N-Acetyl-D-glucosamine binding lectins. A model system for the study of binding specificity

Synthetic glycoconjugates prepared by the direct reductive amination of di-N-acetylchitobiose and tetra-N-acetyl-chitotetraose to poly-l-lysine with sodium cyanoborohydride have been used to explore the binding specificities of the lectins wheat germ agglutinin and Bandeiraea simplicifolia II. These conjugates are effective precipitating antigens for these lectins, and hapten inhibition experiments, employing the per-N-acetylated oligomers of chitin as inhibitors, demonstrate that wheat germ agglutinin and Bandeiraea simplicifolia II lectin have binding sites complementary to three and two contiguous β 1,4-linked N-acetyl-d-glucosamine residues, respectively, in agreement with conclusions reached using other methods. Conjugates prepared by this technique should be useful for examining the binding specificities of other lectins, and the results of a study of the effect of chain length of the hapten on the affinity of the lectin for these conjugates should provide guidance in selection of the hapten most appropriate for these studies.     

Analysis of dual transport systems by means of Hill plots

A method is proposed for calculating the parameters of dual solute transport systems utilizing Hill plots of isotope uptake data. The use of the method is illustrated with synaptosomal uptakes of phenylalanine and leucine and with data that have been reported in the literature for synaptosomal uptakes of taurine and glutamate via dual systems.     

Proteins containing reductively aminated disaccharides: Synthesis and chemical characterization

Synthetic glycoproteins can be prepared by reductive amination of protein and reducing disaccharide in the presence of sodium cyanoborohydride. The reaction proceeds readily in aqueous solutions over a broad pH range to give high degrees of substitution. The degree of substitution can be determined by amino acid analysis, as the secondary amine linkage formed by reductive amination in stable to acid-catalyzed protein hydrolysis conditions. In order to demonstrate that coupling occurs to lysine residues, synthetic α-N-1-(1-deoxyglucitol)-lysine and ?-N-1-(1-deoxyglucitol)-lysine were prepared and compared with bovine serum albumin conjugates of maltose, cellobiose, lactose, and melibiose by amino acid analysis after acid hydrolysis. These studies demonstrate that the expected secondary amine linkages are formed with the ?-amino groups of lysine.     

Chemical modification of Lactobacillus casei thymidylate synthetase with 1-fluoro-2,4-dinitrobenzene and 1,5-difluoro-2,4-dinitrobenzene

Previous studies of the pH dependence of sulfhydryl group modification in thymidylate synthetase (W. A. Munroe, C. A. Lewis and R. B. Dunlap, 1978, Biochem. Biophys. Res. Commun.80, 355–360) suggested that a neighboring general base residue enhanced the nucleophilicity of the catalytic cysteinyl side chain. In an effort to identify the latter residue by active site crosslinking, chemical modification of the enzyme by 1,5-difluoro-2,4-dinitrobenzene was investigated and compared with results of modification by 1-fluoro-2,4-dinitrobenzene. Incubation of enzyme with 1-fluoro-2,4-dinitrobenzene led to rapid inactivation and loss of ability to form ternary complexes. Paper chromatography of the acid hydrolysate of enzyme modified with 1-fluoro-2,4-dinitrobenzene yielded two yellow spots, identified as dinitrophenylenecysteine and dinitrophenylenelysine. Specific active site labeling was indicated by substrate protection with dUMP, by the release of 1.65 of fluoride ion per enzyme dimer during inactivation, and by the fact that 70% of the activity was recovered after incubation of the inactivated enzyme with 2-mercaptoethanol, The results of a similar series of studies with 1,5-difluoro-2,4-dinitrobenzene indicated quite specific active site modification. The equivalents of fluoride ion released during modification, 3.5 per enzyme dimer, and the fact that thiolysis of the totally inactivated enzyme led to a recovery of only 18% of the original activity provided evidence for active site crosslinking with the catalytic cysteine as one of the modification sites. Characterization of the modified enzyme, its yellow acid hydrolysate fragments, and a variety of dinitrophenylene crosslinked models suggested that 1,5-difluoro-2,4-dinitrobenzene had modified the enzyme by crosslinking cysteine and serine residues.     

