The synthesis and N.M.R. spectroscopy of derivatives of 6-amino-6-deoxy-D-galactose-6-15N

Derivatives of 6-amino-6-deoxy-D-galactose-6-¹⁵N have been synthesized by reaction of the 6-deoxy-6-iodo (1) or 6-O-p-tolylsulfonyl derivative of 1,2:3,4-di-O-isopropylidene-α-D-galactopyranose with potassium phthalimide-¹⁵N. The reaction of 1 also yielded an elimination product, 6-deoxy-1,2:3,4-di-O-isopropylidene-β-L-arabino-hex-5-enopyranose. The structures of the 6-amino-6-deoxy-D-galactose derivatives and their precursors were characterized by proton- and ¹³C-n.m.r. spectroscopy, with confirmation of the ¹³C assignments by selective proton decoupling. Selective broadening of the C-1, C-4, C-5, and C-6 resonances of 6-amino-6-deoxy-1,2:3,4-di-O-isopropylidene-α-D-galactopyranose by low concentrations of cupric ion was observed, and studied by computerized measurements of the ¹³C linewidths. The application of this broadening to ¹³C-spectral assignments of amino sugar derivatives is indicated.     

High-performance liquid chromatography of N,O-acylated sialic acids

A rapid, isocratic high-performance liquid chromatographic method for the analysis of N-acetylneuraminic acid, N-glycolylneuraminic acid, and their O-acetylated derivatives is described. Separation of sialic acids and of other monosaccharides as sugar-borate complexes is achieved on an anion-exchange resin. The sialic acids elute as individual peaks after the other sugars tested. The method allows quantitative determination, for example, of amounts of N-acetylneuraminic acid as small as 10 nmol. On cation-exchange resin sialic acids cannot be differentiated, but can be separated from neutral and amino sugars, allowing the determination of as little as 3 nmol of total sialic acids.     

Periodate oxidation studies in the elucidation of the structures of sialic acid containing oligosaccharides

The structures of sialo-oligosaccharide alditols as determined by ¹H-NMR spectrometry together with methylation analysis did not correspond with those derived previously from quantitative periodate oxidation data alone. Possible causes of the discrepancy were explored in the periodate oxidation methodology. No free sialic acid was released by the acidity of the periodate in the course of oxidation at pH 4.5. The anionic properties of the sialic acid residues were therefore utilized to separate the periodate oxidation products and thereby establish the position of the sialic acid in the oligosaccharide chain.     

Arachidonic acid epoxidation: epoxyeicosatrienoic acids are endogenous constituents of rat liver

Epoxyeicosatrienoic acids have been isolated and purified from the livers of male rats. They were identified by gas chromatography-mass spectrometric techniques. These results expand the list of in vivo-produced eicosanoids. Their documented in vitro biological activities suggest a role for them in cell and tissue homeostasis.     

Analysis of the 220-MHz,P.M.R. spectra of some products of the amadori and heyns rearrangements

In order to establish whether p.m.r. spectroscopy is useful for identifying Amadori- and Heyns-rearrangement products, the p.m.r. spectra at 220 MHz of 16 rearrangement products derived from d-glucose or d-fructose and amino acids have been investigated. At pH 3, the protons of the NCH₂ group of N-substituted 1-amino-1-deoxy-d-fructose (Amadori-rearrangement products) resonate at δ 3.25–3.60 in D₂O and are shifted upfield by 0.3–0.6 p.p.m. at pH 9. These protons exchange with deuterium. Also, in D₂O there is an equilibrium of the acyclic, furanose, and pyranose structures, the last being favoured. At pH ? 7, the equilibrium is completely shifted to the β-pyranose form, which adopts exclusively the ²C₅ conformation. At pH 3, the equilibrium favours the β-furanose form. At pH 3, H-1e and H-1a of N-substituted 2-amino-2-deoxy-d-glucoses (Heyns-rearrangement products) resonate at δ 5.55 and 5.04, respectively. At pH 9, the signal for H-2 is shifted upfield by 0.2–0.7 p.p.m. In D₂O solution, these compounds exist as an equilibrium of α- and β-pyranose forms in the ⁴C₁ conformation. The α anomer is stabilised by the amino acid group at position 2. At pH 3, the αβ-ratio is 2–4:1, and, at pH 9, 1.0–1.1:1.     

