Magnetic resonance study of 13C reductively methylated glycophorin and glycophorin glycopeptides

¹³C nuclear magnetic resonance (n.m.r.) spectral data for ¹³C reductively methylated N-terminal tryptic glycopeptides and for ¹³C reductively methylated N-terminal glyco-octapeptides derived from homozygous glycophorins A^M and A^N are presented. Their ¹³C chemical shift data are compared with the previously published ¹³C n.m.r. data for ¹³C reductively methylated homozygous glycophorins A^M and A^N in order to investigate the means of display of the MN blood determinants by these species. The pH dependence of the ¹³C resonances of $\text{N}{}^{\mathrm{\alpha }}\text{,N-[}{}^{13}\text{C}\text{]}\text{dimethyl}$ leucine of glyco-octapeptide A^N and of $\text{N}{}^{\mathrm{\alpha }}\text{,N-[}{}^{13}\text{C}\text{]}\text{dimethyl}$ serine of glyco-octapepti A^M indicated that only a slight structural perturbation occurs at the N-terminus when a large portion of the glycoprotein molecule is removed. However, one structural ‘state’ of ¹³C reductively methylated glycophorin A^M is lost when the glyco-octapeptide A^M is produced. The ¹³C resonance of $\text{N}{}^{\mathrm{\alpha }}\text{,N-[}{}^{13}\text{C}\text{]}\text{dimethyl}$ leucine of glycooctapeptide A^N titrated with a $\text{p}\text{K}{}_{\mathrm{<mtext>a</mtext>}}$ of 7.7 (Hill coefficient $\text{～ 1}$). The ¹³C resonance of $\text{N}{}^{\mathrm{\alpha }}\text{,N-[}{}^{13}\text{C}\text{]}\text{dimethyl}$ serine, on the other hand, exhibited an unusual pH dependence, indicating the existence of some possible steric constraints or hydrogen bonding in this molecule. In comparison to the data obtained for ¹³C-labelled glycooctapeptide A^M molecule, the pH dependence of the chemical shift of the ¹³C resonance of $\text{N}{}^{\mathrm{\alpha }}\text{,N-[}{}^{13}\text{C}\text{]}\text{dimethyl}$ serine of tripeptide tri-L-serine is also presented. Circular dichroism (c.d.) spectra indicated that the reductive methylation technique does not cause a large perturbation of the glycophorin A molecule.     

Structural studies of the M blood group determinant

Structural studies of homozygous glycophorin A^M were undertaken by monitoring the ¹³C methyl resonances of ¹³C reductively methylated glycophorin A^M (contains five $\text{N}{}^{\mathrm{?}}\text{,N-[}{}^{13}\text{C}\text{]}\text{dimethyl}$ Lys residues, and the N-terminal $\text{N}{}^{\mathrm{\alpha }}\text{,N-[}{}^{13}\text{C]}\text{dimethyl}$ Ser residues) in various forms of glycosylation. The results indicate that removal of the α-d-NeuAc residues does not affect the structure about the N-terminal Ser residue. However, removal of the fifteen O-linked oligosaccharide units results in a structural effect about the N-terminal Ser residue. Partial methylation experiments performed on native glycophorin A^M and deglycosylated glycophorin A^M indicate that methylation of the lysine residue(s) may influence the structure about the N-terminal Ser residue, especially in the case of deglycosylated A^M.     

pH titration studies of 13C reductively methylated N-terminal serine of glycophorin AM

The ¹³C resonances of N^α,N-[¹³C]dimethylserine of partially ¹³C reductively methylated glycophorin A^M were monitored as a function of pH at 45°C. For comparison, limited data are also presented for the pH dependence of the ¹³C resonances of N^α,N- [¹³C]dimethylserine of fully ¹³C reductively methylated deglycosylated glycophorin A^M. The ‘major’ component of N^α,N- [¹³C]dimethylserine of glycophorin A^M did not titrate, whereas the ‘minor’ component titrated with a pK_a of 7.80 (Hill coefficient of 0.95). Similar results are also indicated for the N^α,N- [¹³C]dimethylserine resonances of ¹³C reductively methylated deglycosylated glycophorin A^M.     