Immobilization of proteins on aldehyde-activated polyacrylamide supports

A method has been developed for the immobilization of proteins on derivatized polyacrylamide gels. Aminoethyl Bio-Gel P-150 was converted to its stable N-2,3-dihydroxypropyl derivative by borohydride reduction of the Schiff base formed with glyceraldehyde. Periodate oxidation of the modified gel provided a reactive aldehyde, which was subsequently coupled to protein by reductive amination with sodium cyanoborohydride. Coupling efficiencies were found to be >90% for concanavalin A and bovine serum albumin, and the gels contained as much as 5 and 20 mg of protein/ml of gel, respectively. Immobilized concanavalin A retained 89% of its binding capacity and was demonstrated to be chemically stable with variations in pH, and changes in concentrations of Triton X-100 and sodium dodecyl sulfate (at concentrations <0.1%). Bovine β-hexosaminidase and β-glucuronidase, higher molecular weight proteins, were also bound with retention of activity, but with less efficiency. This procedure provides an efficient method for the covalent immobilization of proteins.     

Inhibition of the mono-oxygenase system by butylated hydroxyanisole and butylated hydroxytoluene

The commonly used food-additive antioxidants, butylated hydroxyanisole and butylated hydroxytoluene, are inhibitors of the hepatic microsomal mono-oxygenase system, as assayed by benzpyrene hydroxylase activity and demethylase activities. Generally, butylated hydroxyanisole is a more potent inhibitor than butylated hydroxytoluene. Both inhibitors bind to cytochrome P-450 and induce “type I” binding spectra. Cytochrome P-450 is tentatively assigned as the site of inhibition.     

Immunosuppression by 2′-deoxycoformycin: Studies on the mode of administration

It was previously reported that continuous infusion of 2′-deoxycoformycin (DCF), a potent inhibitor of the enzyme adenosine deaminase (ADase), produces a severe immunodeficiency of both T and B lymphocytes (A. Tedde, M. E. Balis, S. Ikehara, R. Pahwa, R. A. Good, and P. P. Trotta, Proc. Nat Acad. Sci. USA 77, 4899 (1980)). In the present study the immunological and biochemical effects in C57BL/6J mice of daily single intraperitoneal injections of DCF are examined. Five days of daily injections of DCF at 0.4 mg/kg body wt/day produced both immunosuppression and lymphocytotoxicity. Parallel experiments indicated that continuous infusion of DCF at the lower dose of 0.3 mg/kg body wt/day resulted in a more potent depression of immune function and a greater lymphocytotoxicity. In particular we have observed after single injections: (i) A decrease in body weight of 4.7%, compared to 16.2% after continuous infusion; (ii) a decrease in differential lymphocyte count from 90.3% (normal) to 65.6%, compared to 31.4% after continuous infusion, and a decrease in leukocyte count (per mm²) from 5200 (normal) to 3800, compared to 2300 after continuous infusion, while no difference in other blood parameters, including erythrocyte number, hematocrit, and hemoglobin concentration, was observed; (iii) on histological examination, a depletion of T and, to a lesser extent, B lymphocytes, and an atrophy of lymph nodes, spleen, and thymus; and (iv) significant depression in blastogenesis induced by the T-cell mitogens concanavalin A and phytohemagglutinin and, to a lesser extent, by the B-cell mitogen Escherichia coli lipopolysaccharide. ADase was inhibited to a high degree ranging from 84 to 94% in lymphoid organs and erythrocytes from animals receiving DCF by either mode of administration. These data provide further direct support for the importance of ADase to proper lymphocyte function, and suggest that continuous infusion may be the preferred mode of administration in clinical trials of DCF as an antileukemic agent.     

Coordinate regulation of adenylate cyclase, protein kinase, and specific enzyme synthesis by cholera toxin in hormonally unresponsive hepatoma cells

