphatase activity and could be significantly inhibited by 1mm NAD. However, a residual phosphate deposition due to cleavage of NAD could not be suppressed. Adenylate cyclase activity could be markedly activated by 5×10^–5 m forskolin, an activator of the catalytic subunit of the enzyme, and inhibited by 1mm 25-dideoxyadenosine, a specific inhibitor of adenylate cyclase. Adenylate cyclase was localized predominantly in the sinusoidal part of the plasma membrane, while ATP-pyrophosphatase seemed to be restricted to the canalicular part. It is concluded that at least three parallel assays are necessary for routine histochemical demonstration of adenylate cyclase, namely (1) basal activity (2) activation by forskolin and (3) inhibition by 25-dideoxyadenosine, to demonstrate a specific enzyme reaction.     

Sugar conformation of a stereospecific 2′-R or 2′-S deuterium-labeled DNA decamer studied with proton-proton J coupling constants

The sugar conformation of a DNA decamer was studied with proton-proton ³J coupling constants. Two samples, one comprising stereospecifically labeled 2-R-²H for all residues and the other 2-S-²H, were prepared by the method of Kawashima et al. [J. Org. Chem. (1995) 60, 6980–6986; Nucleosides Nucleotides (1995) 14, 333–336], the deuterium labeling being highly stereospecific 99% for all 2-²H, 98% for 2-²H of A, C, and T, and 93% for 2-²H of G). The ³J values of all H1-H2 and H1-H2 pairs, and several H2-H3 and H2-H3 pairs were determined by line fitting of 1D spectra with 0.1–0.2 Hz precision. The observed J coupling constants were explained by the rigid sugar conformation model, and the sugar conformations were found to be between C3-exo and C2-endo with _m values of 26° to 44°, except for the second and 3 terminal residues C2 and C10. For the C2 and C10 residues, the lower fraction of S-type conformation was estimated from J_H1H2 and J_H1H2 values. For C10, the N–S two-site jump model or Gaussian distribution of the torsion angle model could explain the observed J values, and 68% S-type conformation or C1-exo conformation with 27° distribution was obtained, respectively. The differences between these two motional models are discussed based on a simple simulation of J-coupling constants.     

Interaction among anion,cation and glucose transport proteins in the human red cell

Summary The time course of binding of the fluorescent stilbene anion exchange inhibitor, DBDS (4,4-dibenzamido-2,2-stilbene disulfonate), to band 3 can be measured by the stopped-flow method. We have previously used the reaction time constant, _DBDS, to obtain the kinetic constants for binding and, thus, to report on the conformational state of the band 3 binding site. To validate the method, we have now shown that the ID₅₀ (0.3±0.1 m) for H₂-DIDS (4,4-diisothiocyano-2,2-dihydrostilbene disulfonate) inhibition of _DBDS is virtually the same as the ID₅₀ (0.47±0.04 m) for H₂-DIDS inhibition of red cell Cl^– flux, thus relating _DBDS directly to band 3 anion exchange. The specific glucose transport inhibitor, cytochalasin B, causes significant changes in _DBDS, which can be reversed with intracellular, but not extracellular,d-glucose. ID₅₀ for cytochalasin B modulation of _DBDS is 0.1±0.2 m in good agreement withK _D =0.06±0.005 m for cytochalasin B binding to the glucose transport protein. These experiments suggest that the glucose transport protein is either adjacent to band 3, or linked to it through a mechanism, which can transmit conformational information. Ouabain (0.1 m), the specific inhibitor of red cell Na⁺,K⁺-ATPase, increases red cell Cl^– exchange flux in red cells by a factor of about two. This interaction indicates that the Na⁺,K⁺-ATPase, like the glucose transport protein, is either in contact with, or closely linked to, band 3. These results would be consistent with a transport proteincomplex, centered on band 3, and responsible for the entire transport process, not only the provision of metabolic energy, but also the actual carriage of the cations and anions themselves.     

The synthesis of yellow tetrazolium

Summary The syntheses of 4,4-biphenyl tetrazonium chloride monohydrate, of 1,1-di-(3-nitrophenyl)-3,3-dimethyl-5,5-(4,4-biphenylene)-diformazan, and of yellow tetrazolium [2,2-di-(3-nitrophenyl)-5,5-dimethyl-3,3-(4,4-biphenylene)-ditetrazolium chloride] are described. Solutions of the pure formazan in pyridine absorb visible light maximally at 455 nm; ₄₅₅ is 54,050. Yellow tetrazolium has been specifically designed to meet the requirement of a new technique in quantitative cytochemistry, which involves the consecutive use of two bistetrazolium salts on the same tissue section.I thank Professor L. Young of St. Thomas's Hospital Medical School and Professor C. Long of the Royal College of Surgeons for their interest and encouragement. I am greatly indebted to a large number of friends and colleagues for helpful discussion, and to Dr. A. Deavin and Miss A. Banks for exploratory experiments which involved column chromatography. Microanalyses were carried out by Mr. G. S. Crouch at the London School of Pharmacy.