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.     

C n.m.r. study of the pH behaviour of N-methylated peptides related to the N-terminus of glycophorins

¹³C nuclear magnetic resonance spectroscopy (¹³C n.m.r.) was used to determine the pH titration parameters for the N-terminal N,N-[¹³C]dimethylamino and N,N-[¹³C]monomethylamino groups of glycophorins A^M and A^N, and some 28 related glycoproteins, glycopeptides and peptides. The results show that glycosylation of the Ser and Thr residues at positions 2, 3 and 4 of the glycophorins have a pronounced effect on the titration parameters. Substitution of amino acids 4 and 5 in the glycophorin sequence appears to minimally affect our titration parameters. Internal hydrogen-bonding involving the N-terminal Ser residue may explain some of the unusual pH titration results observed for glycophorin A^M.     

13C-nuclear magnetic resonance study of glycophorins AM and AN modified with various pyrylium salts

The environment of the N-terminal amino groups of glycophorins A^M and A^N has been studied using¹³C-NMR spectroscopy and pyrylium salts as amino-blocking agents. The extent of amino blocking was monitored by¹³C-reductive methylation of the residual free amino groups. The pyrylium ions reacted with the N-terminal amino groups of the two glycophorins at almost identical rates, which is thought to indicate that the overriding steric bulk of the pyrylium salt may determine the rate of the reaction. The difference in the rates of modification of lysine residues of glycophorins A^M and A^N by the pyrylium ions did indicate that there may exist an environmental difference around the lysine residues between the two glycophorins. This environmental difference may result from solution aggregation of the glycophorin A molecules or from some differences in the pK_a values of the five lysine residues found in glycophorins A^M and A^N.     

13C-nuclear magnetic resonance study of glycophorins AM and AN modified with various pyrylium salts

The environment of the N-terminal amino groups of glycophorins A^M and A^N has been studied using¹³C-NMR spectroscopy and pyrylium salts as amino-blocking agents. The extent of amino blocking was monitored by¹³C-reductive methylation of the residual free amino groups. The pyrylium ions reacted with the N-terminal amino groups of the two glycophorins at almost identical rates, which is thought to indicate that the overriding steric bulk of the pyrylium salt may determine the rate of the reaction. The difference in the rates of modification of lysine residues of glycophorins A^M and A^N by the pyrylium ions did indicate that there may exist an environmental difference around the lysine residues between the two glycophorins. This environmental difference may result from solution aggregation of the glycophorin A molecules or from some differences in the pK_a values of the five lysine residues found in glycophorins A^M and A^N.     

Induction of a relatively fast transbilayer movement of phosphatidylcholine in vesicles. A 13C NMR study

[$\text{N-}{}^{13}\text{CH}{}_3$] Phosphatidylcholines are introduced into the outer monolayer of phosphatidylcholine vesicles with the phosphatidylcholine exchange protein from bovine liver. The transbilayer distribution of the [$\text{N-}{}^{13}\text{CH}{}_3$] phosphatidylcholine is measured with ¹³C NMR. The transbilayer movements of $\text{[N-}{}^{13}\text{CH}{}_3\text{]-}\text{dioleoyl}$ phosphatidylcholine and [$\text{N-}{}^{13}\text{CH}{}_3$] dimyristoyl phosphatidylcholine at 30°C in vesicles composed of these phosphatidylcholines are extremely slow processes with estimated half-times of days. [$\text{N-}{}^{13}\text{CH}{}_3$] Dioleoyl phosphatidylcholine introduced into dimyristoyl phosphatidylcholine vesicles migrates from the outer to the inner monolayer with a half-time of less than 12 h. The data suggest that differential changes in the lateral packing of the two monolayers might be a driving force for transbilayer transport of phospholipids.     