Since none of the hormones which activate adenylate cyclase in other tissues have been found to activate adenylate cyclase or to induce tyrosine aminotransferase in cultured Reuber hepatoma cells (H35), despite the stimulatory effects of cyclic AMP derivatives on the latter enzyme, we tested the ability of cholera toxin to influence these processes. At low concentrations cholera toxin was found to mimic the ability of cyclic AMP derivatives to selectively stimulate the synthesis of the aminotransferase. Adenylate cyclase and protein kinase activity were also enhanced, but only after a lag period as in other systems. Specific phosphorylation of endogenous H₁ histone was also shown to be increased by cholera toxin treatment. The increase in tyrosine aminotransferase activity is due to an increase in de novo synthesis as shown by radiolabeling experiments utilizing specific immunoprecipitation. The activity of another soluble enzyme induced by dibutyryl cyclic AMP, PEP carboxykinase, was also stimulated by exposure of H35 cells to cholera toxin. Combinations of cholera toxin and dexamethasone led to greater than additive increases in the activity of both the aminotransferase and carboxykinase. Close coupling of cyclic AMP production with protein kinase activation and enzyme induction was suggested by the observation that the ED₅₀ values for the stimulation of adenylate cyclase, cyclic AMP production, protein kinase, and tyrosine aminotransferase activities were found to be the same (5–7 ng/ml) within experimental error. The results indicate that the adenylate cyclase system in H35 cells is functionally responsive and they support the suggestion that activation of protein kinase is functionally linked to induction of specific enzymes.     

Spontaneous proliferation in unfractionated spleen cell cultures: autologous mixed-lymphocyte reactions (AMLR) which can be differentially regulated by prostaglandins and lymphokines

The present studies were undertaken to define the contribution of the autologous or syngeneic mixed-leukocyte reactions (AMLR/SMLR) to the cellular proliferation observed in unfractionated spleen cell cultures. Proliferation was studied in whole, untreated 6-day murine spleen cell cultures supplemented with syngeneic serum. These cultures exhibited relatively low but significant levels of cellular proliferation as measured by uptake of radioactive thymidine ([3H]TdR). Treatment of spleen cells with monoclonal anti-Thy 1.2 antibody and complement before culture, the addition of specific anti-I-A monoclonal antibodies to the cultures or removal of Ia+ adherent cells before initiation of culture all inhibited the proliferative response significantly. Thus, the autologous proliferation of untreated and unfractionated spleen cells manifests the main characteristics of the AMLR/SMLR, namely, its dependence on T (responder) and Ia+ (stimulator) cells and specific inhibition by anti-I-A antibodies. A marked augmentation in cellular proliferation was observed in unfractionated spleen cell cultures treated for the initial 24 hr of culture with 5 X 10(-6) M indomethacin, an inhibitor of prostaglandin synthesis. Conversely, the addition of 7 X 10(-9) M prostaglandin E1 (PGE1) to these cultures depressed cellular proliferation. This suppression of autologous splenic cell proliferation induced by PGE1 could be partially reversed by the addition of concanavalin A-induced lymphokine (LK) preparations early in the culture. These findings indicate that (a) the proliferation of unfractionated spleen cell cultures occurring in the absence of exogenous stimulatory signals is due largely to an ongoing AMLR, and (b) biologically active mediators with opposing influences, namely, prostaglandins and immunostimulatory LK, participate in the regulation of the AMLR.     

Characterization of interleukin 2-dependent cytotoxic T-cell clones. IV. Production of alpha, beta and gamma interferons and interleukin 2 by Lyt-2+ T cells

The production of alpha, beta and gamma interferons (IFN) and interleukin 2 (IL-2) by Lyt-2+-dependent cytotoxic T-cell lines/clones was investigated. Cloned and uncloned T-cell lines specific for H-2Dd or the unique RL male 1 leukemia antigen were studied. After infection with Sendai virus (SV) or Newcastle disease virus (NDV) all cell lines produced IFN-alpha and -beta. Induction of IFN-gamma was attempted with the mitogens Con A, PHA, PWM, SEA, and SEB, with poly(I:C), with antibodies Lyt-1.2, -2.2, and Thy-1.2, or with the target cells Meth A (H-2Dd+) and RL male 1. All mitogens were effective inducers. However, the antibodies and poly(I:C) were not. One uncloned RL male 1-specific cell line CTLL-RP, produced IFN-gamma after induction with RL male 1. Production of IFN-alpha, beta depended on IL-2, whereas production of IFN-gamma did not, although addition of highly purified IL-2 increased IFN-gamma production even in the absence of other inducers. Crude IL-2 inhibited the production of IFN-gamma but not IFN-alpha, beta. In response to mitogens, some T-cell clones also produced IL-2. The results demonstrate that Lyt-2+ cells can produce a broad spectrum of lymphokine activities after appropriate stimulation. Their availability now affords us the opportunity to study the regulation of lymphokine production at the clonal level.     