Conformational studies on pertrimethylsilyl derivatives of some 6-deoxyaldohexopyranoses and of four less-common aldohexopyranoses by 220-MHz P.M.R. spectroscopy. Substituent and configurational effects on the chemical shifts of the ring protons

The complete interpretation of 220-MHz p.m.r. spectra and the accurate chemical shifts and coupling constants, obtained after computer simulation of the spectra, of the per-O-trimethylsilyl (Me₃Si) derivatives of a number of 6-deoxy-aldohexopyranoses and of β-D-altro-, β-D-allo-, and α- and β-D-talo-pyranose are given. By means of an adapted Karplus equation, the structure of the derivatives has been studied in detail. All of the pyranoid rings occur in the ⁴C₁(D) or ¹C₄(L) chair conformation. The preferred conformation of the C-5—CH₂OSiMe₃ group in the four aldohexopyranoses was found to be dependent on the configuration at C-4. By comparison of Me₃Si-aldohexopyranoses with the corresponding 6-deoxy analogues, it was found that the 6-OSiMe₃ group has no marked effect on the conformation of thering. The influence of this group on the chemical shifts of the ring protons is discussed in terms of electric field and inductive effects. Rules are presented for the estimation of the chemical shifts of the ring protons of Me₃Si-aldohexopyranoses and Me₃Si-6-deoxyaldohexopyranoses.     

The binding of antifibrinolytic amino acids to kringle-4-containing fragments of plasminogen

A monoclonal antibody to human plasminogen, 10-F-1, was found to interact with the lysine-binding site (LBS) on the kringle 4 (K 4) region of the molecule. This observation has been employed to measure the binding of various antifibrinolytic amino acid analogs of ?-aminocaproic acid (?ACA) to its site on K 4 in appropriate elastolytic-derived fragments of human plasminogen and to other species of plasminogen to which antibody 10-F-1 cross-reacts. By analysis of the concentration dependence of ?ACA displacement of [¹²⁵I]10-F-1 from human Glu₁Pg, a K_D for ?ACA of 7.1 ± 1.0 mm was calculated. Similar experiments with K 4-containing fragments of Glu₁Pg, viz., Lys₇₇Pg, K 4, Lys₇₇H and Val₃₅₄Pg, yielded K_D values of 6.6 ± 1.0, 7.5 ± 1.0, 6.6 ± 1.0, and 12.0 ± 2.0 mm, respectively. When baboon, goat, monkey, rabbit, and sheep plasminogens were substituted for human plasminogen, the K_D values calculated ranged from 2.1 to 7.1 mm. The K_D values for several analogs of ?ACA, i.e., 4-aminobutyric acid, 5-aminopentanoic acid, 8-aminooctanoic acid, l-lysine, and trans-aminomethyl cyclohexane-1-carboxylic acid, were measured to the K 4 region of Lys₇₇Pg. The values obtained were 11.3 ± 1.5, 9.0 ± 1.0, 71.0 ± 10, 38.0 ± 5.0, and 1.1 ± 0.4 mm, respectively. Additionally, the K_D of trans-aminomethylcyclohexane-1-carboxylic acid towards the K 4 region of Glu₁Pg, Lys₇₇Pg, and isolated K 4 was found to be 2.4 ± 0.5, 1.1 ± 0.3, and 2.0 ± 0.6 mm, respectively. These studies show directly that the LBS on the K 4 domain of plasminogen represents one of its 4–5 weak binding sites and that this site can be specifically probed with the use of monoclonal antibody 10-F-1. Furthermore, it appears as though this site is conserved in several important proteolytic fragments of plasminogen, providing additional evidence that these fragments exist as independent domains in the native molecule. Finally, this weak LBS on the K 4 domain of human plasminogen is also present in other species of plasminogen.     

A new approach to the characterization of the B and A forms of DNA by I.R. spectroscopy

The RNA conformational changes of B, A and C forms are reflected in the infrared absorption spectra in the region of 800 cm^?1 to 900 cm^?1 and allow one to investigate unoriented samples. The transition to the A form is characterized by the appearence of bands at about 870 cm^?1 and at 813 cm^?1 whereas the B and the C forms exhibit a band at 837 cm^?1, these bands undoubtedly arise from phosphate diester stretching vibrations and yield information about backbone conformation. The presence of these infrared bands provides a criterion for testing the simultaneous presence of two coexisting forms of DNA. It represents a useful method for structural studies of nucleic acid complexes such as protein-DNA for which it is difficult to obtain orientation.     

Affinity labeling of spinach ferredoxin-NADP+ oxidoreductase with periodate-oxidized NADP+

Periodate-oxidized NADP⁺ (dialdehyde-NADP⁺) inactivated soluble ferredoxin-NADP⁺ oxidoreductase and combined covalently to the enzyme. This inactivation was first order with respect to dialdehyde-NADP⁺ and followed saturation kinetics, indicating that the enzyme initially forms a reversible complex with the inactivator. NADP⁺ afforded complete protection against inactivation, while spinach ferredoxin was uneffective. In the presence of exogenous ferredoxin and illuminated thylakoids, the nucleotide analog functioned as a coenzyme for the reductase, although with rather lower efficiency than NADP⁺. It also acted as a competitive inhibitor with respect to NADPH in diaphorase activity. Incorporation of radioactivity from periodate-oxidized [³H]NADP⁺ gave a stoichiometry of 0.85 mol of reagent/mol of reductase, indicating that the modification of a single residue in the flavoprotein is responsible for the loss of enzymatic activity.     

The fluorescence of fluorescamine-amino acids.

Fluorescamine reacts with various amino acids to yield solutions exhibiting different amounts of fluorescence, and the fluorogenic reaction does not go to completion. To investigate these phenomena, we measured the lifetimes and quantum yields of the fluorescamine-amino acids and studied their rates of formation by stopped-flow fluorescence and transmission measurements. The quantum yields were similar (0.11 ± 0.03), as were the lifetimes (3.5 ± 0.5 ns). The only exceptions were the derivatives of tryptophan and cysteine, which were internally quenched. It was concluded that the chemical yields of the fluorescamine-amino acids varied greatly. Kinetic experiments showed wide variations in rates, with some amino acids requiring several seconds for reaction under some conditions. Proline, on the other hand, reacted rapidly, with a second order rate constant of 6.2 × 10^?4 liter mol^?1 s^?1. Sequential additions of fluorescamine to amino acids were more efficient in producing the fluorescent product than the same amount of reagent added all at once; this suggested that fluorescamine was inactivated by a concentration-dependent process. A mechanism to explain the low chemical yields is proposed in which both base and amine catalyze the inactivation of fluorescamine.     

Fractionation of sialoglycoproteins on an immobilized sialic acid-binding lectin

Biochemically important monosaccharides were rapidly separated by automated chromatography of their borate complexes on columns packed with newly developed anion-exchange resins, and sensitively detected by fluorimetric postlabeling with 2-cyanoacetamide. Optimization studies for stepwise, gradient, and single-buffer elutions are described, and the reproducibility of the determination of such monosaccharides is discussed. A few examples of the application of this rapid, sensitive automated analysis are also presented.     

Human autologous rosette-forming cells: II. Specificity of the binding between lymphocytes and erythrocytes

The relationship between autorosettes and allorosettes was investigated using a mixed rosette assay in which the origin of the erythrocytes was assessed by the fluorescein isothiocyanate (FITC) labeling of one type of erythrocyte. The data show that auto- and allorosettes belong to the same T-cell subset: (1) in most of the subjects, the percentages of T cells binding autologous red blood cells (auto-RBC) are equivalent to those binding allogeneic RBC (allo-RBC); (2) the percentage of rosettes formed after the simultaneous addition of auto- and allo-RBC is similar to that of autorosettes alone or allorosettes alone; and (3) nearly 80% of the resetting cells bind both types of RBC as directly visualized in the mixed rosette assay. The experiments in which the lymphocytes are resetted first with one type of RBC, and then with the other type support the finding that auto- and allo-RBC may bind to the lymphocytes through a single receptor which exhibits a varying affinity for RBC according to their origin.     

Simultaneous determination of [2-15N]- and [5-15N]glutamine with gas chromatography-mass spectroscopy: applications to nitrogen metabolic studies

A gas chromatographic-mass spectrometric method for analysis of L-[2-15N]- and L-[5-15N]glutamine is described. The method is based on direct acylation of glutamine with trifluoroacetic anhydride and the formation of the N,N-bis-trifluoroacetyl-L-glutamine derivative. This simple and sensitive method is capable of detecting approximately 0.5 atom% excess 15N in as little as 10 microliter of plasma with a mean coefficient of variance of 11.6%. The method was applied to determine the appearance of 15N enrichment in plasma amino-N and amide-N of glutamine in a healthy adult volunteer during a constant infusion of 15NH4Cl. A plateau level of 3.7 and 2.6 atom% excess was observed in amide-N and amino-N, respectively, at 1 and 2 h after 15NH4Cl infusion was started.     

Differences in sialic acid contents of low cancer cells,high cancer cells and normal mouse lung counterparts

Sialic acid contents of low cancer (P 4 BIS) high cancer (P 4 BIS T) cells and their normal (PB) mouse lung counterparts have been determined. This content is 5 to 10 fold higher for cells in logarithmic phase growth than for confluent cells, as well for normal cells as for transformed derived cells lines. Growing normal PB cells contain a large amount of sialic acid (21.2 μg/10⁶ cells): it is reported that cellular sialic acid content decreases dramatically with the tumor producing capacities of the cells (3.4 μg/10⁶ P 4 BIS cells; 2.1 μg/10⁶ P 4 BIS T cells).It has been found in conditions which maintain cell viability that transformed neuraminidase treated cells or trypsin treated cells liberate large percentages of sialic acid, or sialoglycoproteins, whereas small percentages are liberated from normal cells, indicating that transformed cell surface glycoproteins may be reached more easy by enzymes that normal cells: in that aspect low cancer cells (P 4 BIS) appear transitory between normal (PB) and high cancer cells (P 4 BIS T) in the same way they are transitory in tumor producing capacities.     

Identification of novel calcium binding proteins of heart and brain 100,000 x g supernatant

The chelex competitive calcium binding assay has been used to assay the calcium binding activity of the 100,000 X g supernatant of bovine heart and brain. Chromatography of brain 100,000 X g supernatant on diethylamino-ethyl (DEAE) cellulose reveals the presence of two peaks of calcium binding activity, peak I eluting at about 0.05 M NaCl and peak II at about 0.18 M NaCl. Chromatography of peak I on Sephadex G-150 resolves a major and a minor peak of calcium binding activity, at Mr 40,000 and Mr 150,000. Chromatography of peak II (0.18 M NaCl) on Sepharose 6B produces two peaks of calcium binding activity, a broad peak of calcium binding activity composed of two molecular weight species of Mr 230,000 and Mr 420,000, and a sharp peak of calcium binding activity with Mr 75,000. Chromatography of the 100,000 X g supernatant of bovine heart on DEAE Cellulose reveals two peaks of calcium binding activity. Chromatography of the lower ionic strength peak on Sephadex G-150 resolved major and minor peaks of calcium binding activity at Mr 65,000 and 150,000, respectively. The results of this study suggest the presence of several calcium binding proteins, other than calmodulin, in these tissues.     

13C-N.M.R.-spectral study of the pH behavior of reductively [13C]methylated,glycophorin a glyco-octapeptides and a related glycopentapeptide

The pH dependence of the labeled-carbon resonances of reductively [¹³C] methylated compounds tri-l-Ser, glyco-octapeptide A^M, asialoglyco-octapeptide A^M, glyco-octapeptide A^N, asialoglyco-octapeptide A^N, and a glycopentapeptide was investigated. The results are discussed relative to those previously observed for reductively [¹³C]methylated, intact glycophorins A^M and A^N, and in terms of the mode of display of the MN blood-group specificities by these related glycoproteins. The results indicated that the α-d-NeuAc groups appear to affect the pH-titration results of glyco-octapeptides A^M and A^N. Moreover, comparison of the pH-titration results for reductively [¹³C]methylated glyco-octapeptide A^M and reductively [¹³C]methylated asialoglyco-octapeptide A^M with those of a reductively [¹³C]methylated glycopentapeptide and reductively [¹³C]methylated tri-l-Ser indicated that the other carbohydrate residues present (α-d-GalNAc and β-d-Gal) may also affect the pH-titration results. The reductive-methylation modification appears to affect the chemical shifts of the carbohydrate and peptide carbon atoms of the glycopentapeptide minimally.     

Positive cooperativity in binding by albumin: The system bovine serum albumin and alizarin yellow G cobinding by salicylic acid

The binding of alizarin yellow G—an azo derivative of salicylic acid—by bovine serum albumin has been investigated using the method of equilibrium dialysis. Six strong and a number of additional, weak binding sites have been found to be present. The system is characterized by strong positive cooperativity between the first and second sites. Six binding constants have been determined on the basis of a simplified mathematical model. The results are ~2 × 10⁴m^?1 for the first binding site, 6 × 10⁵m^?1 for the second, and between 4 × 10⁴ and 10⁵m^?1 for the rest. The phenomenon is discussed in terms of the existence of various conformers or of the conformational adaptability of albumin. Cobinding by salicylic acid does not displace alizarin yellow G but induces a conformational change in the protein which affects the absorption spectrum of the bound dye. As expected for this kind of heterotropic interaction, the spectrum of the system albumin-salicylic acid is similarly affected by the cobinding of alizarin yellow G.