Identification and partial characterization of plasma membrane polypeptides of Trypanosoma brucei

A plasma membrane-enriched vesicle fraction has been prepared from Trypanosoma brucei by sonication and differential centrifugation on sucrose gradients. This fraction is enriched 5-fold in the plasma membrane marker enzymes adenyl cyclase (EC 4.6.1.1) and ouabain-inhibitable, ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}$)-dependent adenosine triphosphatase (EC 3.6.1.3). It is also enriched up to 14-fold in iodinated surface proteins, and up to 4-fold in [³H]mannose-labeled glycoproteins, of which the major variable surface coat glycoprotein is the main constituent. Proteins of the plasma membrane fraction and other subcellular fractions have been identified by electrophoretic analysis in sodium dodecyl sulfate-polyacrylamide gradient slab gels. Several high molecular weight surface glycopeptides have been selectively investigated and partially characterized by a combination of metabolic labeling with [³H]mannose, lactoperoxidase-catalyzed surface iodination, and affinity chromatography on Con A-Sepharose. In addition to the major variable surface coat glycoprotein (estimated $\text{M}{}_{\mathrm{<mtext>r</mtext>}}\text{= 58 000}$), there are several minor surface glycopeptides ($\text{M}{}_{\mathrm{<mtext>r</mtext>}}\text{= 76 000, 86 000}\text{and}\text{92 000–100 000}$) which are apparent extrinsic membrane components, and two surface glycopeptides ($\text{M}{}_{\mathrm{<mtext>r</mtext>}}\text{= 42 000}\text{and}\text{130 000}$) which are intrinsic membrane components.     

Magnetic resonance study of 13C-enriched glycophorin A reconstituted into phospholipid vesicles

$\text{[S-[}{}^{13}\text{C}\text{]}\text{methylmethionine-8 and -81]glycophorin}$ A was reconstituted into l-α-phosphatidyl choline vesicles. Results indicate that the $\text{S-[}{}^{13}\text{C}\text{]}\text{methylmethyionine}\text{-81}$ residue in the phospholipid bilayer has limited mobility and is not susceptible to dealkylation, whereas the opposite effects are indicated for the $\text{S-[}{}^{13}\text{C}\text{]}\text{methylmethionine}\text{-8}$ residue.     

C-n.m.r. spectral study of C reductively methylated glycopeptides derived from glycophorin A

¹³C-n.m.r. spectral data for ¹³C reductively methylated intact homozygous and heterozygous glycophorins A were compared with the ¹³C-n.m.r. spectral data for the ¹³C reductively methylated homozygous and heterozygous N-terminal glycopeptides derived from the trypsin digest of glycophorin A. The results indicate that pronounced aggregation of this glycoprotein in solution does not affect the structural differences that we have previously observed for glycophorins A^M and A^N at and/or near the N-terminal amino acid. Moreover, the data suggest that two structural states exist for glycophorin A^M.     

Inhibition of anion transport in the red blood cell by anionic amphiphilic compounds I. Determination of the flufenamate-binding site by proteolytic dissection of the band 3 protein

Flufenamate, a non-steroidal anti-inflammatory drug, is a powerful inhibitor of anion transport in the human erythrocyte ($\text{I}{}_{50}\text{= 6·10}{}^{\mathrm{?7}}\text{M}$). The concentration dependence of the binding to ghosts reveals two saturable components. [¹⁴C]Flufenamate binds with high affinity ($\text{K}{}_{\mathrm{<mtext>d</mtext>}}{}_1\text{= 1.2·10}{}^{\mathrm{?7}}\text{M}$) to 8.5·10⁵ sites per cell (the same value as the number of band 3 protein per cell); it also binds, with lower affinity ($\text{K}{}_{\mathrm{<mtext>d</mtext>}}{}_2\text{= 10}{}^{\mathrm{?4}}\text{M}$) to a second set of sites (4.6·10⁷ per cell). Pretreatment of cells with 4-acetamido-4′-isothiocyanostilbene-2,2′-disulfonic acid (SITS), a specific inhibitor of anion transport, prevents [¹⁴C]flufenamate binding only to high affinity sites. These results suggest that high affinity sites are located on the band 3 protein involved in anion transport. Extracellular chymotrypsin and pronase at low concentration cleave the 95 kDa band 3 into 60 kDa and 35 kDa fragments without affecting either anion transport or [¹⁴C]flufenamate binding. Splitting by trypsin at the inner membrane surface of the 60 kDa chymotryptic fragment into 17 kDa transmembrane fragment and 40 kDa water-soluble fragment does not affect [¹⁴C]flufenamate binding. In contrast degradation at the outer membrane surface of the 35 kDa fragment by high concentration of pronase or papain decreases both anion transport capacity and number of high affinity binding sites for [¹⁴C]flufenamate. Thus it appears that 35 kDa peptide is necessary for both anion transport and binding of the inhibitors and that the binding site is located in the membrane-associated domain of the band 3 protein.     

A simple procedure for resolution of Escherichia coli RNA polymerase holoenzyme from core polymerase.

The association constant, K_A, for myosin subfragment-1 binding to actin was measured as a function of ionic strength [KCl, LiCl, and tetramethylammonium chloride (TMAC)]and temperature by the method of time-resolved fluorescence depolarization. The following thermodynamic values were obtained from solutions of 0.20 × 10^?6m S-1, 1.00 × 10^?6m actin in 0.15 m KCl, pH 7.0, at 25 °C: ΔG ° = ?39 ± 1 kJ M^?1, ΔH⁰ = 44 ± 2 kJ M^?1 and $\text{ΔS}{}^0\text{= 0.28 ± 0.01 kJ}\text{M}{}^{\mathrm{?1}}{}^0\text{K}{}^{\mathrm{?1}}$. For measurements in KCl (0.05 to 0.60 m), In $\text{K}{}_{\mathrm{a}}\text{= ?8.36 (KCl)}{}^{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$. Thus, the binding is endothermic and strongly inhibited by high ionic strength. When KCl was replaced by LiCl or TMAC the ionic effects on the binding were cation specific. The nature of actin-(S-1) binding in the rigor state is discussed in terms of these results.     

Non-sterol metabolism of mevalonate in vitro: artifacts and realities.

Commercial [5-¹⁴C]mevalonate is shown to contain several radioactive impurities, which give artifactually high amounts of Hyamine bound, volatile acidic radioactivity when incubated with killed or living rat renal cortex slices, as compared with [5-¹⁴C]mevalonate purified either by liquid-liquid partition chromatography or through the enzymically generated $\text{R}\text{-5-phospho-[5-}{}^{14}\text{C]mevalonate}$ by ion-exchange chromatography. The artifactual ${}^{14}\text{CO}{}_2$ results were not diluted by incubation with increasing amounts of unlabelled mevalonate, whereas the ${}^{14}\text{CO}{}_2$ and [${}^{14}\text{C}$]cholesterol produced by rat renal cortex slices incubated with purified [5-¹⁴C]mevalonate were both diluted to the same extent by unlabelled mevalonate. It is concluded that $\text{R}\text{[5-}{}^{14}\text{C]mevalonate}$ is genuinely oxidized to ${}^{14}\text{CO}{}_2\text{in}\text{vitro}$, and that purification of substrate before its use is necessary. Production of ${}^{14}\text{CO}{}_2$ and various [${}^{14}\text{C}$]lipids from purified [5-¹⁴C]mevalonate, as a function of time and substrate concentration, by renal cortex and liver slices, is described.     

Dopamine-stimulated cyclic AMP and binding of [3H]dopamine in acini from dog pancreas

Dispersed acini from dog pancreas were used to examine the ability of dopamine to increase cyclic AMP cellular content and the binding of [³H]dopamine. Cyclic AMP accumulation caused by dopamine was detected at 1·10^?8 M and was half-maximal at $\text{7.9±3.4·10}{}^{\mathrm{?7}}\text{M}$. The increase at 1·10^?5 M, (7.5-fold) was equal to the half-maximal increase caused by secretin at 1·10^?9 M. Haloperidol, a dopaminergic receptor antagonist inhibited cyclic AMP accumulation caused by dopamine. The IC₅₀ value for haloperidol, calculated from the inhibition of cyclic AMP increase caused by 1·10^?5 M dopamine was $\text{2.3±0.9·10}{}^{\mathrm{?6}}\text{M}$. Haloperidol did not alter basal or secretin-stimulated cyclic AMP content. [³H]Dopamine binding was studied on the same batch of cells as cyclic AMP accumulation. At 37°C, it was rapid, reversible, saturable and stereospecific. The $\text{K}{}_{\mathrm{<mtext>d</mtext>}}$ value for high affinity binding sites was $\text{0.43±0.1·10}{}^{\mathrm{?7}}\text{M}$ and $\text{4.7±1.6·10}{}^{\mathrm{?7}}\text{M}$ for low affinity binding sites. The concentration of drugs necessary to inhibit specific binding of dopamine by 50% was $\text{1.2±0.4·10}{}^{\mathrm{/t-7}}\text{M}$ noradrenaline, 2·10^/t-7 M epinine, $\text{4.1±1.8·10}{}^{\mathrm{/t-6}}\text{M}$ fluphenazine, $\text{8.0±1.6·10}{}^{\mathrm{/t-6}}\text{M}$ haloperidol, $\text{4.2±1.2·10}{}^{\mathrm{?6}}\text{M}$cis-flupenthixol, $\text{2.7±0.4·10}{}^{\mathrm{?5}}\text{M}$trans-flupenthixol, $\text{>1·10}{}^{\mathrm{?5}}\text{M}$ apomorphine, sulpiride, naloxone and isoproterenol.     

Modulation of human erythrocyte Ca2+/Mg2+ ATPase activity by phospholipase A2 and proteases. A comparison with calmodulin

The human erythrocyte membrane $\text{Ca}{}^{\mathrm{2+}}\text{Mg}{}^{\mathrm{2+}}$ ATPase responded to the presence of an acidic phospholipase A₂ and to low levels of trypsin (and chymotrypsin) in much the same way as it did to calmodulin isolated from human erythrocytes. The increased concentration of ATP hydrolyzed in 1 hour was similar to that observed when calmodulin had been added to a suspension of membranes during the assay. The observations reported here strongly suggest that activation of the $\text{Ca}{}^{\mathrm{2+}}\text{M}{}^{\mathrm{2+}}$ ATPase can proceed by introducing apparently distinct perturbations either to the protein or to phospholipid domains of the erythrocyte membrane.     

pH-Abhängigkeit der 13C-15N-kopplungskonstanten 15N-hochmarkierter aminosäuren,gewonnen aus algenmassenkulturenpH dependence of 13C-15N coupling constants of highly 15N-enriched amino acid isolated from mass cultivation of algae

The alga Ankistrodesmus braunii was grown with [¹⁵N]nitrate under the optimized conditions of a large-scale mass cultivation. 19.7% of the dried algae were isolated as a mixture of amino acids. The ¹⁵N-labelled amino acids (¹⁵N content up to 98%) were separated by ion exchange chromatography using pyridine acetate gradients. The ¹⁵N content of the analytically pure amino acids was determined by combined gas-liquid chromatography-mass spectrometry of the trifluoroacetylated methylesters and by emission spectroscopy in the ¹⁵N analysator. Using pulse Fourier transform ¹³C nuclear magnetic resonance, the pH dependence of the ¹³C-¹⁵N coupling constants of Asp, Pro, Ser, Glu, Gly, Ala, Val, Ile and Leu was determined in aqueous solutions. Increasing coupling constants were found with pH and decreasing electron density, respectively. The relation of Binsch et al. (Binsch, G., Lambert, J.B., Roberts, B.W. and Roberts, J.D. (1964) J. Am. Chem. Soc. 86, 5564–5570) between the coupling constant and the product of the S-part of the ¹³C and ¹⁵N hybridization $\text{S}{}_{\mathrm{C}}\text{·}\text{S}{}_{\mathrm{N}}\text{= 80 · J (}{}^{13}\text{C}\text{-}{}^{15}\text{X}\text{)}$ fits best in acidic medium. The magnitude of the coupling constants correlates well with the electron densities calculated by Del Re et al. (Del Re, G., Pullman, B. and Yonezawa, T. (1963) Biochim. Biophys. Acta 75, 153–182). The recording of ¹³C nuclear magnetic resonance spectra over the entire pH range revealed no change in the sign of the ¹³C-¹⁵N coupling constants of the amino acids.     

Studies on the metabolism of guanidine compounds in mammals

[¹⁴C]Guanidine was observed in the urine after subcutaneous administration to rats of l-[guanidino-¹⁴C]arginine or l-[guanidino-¹⁴C]canavanine. [${}^{14}\text{C}$]Hydroxyguanidine was additionally detected in the urine after injection of dl-[guanidino-¹⁴C]canavanine. These ${}^{14}\text{C}$ metabolites were characterized by high-voltage electrophoresis and paper chromatography, by enzymatic conversion of [¹⁴C]hydroxyguanidine to [¹⁴C]guanidine, and by repeated recrystallization of isolated urinary [${}^{14}\text{C}$]guanidine as the picrate salt with no significant loss of specific activity. These experiments demonstrate that both l-arginine and l-canavanine can serve as precursors of guanidine in the rat.     

A stereochemical study on the metabolism of isobutyrate in Pseudomonas putida

Ammonium[2-³H,1-¹⁴C]isobutyrate was converted by Pseudomonas putida ATCC 21244 into S(+)-β-hydroxyisobutyric acid (β-HIBA) with loss of the α-tritium atom. The recovered isobutyrate had the same ${}^3\text{H}{}^{14}\text{C}$ as the starting material. Ammonium (2S)-[3-¹³C]isobutyrate was synthesized and converted by P. putida into β-HIBA. The ¹³C-nmr of the corresponding methyl ester benzoate showed ¹³C enrichment in the hydroxymethyl carbon atom. The results therefore indicate that isobutyrate metabolism in this organism proceeds via an unsaturated intermediate (probably methacrylyl-CoA) formed by dehydrogenation of the 2-pro-S-methyl group of the precursor (isobutyryl-CoA). Hydration of the intermediate proceeds with addition of a proton at C-2 from the same side as the hydrogen removed in the dehydrogenation.     

The ferritin content of human red blood cells during the replacement of embryonic cells by fetal cells

Mature human embryonic erythrocytes (hemoglobin is ≥ 90% of the cellular protein) contained at least 20 times as much ferritin as human adult erythrocytes, suggesting the possibility that the embryonic red cells participate in iron storage as they do in other embryonic or larval vertebrates. The ferritin content of mature red cells in the circulation declined when fetal red cells replaced embryonic red cells; the cell replacement was monitored by the disappearance of embryonic ε-chains and the appearance of the fetal globin chains, γ^A and γ^G. A constant ratio of 0.67 was obtained for $\text{γ}{}^{\mathrm{<mtext>G</mtext>}}\text{γ}{}^{\mathrm{<mtext>A</mtext>}}\text{+ γ}{}^{\mathrm{<mtext>G</mtext>}}$ from the first detectable appearance (4 weeks after conception) until 13 weeks, a value which is similar to the value previously obtained at 20 weeks gestation and birth but higher than that observable in adults; thus, both γ^G and γ^A chains are produced in similar amounts throughout gestation. The high levels of ferritin in normal human embryonic erythrocytes emphasize the similarity of erythropoiesis in human embryos and other vertebrates. In addition, the results show that red cell ferritin can be used as a marker for studying the mechanism of induction of embryonic erythropoiesis in cultured cell lines, such as K562 from human chronic myelocytic leukemia, and that ferritin content may also serve as a marker for cellular transformations involving reversions to embryonic erythropoiesis.