NADPH-cytochrome c reductase in outer membrane of kidney mitochondria. Purification and properties.

NADPH-cytochrome c reductase of vitamin D₃-deficient chick kidney mitochondria has been purified approximately 1100-fold to a specific activity of 788 nmol cytochrome c reduced/min/mg protein. Analytical gel electrophoresis of the purified enzyme revealed two bands when stained for protein, but only the more anodic band demonstrated NADPH-tetrazolium reductase activity. The relative ease of solubilization of the reductase without the use of lipases, proteases, or detergents was the first line of evidence that suggested a novel mitochondrial localization for this previously unreported NADPH-linked cytochrome c reductase. The apparent properties of the reductase suggest that the enzyme is a component of kidney mitochondrial outer membrane. The kinetic determination of Michaelis constants with respect to NADPH, cytochrome c, and NADH gave the following values: K_mNADPH = 1.7 μM, K_mcytc = 3.4 μM, and K_mNADH = 20 mM. These constants were different from those of the intact kidney microsomal reductase: K_mNADPH = 5.5 μM, K_mcytc = 10.5 μM, and K_mNADH = 13.3 μM. The mitochondrial as well as the intact microsomal reductases exhibited a ping-pong kinetic mechanism for NADPH-mediated cytochrome c reduction. Spectrofluorometric measurements demonstrated the presence of equimolar amounts of FAD and FMN. The oxidized enzyme has absorption maxima at 280 and 450 nm with a shoulder at 415 nm. Upon reduction with NADPH a distinct loss in the 450-nm absorption was observed. Ouchterlony immunodiffusion studies with rabbit antiserum to chick renal mitochondrial ferredoxin did not reveal cross-reactivity when the purified reductase was the antigen. This excludes the involvement of a ferredoxin-type iron-sulfur protein in the NADPH-mediated reduction of cytochrome c by the purified reductase.     

In vitro correction of erythrocyte glucose 6-phosphate dehydrogenase (G6PD) deficiency.

The permeability characteristics of egg phosphatidylcholine (EPC) vesicles to Eu³⁺ has been examined by ³¹P-nmr, in various mixed lipid systems. It has been found that incorporation of small amounts of sodium taurocholate (NaTC) in the EPC vesicle greatly increased the vesicular permeability to Eu³⁺. Incorporation of cholesterol in the EPC vesicle significantly inhibits the ability of NaTC to induce permeability alterations in this mixed system. It has been reported that at low EPC:NaTC ratios (2:1-0.65:1), mixed micelles of the components are formed [N. A. Mazer, R. F. Kwasnick, M. C. Carey, and G. B. Benedek, 1977, in Micellization, Solubization, and Microemulsions (Mittal, K. L., ed.) Vol. 1, pp. 483-402, Plenum Press, New York]. By examination of the ³¹P-nmr linewidths of EPC, at various ratios of EPC:NaTC, it is possible to follow the decrease in size of the EPC vesicle, as it becomes incorporated into the smaller NaTC micelles. The ³¹P{¹H} nuclear Overhauser enhancement of the simple EPC vesicle is not significantly different from this same parameter measured for EPC, when it exists in mixed micellar systems with NaTC. This indicates that there must be considerable internuclear head group interactions of EPC molecules in EPC-NaTC mixed micelles. This argues for sequestering of EPC in such micelles.     

Formation and stability of the complex formed between human antithrombin-III and thrombin

The inhibition of thrombin by antithrombin-III involves formation of a 1:1 covalent complex between protease and inhibitor and concomitant cleavage of the antithrombin-III peptide chain after Arg-385. The resultant fragment remains connected to the complex via a disulfide bond. This complex spontaneously breaks down into a fragment of approximately 55,000 daltons and smaller peptides. Breakdown is prevented by the presence of hydroxylamine or diisopropylflurophosphate, or by denaturation with urea. It occurs even if the purified complex is treated with diisopropylflurophosphate prior to purification, and can be greatly accelerated by the presence of small amounts of active thrombin. The initial sites of proteolytic attack on the complex are after Arg-13 of the thrombin A chain and Arg-68 of the thrombin B chain. These data indicate that active thrombin can be released from the antithrombin-thrombin complex, and that thrombin becomes more susceptible to proteolytic attack when complexed with antithrombin.     

Endo-(1→4)-β-d-glucanases from sclerotium rolfsii. Purification,substrate specificity,and mode of action

Three purified endo-(1→4)-β-d-glucanases (EC 3.2.1.4), A, B, and C, from Sclerotium rolfsii culture filtrates showed homogeneity in disc-gel electrophoresis and in analytical isoelectric-focusing in polyacrylamide gel. The three endo-d-glucanases are glycoproteins, endo B and endo C being composed of a single polypeptide chain, and endo A of two dissimilar polypeptide chains that are covalently bound by a disulfide bridge. Endo B and endo C do not contain half-cystine residues. With carboxymethylcellulose as substrate, the liquifying activity of the three enzymes was inhibited by cellobiose but not by d-glucose. The specificity of the enzymes is restricted to β-(1→4) linkages, but they showed some differences in the mode of attack on cellodextrins, phosphoric acid-swollen cellulose, and lichenan to give cellobiose, cellotriose, and small proportions of d-glucose. Endo B in addition showed endo-d-xylanase activity.     

Comparison of cytochromes P-450 with high activity toward benzo[a]pyrene purified from liver microsomes of beta-naphthoflavone and 3-methylcholanthrene-pretreated rats

Cytochromes P-450 with high activity toward benzo[a]pyrene were isolated from liver microsomes of rats treated with either β-naphthoflavone or 3-methylcholanthrene and examined for similarity using several physical and catalytic criteria. The β-naphthofla-vone-inducible cytochrome P-446 and the 3-methylcholanthrene-inducible cytochrome P-448 have the same subunit molecular weight (56,000 ± 1000) and electrophoretic mobility. Antibodies prepared to either form cross-react with each form without spurring in Ouchterlony double-diffusion experiments suggesting immunochemical identity. After proteolytic digestion with Staphylococcus aureus SV-8 protease and electrophoresis, both Cytochromes P-450 show the presence of the same bands. Both cytochromes have the same absorption maximum (446.5 ± 0.5 nm) in the CO-reduced absolute spectrum. The catalytic activity toward benzo[a]pyrene of cytochrome P-446 is somewhat greater than that of cytochrome P-448. Thus, all the physical evidence suggests identity of the two cytochromes. The significance of the difference in catalytic activity remains to be defined.     

Localization of the disulfide bond in human antithrombin III required for heparin-accelerated thrombin inactivation

Heparin accelerates the rate of inhibition of thrombin by antithrombin III. Reduction of one of the three antithrombin disulfide bonds with dithiothreitol under mild conditions abolishes this rate-enhancing effect without affecting the rate of reaction in the absence of heparin. Alkylation of mildly reduced antithrombin III with [³H]iodacetic acid followed by digestion with cyanogen bromide yielded two major labeled peptides. The smaller peptide, containing Cys-422, was identified as extending from Gly-414 to the C-terminus, Lys-424. Our data are consistent with the larger labeled peptide being the one extending from Glu-104 to Met-243 and containing Cys-239. Cys-422 has been shown by others to be linked to Cys-239. These data indicate that the sensitive disulfide bond in antithrombin III extends between Cys-239 and Cys-422; the site at which thrombin cleaves the antithrombin III is between these two half-cystines.     

Purification of two forms of kanamycin acetyltransferase from Escherichia coli

Kanamycin acetyltransferase acylates aminoglycoside antibiotics using acetyl-CoA, and thereby conveys bacterial resistance to several clinically important antibiotics, notably amikacin. The enzyme was quantitatively and reproducibly released from Escherichia coli W677 harboring plasmid pMH67 by a modified osmotic shock procedure (bacterial cells are incubated overnight in sucrose and again without sucrose before onset of osmotic shock). The enzyme was purified by dye-ligand chromatography on Affi-Gel Blue in addition to antibiotic affinity chromatography on neomycin-Sepharose-4B. The activity did not increase with subsequent chromatography on ion-exchange, hydrophobic, or molecular-exclusion gels. However, both dye-ligand and molecular-exclusion chromatography, as well as disc-gel electrophoresis, separated the purified enzyme equally into two active protein fractions. Based on the more active of the two forms, the purification was 112-fold with a specific activity of 1.9 IU/mg. The less-active form has an unusual absorbance spectrum, with a maximum near 255 nm, which cannot be explained by the amino acid composition. Chromatography of this form alone regenerated both forms, suggesting that the enzyme is noncovalently conjugated to an uncharged chromophore, such as a lipid. The purified enzyme has a very sharp pH optimum at 5.5 with a plateau on the alkaline side, but is most stable between pH 8.5 and 9.5. Data from electrophoresis in the presence of sodium dodecyl sulfate and gel-filtration on Ultrogel AcA 44 are consistent with a tetrameric protein of 60-70,000 Da.     

Colorimetric determination of acetaldehyde in the presence of formaldehyde

A procedure is described that enables use of the p-phenylphenol color reaction to determine acetaldehyde in the presence of formaldehyde. The sample is first treated with an acidic 2,4-pentanedione reagent, which selectively removes formaldehyde. The method is applicable to blochemical reactions using tissue preparations.     

Collagen lysyl hydroxylation occurs within the cisternae of the rough endoplasmic reticulum.

Resolution of the heavy microsomal fraction of lung tissue by Ficoll density gradient centrifugation yielded a rough endoplasmic reticulum microsomal fraction containing the highest specific activity of detergent-released lysyl hydroxylase. This same microsomal fraction was previously shown to contain the highest specific activity of detergent-released prolyl hydroxylase activity. When hydroxylation was inhibited during the biosynthesis of collagen, this microsomal fraction contained lysine-rich, hydroxylysine-deficient, collagenase-digestible substrate that could be hydroxylated in the absence of detergent. The results indicate coordinate localization of both prolyl and lysyl hydroxylation reactions within the cisternae of the rough endoplasmic reticulum.     

Substrate specificity of Gaucher spleen phosphoprotein phosphatase

The spleen in Gaucher's disease contains elevated levels of two distinct acid phosphatases. One of the isoenzymes, a tartrate-resistant type 5 acid phosphatase which we have designated SP_II acid phosphatase, possesses considerable phosphoprotein phosphatase activity. The enzyme dephosphorylates phosvitin and casein at specific rates (V) of 38.6 and 45.0 units/mg, respectively. The dephosphorylation of the oligophosphoproteins as well as various fragments of phosvitin, histories, and monophosphopeptides was studied kinetically. Positive cooperativity (Hill coefficient = 1.3–2.0) was observed for the dephosphorylation of phosvitin and casein as well as for the dephosphorylation of fragments of phosvitin which contained as few as two vicinal phosphoserine residues. In contrast, the hydrolysis of phosphomonoesters such as o-phosphorylserine or various monophosphopeptides exhibited typical Michaelis-Menten kinetics. Cooperativity appears to depend upon the substrate rather than the enzyme. The cooperativity of dephosphorylation was not affected by altering the secondary structure of phosvitin from a random to β conformation or by acetylation of the protein; however, acetylated phosvitin was dephosphorylated more rapidly (V = 50.8 units/mg) than native phosvitin indicating that the very basic phosphatase enzyme (pI = 8.5) prefers more acidic phosphoproteins as substrates rather than basic proteins such as histone (V= 0.0013 unit/mg). A monophosphohexa-peptide (V = 0.47 unit/mg) and monophosphoheptapeptide (V = 0.18 unit/mg) proved to be much poorer substrates than phosvitin, and monophosphoproteins such as glycogen phosphorylase, phosphorylase kinase, and glycogen synthase were not dephosphorylated by the enzyme. Although the phosphatase is active on monophosphopeptides and the presence of flanking amino acids considerably decreases the K_m of the enzyme for the phosphoserine residue (up to 100-fold), the enzyme appears to prefer peptide or protein substrates that contain two or more phosphoserine residues in close proximity. Finally, previous results showing the spleen phosphatase to be composed of 16,000- and 20,000-dalton subunits were apparently due to proteolysis during isolation since when 1.0 mm phenylmethylsulfonyl fluoride was included in the isolation media, the enzyme appeared as a single 35,000-dalton species when subjected to polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate.