Microbial metabolism of pyridinium compounds. Radioisotope studies of the metabolic fate of 4-carboxy-1-methylpyridinium chloride

Extracts of Achromobacter D formed CO(2), methylamine, succinate and formate as metabolic end-products from N-methylisonicotinic acid (4-carboxy-1-methylpyridinium chloride). The origin of the CO(2) in the 4-carboxyl group and of the methylamine in the N-methyl group of N-methylisonicotinate was demonstrated with carboxyl-(14)C- and N-Me-(14)C-labelled substrates respectively. The carbon skeletons of formate and succinate were shown to arise from the C-2 and the C-3-C-6 atoms of the heterocyclic ring respectively by using N-methyl[2,3-(14)C(2)]isonicotinate. This result is consistent with ring cleavage by the organism between C-2 and C-3.     

Identification of the thiol ester linked lipids in apolipoprotein B

Human plasma low-density lipoproteins of 1.032-1.043 g/mL density were totally delipidized. The reduced and carboxymethylated apolipoprotein B was incubated with 50 mM [14C]methylamine at pH 8.5 at 30 degrees C. Covalent incorporation of [14C]methylamine was observed with concomitant generation of new sulfhydryl groups, which could be blocked with [3H]- or [14C]iodoacetic acid. One type of the [14C]methylamine-modified products was separated from the protein and was found to be lipid in nature. Its Rf on thin-layer chromatography (TLC) was similar to that of the synthetic N-methyl fatty acyl amides. After purification with TLC and transesterification in 3 N methanolic HCl, methyl esters of C16 and C18 fatty acids at 1:1 ratio were identified by gas-liquid chromatography. The transesterification method was verified with the known N-methyl fatty acyl amides. These results suggest the presence of labile thiol ester linked palmitate and stearate in apolipoprotein B. Under mild alkaline conditions, the thiol ester bonds are broken by methylamine and form N-methyl fatty acyl amides and release new-SH groups. Intramolecular thiol ester bonds linked between cysteine side chains and acidic amino acid residues were also found present, which will be reported separately.     

Chemical modification of membrane proteins in relation to inhibition of anion exchange in human red blood cells

Mono-, di-, and trisulfonic acids, including 4,4′-diacetamido stilbene-2,2′-disulfonic acid (DAS) and 2-(4′-amino phenyl)-6-methylbenzene thiazol-3′,7-disulfonic acid (APMB) produce a reversible inhibition of sulfate equilibrium exchange in human red cells. A study of the sidedness of the action of a number of these sulfonic acids in red cell ghosts revealed that some, like DAS, inhibit only at the outer membrane surface while others, like APMB, inhibit at either surface. This finding suggests that at least two different types of membrane sites are involved in the control of anion permeability. The nature of the anion permeability controlling sites in the outer cell surface was investigated by studying the effects of DAS on the inhibition by dinitrofluoro-benzene (DNFB) of anion equilibrium exchange and on the binding of DNFB to the proteins of the red blood cell membrane. After exposure to DNFB in the presence of DAS for a certain period of time, there was a reduction of both the inhibitory effect of DNFB on sulfate exchange and the binding of DNFB to the protein in band 3 of SDS polyacrylamide gel electropherograms (nomenclature of Steck, J. Cell. Biol., 62: 1, 1974). Since binding to other membrane proteins was not affected, this observation supports the assumption that the protein in band 3 plays some role in anion transport. In accordance with the absence of an inhibitory effect at the inner membrane surface, internal DAS does not affect DNFB binding to the protein in band 3. DAS protected the anion exchange system not only against inhibition by DNFB but also by m-isothiocyanato benzene sulfonic acid. In contrast to DAS, the equally inhibitory phlorizin does not reduce the rate of dinitrophenylation of the protein in band 3. This suggests that either not all inhibitors of anion exchange exert their action by a combination with sites on the protein in band 3 or that in spite of the described evidence this protein is not involved in the control of anion movements. The effect of the irreversibly binding inhibitor 4-acetamido-4′-isothiocyanato-stilbene-2,2′-disulfonic acid (SITS) on DNFB binding to the protein in band 3 was studied in an attempt to differentiate DNFB binding related to inhibition of anion permeability from DNFB binding which is not involved. At least three distinguishable populations of DNFB binding sites were found: (1) binding sites common for DNFB and SITS which are probably related to inhibition, (2) other common sites which are not related to inhibition and (3) different sites whose dinitrophenylation is not affected by SITS. The number of sites in population (1) was estimated to be 0.8–1.2 ± 10⁶/cell. A study of the concentration dependence of the inhibition of anion equilibrium exchange with 4,4′-isothiocyanato-2,2′-stilbene disulfonic acid (DIDS) and APMB further suggests that among the sites in population (1) a major fraction is susceptible to modification by APMB and DIDS while the rest is only susceptible to DIDS. It remains undecided whether these differences of susceptibility reflect differences of accessibility or reactivity.     

Orally supplemented Lactobacillus acidophilus strain L‐92 inhibits passive and active cutaneous anaphylaxis as well as 2,4‐dinitroflurobenzene and mite fecal antigen induced atopic dermatitis‐like skin lesions in mice

Oral supplementation of lactic acid bacteria is a potential approach to the prevention and manipulation of allergic diseases such as atopic dermatitis. Our previous report showed that heat‐killed Lactobacillus acidophilus strain L‐92 (L‐92) possessed anti‐allergic properties, although its physiological function in atopic dermatitis has largely remained undefined. To evaluate the anti‐allergic efficacy of L‐92, we used four experimental animal models with the major features of atopic dermatitis and compared the results to those of clinically active drugs. ICR mice were passively sensitized by anti‐dinitrophenyl mouse monoclonal IgE for passive cutaneous anaphylaxis (PCA), and BALB/c mice were actively sensitized by ovalbumin for active cutaneous anaphylaxis (ACA). Allergic reaction was induced by repeated exposure to 2,4‐dinitroflurobenzene (DNFB) and mite (Dermatophagoides farinae) fecal allergen, in BALB/c and NC/Nga mice, respectively. Orally administrated L‐92 significantly inhibited the vascular permeability increase in both PCA and ACA, and the elevation of ovalbumin‐specific IgE titer in ACA. Moreover, repeated applications of DNFB and mite fecal antigen onto the BALB/c and NC/Nga mouse ear, respectively, caused clinical symptoms similar to atopic dermatitis such as ear swelling, scratching behavior and elevation of total serum IgE levels that were also moderately suppressed by L‐92. In addition, L‐92 treated mice exhibited lower levels of mast cells, eosinophil infiltration and Th1/Th2 cytokine expression. Our results, therefore, suggest that oral administration of L‐92 might be useful for alleviating allergic symptoms.     

Phase transition properties of 1,2- and 1,3-diacylphosphatidylethanolamines with modified head groups

The phase transition properties of dilute aqueous suspensions of "nonhydrated" (i.e., lipid suspensions which had not been heated above room temperature or above the main phase transition temperature of the fully hydrated lipid, whichever was lower) and hydrated 1,2(alpha)- and 1,3(beta)-dipalmitoylphosphatidylethanolamines with modified head groups have been determined by high-sensitivity differential scanning calorimetry at a scan rate of 0.1 K min-1. In both the 1,2 and 1,3 series, the head-group modifications of the phosphoethanolamine moiety included N-methyl, N,N-dimethyl, and N,N,N-trimethyl (phosphocholine). In the 1,2 series, additional modifications were dinitrophenyl, trinitrophenyl, N-(dinitrophenyl)aminocaproyl, N-(trinitrophenyl)aminocaproyl, and N-4-nitro-2,1,3-benzoxadiazole. Also included in this study were 1,2-dihexadecylphosphatidylethanolamine and the corresponding N-methyl-substituted lipid. In general, increasing bulkiness of the head-group substituent caused increasing lowering of the transition temperature, the most extreme cases among the hydrated lipids being the 45 degrees C lowering produced by the N-(dinitrophenyl)aminocaproyl substitution and its trinitrophenyl analogue in the 1,2 series. No simple trend is evident in the changes produced in the calorimetric enthalpy of transitions.     

The bacterial oxidation of N-methylisonicotinate, a photolytic product of Paraquat

Two bacteria have been isolated that are capable of oxidizing N-methylisonicotinate, a photodegradation product of Paraquat (1.1'-dimethyl-4,4'-bipyridylium ion). N-Methylisonicotinate-grown cells of strain 4C1, a Gram-positive rod, oxidized 2-hydroxy-N-methylisonicotinate without lag. Cell-free extracts of these cells converted 2-hydroxyisonicotinate into 2,6-dihydroxyisonicotinate; the reaction did not require molecular oxygen. Maleamate was deamidated and maleate isomerized to fumarate by soluble enzyme systems. [(14)C]Formaldehyde was isolated as the dimedone derivative from the supernatant of a cell suspension oxidizing N-[(14)C]methylisonicotinate, and no [(14)C]-methylamine was detected. Whole cells incubated with N-methyl[carboxy-(14)C]isonicotinate released 95% of the radioactivity as (14)CO(2). The second bacterium, strain 4C2, a Gram-negative rod, did not oxidize any of the mono- or di-hydroxypyridines or their N-methyl derivatives that were available or could be synthesized; nor did cell-free extracts oxidize any of these compounds. Methylamine was oxidized by whole cells without lag; cell-free extracts converted methylamine into formaldehyde when a soluble enzyme system requiring an electron acceptor was used; formaldehyde was oxidized to formate and formate to CO(2) by enzyme systems requiring NAD(+).     

Presence of covalently attached fatty acids in rat apolipoprotein B via thiolester linkages

Thiolester linked lipids in rat apolipoprotein B (ApoB) were examined by incubating reduced and carboxymethylated ApoB in 6 M urea buffer with [14C]methylamine at pH 8.5, 30 degrees C. It was observed that [14C]methylamine was covalently incorporated into ApoB, and there was a [14C]methylamine modified product which was lipid in nature. After extraction with organic solvents, the [14C]methylamine labeled product showed its Rf on TLC to be similar to that of the synthetic N-methyl fatty acyl amide. After purification on TLC and transesterification with 3 N methanolic HC1, methyl esters of C16:0, C18:0 and C18:1 fatty acids were identified by gas-liquid chromatography. These results suggest that rat ApoB, similar to human ApoB, contained covalently linked fatty acids through the high energy, labile thiolester bonds.     

Quantitative determination and regional distribution of pipecolic acid in rodent brain

A rapid and sensitive method for the quantitative determination of pipecolic acid (PA), one of the three cyclic secondary imino acids present in mammalian brain is described. The quantification and identification of PA are accomplished in rat and mouse brain using high performance liquid chromatography with electrochemical detection (LCEC) and nipecotic acid (NPA) as an internal standard. The cyclic imino acids are derivatized with 2,4-dinitrofluorobenzene (DNFB) to dinitrophenyl derivatives. The remaining time for LCEC analysis is less than 30 min and the limit of sensitivity is in the lower picomole range. The levels of PA found in rat and mouse brain are comparable to those reported using gas chromatography/mass spectrometry. The regional distribution of PA shows higher concentrations of PA in hypothalamus, pons-medulla oblongata and cerebellum. The present results demonstrate that LCEC is sensitive enough to determine endogenous levels of PA in mg amounts of rodent brain tissue. Due to its simplicity and rapidity, the technique represents an alternative to existing methods. This method can also be used for determination of PA in CSF, blood or urine of hyperipecolic patients.     

Nitrogen assimilation in facultative methylotrophic bacteria

A study was done of the pathways of nitrogen assimilation in the facultative methylotrophsPseudomonas MA andPseudomonas AM1, with ammonia or methylamine as nitrogen sources and with methylamine or succinate as carbon sources. When methylamine was the sole carbon and/or nitrogen source, both organisms possessed enzymes of the glutamine synthetase/glutamate synthase pathway, but when ammonia was the nitrogen sourcePseudomonas AM1 also synthesized glutamate dehydrogenase with a pH optimum of 9.0, andPseudomonas MA elaborated both glutamate dehydrogenase (pH optimum 7.5) and alanine dehydrogenase (pH optimum 9.0). Glutamate dehydrogenase and glutamate synthase from both organisms were solely NADPH-dependent; alanine dehydrogenase was NADH-dependent. No evidence was obtained for regulation of glutamine synthetase by adenylylation in either organism, nor did glutamine synthetase appear to regulate glutamate dehydrogenase synthesis.     

Isolation and visualisation of alkali stable protein/DNA complexes

Distinct polypeptides, 54,000–68,000 daltons in size, are alkali-stably bound to eukaryotic DNA. DNA fragments several hundred base pairs in length associated with these polypeptides are preferentially retained on glass fibre filters from solutions containing 1 M sodium chloride. About 50 percent of the protein/DNA complexes present in total DNA are retained on filters together with about 2 percent of the DNA. This preferential binding is demonstrated (a) by the ratio of ³H and ³⁵S radioactivity retained on filters after filtration of DNA from [³H]thymidine and L-[³⁵S]methionine labelled cells, (b) radioiodination of the material retained on filters and passing filters respectively and (c) by electron microscopical visualisation of the polypeptide component in the complexes after chemical modification with dinitrofluorobenzene (DNFB) followed by incubation with dinitrophenyl (DNP) specific antibodies.     

Pathway and rate-limiting step of glyphosate degradation by Aspergillus oryzae A-F02

Aspergillus oryzae A-F02, a glyphosate-degrading fungus, was isolated from an aeration tank in a pesticide factory. The pathway and rate-limiting step of glyphosate (GP) degradation were investigated through metabolite analysis. GP, aminomethylphosphonic acid (AMPA), and methylamine were detected in the fermentation liquid of A. oryzae A-F02, whereas sarcosine and glycine were not. The pathway of GP degradation in A. oryzae A-F02 was revealed: GP was first degraded into AMPA, which was then degraded into methylamine. Finally, methylamine was further degraded into other products. Investigating the effects of the exogenous addition of substrates and metabolites showed that the degradation of GP to AMPA is the rate-limiting step of GP degradation by A. oryzae A-F02. In addition, the accumulation of AMPA and methylamine did not cause feedback inhibition in GP degradation. Results showed that degrading GP to AMPA was a crucial step in the degradation of GP, which determines the degradation rate of GP by A. oryzae A-F02.     

Human T lymphocytes become glucocorticoid-sensitive upon immune activation

A murine model for Transfer Factor (TF) was used in an attempt to identify the nature of its antigen-specific component. TF was prepared from lymph node cells of CBA/Ca/T6 mice sensitized 30 days previously with 2,4-dinitrofluorobenzene (DNFB). To assay for the specific component of TF, 2 × 10⁷ lymphocyte equivalents were injected intravenously into normal syngeneic recipients. Lymph node cells obtained 18–24 hr later gave a positive response in the macrophage migration inhibition (MMI) test in the presence of the soluble analog of DNFB (sodium 2,4-dinitrobenzenesulfonate). The activity of TF was abrogated by absorption with anti-Ia sera including both an Ia alloantiserum (A.TH anti-A.TL) and a xenogeneic rabbit anti-serum which exclusively recognizes carbohydrate-defined Ia antigens. Analysis by paper chromatography using the technique for purification of carbohydrate-defined Ia antigens revealed that MIF production was obtained exclusively with those fractions known to contain Ia antigenic activity. In addition, pretreatment of TF with insoluble conconavalin A (Con A) which has an affinity for carbohydrate-defined Ia antigens resulted in removal of its activity. Taken together these findings pointed to the presence in TF of I-region gene products. Absorption with antibody directed against the dinitrophenyl determinant abolished the capacity of TF to stimulate macrophage inhibition factor production suggesting that it might also contain antigen fragments possibly in association with Ia. No evidence was, however, obtained for H-2 restriction of the action of TF in vivo since it was found to exert an effect in a variety of strain combinations including A.TH and Balb/c which share no known common I-region specificities. Parallel experiments were carried out with the lymphocyte transformation assay since this is known to be a measure of the nonspecific components in TF. Pretreatment with mouse allo-anti-Ia^k serum directed against both protein-and carbohydrate-defined Ia antigens caused a partial reduction in the proliferative response. In contrast no change in response was observed when the TF was absorbed with insoluble Con A or anti-DNP serum. Furthermore, lymphocyte transformation was obtained with only one of the three paper chromatography fractions positive in the MMI assay as well as two other different fractions. Taken together, these findings permitted a distinction to be made between specific and nonspecific components of TF and indicated that the specificity of TF could be explained in terms of the presence of I-region gene coded products possibly in association with antigen fragments.     

Uptake of methylamine via an inducible,energy-dependent transport system in the facultative methylotroph Arthrobacter P1

Cytoplasmic membrane vesicles were prepared by a lysozyme-salt treatment from Arthrobacter P1 grown on methylamine as the carbon and energy source. In the presence of an ascorbate-phenazine methosulphate electron donor system, these vesicles accumulated methylamine in unmodified form by an inducible transport system. This system has a high affinity for methylamine (K_app=20–25 M). The effect of the ionophores valinomycin and nigericin combined with membrane potential () and pH-gradient (pH) measurements demonstrated that methylamine uptake is electrogenic and driven by the . Optimal activity is observed at pH 6.5 and 30°C. Methylamine uptake was not affected by the presence of ammonium ions but was inhibited by the primary amines ethylamine (competitively), propylamine, butylamine and benzylamine. In addition, formaldehyde and acetate, at a concentration of 1 mM, inhibited methylamine uptake almost completely. These compounds were shown to be non-competitive inhibitors. A strong inhibition observed in the presence of plumbagin could be relieved by addition of dithiothreitol. This indicates that the oxidation-reduction state of, probably, carrier dithiol-disulfide-groups is an important factor in methylamine translocation in Arthrobacter P1.     

Effect of Chromatographic Conditions on Enantioseparation of Bovine Serum Albumin Chiral Stationary Phase in HPLC and Thermodynamic Studies

Twelve chiral compounds were enantiomerically resolved on bovine serum albumin chiral stationary phase (BSA‐CSP) by high‐performance liquid chromatography (HPLC) in reversed‐phase modes. Chromatographic conditions such as mobile phase pH, the percentage of organic modifier, and concentration of analyte were optimized for separation of enantiomers. For N‐(2, 4‐dinitrophenyl)‐serine (DNP‐ser), the retention factors (k) greatly increase from 0.81 to 6.23 as the pH decreasing from 7.21 to 5.14, and the resolution factor (R_s) exhibited a similar increasing trend (from 0 to 1.34). More interestingly, the retention factors for N‐(2, 4‐dinitrophenyl)‐proline (DNP‐pro) decrease along with increasing 1‐propanol in mobile phase (3%, 5%, 7% and 9% by volume), whereas the resolution factor shows an upward trend (from 0.96 to 2.04). Moreover, chiral recognition mechanisms for chiral analytes were further investigated through thermodynamic methods. Chirality 25:487–492, 2013. © 2013 Wiley Periodicals, Inc.     

Electron microscopy of α2-macroglobulin with a thiol ester bound ligand

In order to covalently bind the hydrolyzed thiol ester groups of the human α₂-macroglobulin (α2M) transformed by methylamine, the phospholipase A2 (PLA2), a small enzyme (M_r = 13 000) from Naja nigricollis snake venom was activated by succinimidyl 4-(maleimidomethyl)cyclohexane-1-carboxylate (SMCC). Average images determined from electron micrographs of the methylamine-transformed α2M, with and without activated PLA2, were determined by image processing and compared. A localization of the PLA2 was achieved by subtracting the average image of α2M transformed by methylamine from that containing PLA2. The results are consistent with previous work showing the central localization of chymotrypsin trapped in α2M. They also suggest that the four thiol esters are located near the center of the α2M. molecule.     

Regulation of methylotrophic and heterotrophic metabolism in Arthrobacter P1. Growth on mixtures of methylamine and acetate in batch and continuous cultures

Incubations of Arthrobacter P1 in batch culture in media with mixtures of acetate and methylamine resulted in sequential utilization of the two carbon substrates, but not in diauxic growth. Irrespective of the way cells were pregrown, acetate was the preferred substrate and subsequent studies showed that this is due to the fact that acetate is a strong inhibitor of the methylamine transport system and amine oxidase in Arthrobacter P1. An analysis of enzyme activities in cell-free extracts showed that synthesis of amine oxidase occurred already in the first growth phase with acetate, whereas rapid synthesis of hexulose phosphate synthase was only observed once methylamine utilization started. It is therefore concluded that in Arthrobacter P1 the synthesis of the enzymes specific for methylamine oxidation is not regulated co-ordinately with those involved in formaldehyde fixation, but induced sequentially by methylamine and formaldehyde, respectively.During growth of Arthrobacter P1 on the same mixture in carbon- and energy source-limited continuous cultures both substrates were used simultaneously and completely at dilution rates below the _max on either of these substrates. Addition of methylamine, in concentrations as low as 0.5 mM, to the medium reservoir of an acetate-limited continuous culture (D=0.10 h^-1) already resulted in synthesis of both amine oxidase and hexulose phosphate synthase. In the reverse experiment, addition of acetate to the medium reservoir of a methylamine-limited continuous culture (D=0.10 h^-1), acetate was initially only used as an energy source. Synthesis of the glyoxylate cycle enzymes, however, did occur at acetate concentration in the feed above 7.5–10 mM. This indicates that at acetate concentrations below 10 mM the metabolism of the C₁ substrate methylamine is able to cause a complete repression of the synthesis of the enzymes involved in carbon assimilation from the C₂ substrate acetate.Abbreviations HPS Hexulose phosphate synthase - MS mineral salts - RuMP ribulose monophosphate     

High-performance liquid chromatographic method for the quantitative analysis of a synthetic copolymer with antitumor activity (copovithane) and methylamine in human blood plasma and urine

A method for the determination of a synthetic polymeric compound with antitumor activity (copovithane) and methylamine in blood plasma and urine is described. Copovithane is prepared by radical polymerisation of a diurethane with N-vinylpyrrolidone.The method is based on high-performance liquid chromatography of the methylamine hydrochloride which arises during the hydrochloric acid hydrolysis of the parent substance. The methylamine hydrochloride is converted to the trinitrobenzenesulphonyl derivative for the purpose of chromatographic detection.The limit of determination for copovithane in blood plasma is 1.2 mg/l and in urine 1.5 mg/day. The determination limit for methylamine in blood plasma is 0.2 mg/1 and in urine 0.3 mg/day. The imprecision is dependent on the sample, and amounts to ± 6.8% for blood plasma and ± 6.4% for urine.     

Protein cross-links: universal isolation and characterization by isotopic derivatization and electrospray ionization mass spectrometry.

A general method of unequivocally identifying and obtaining sequence information on cross-linked peptides derived by proteolytic digestion of cross-linked proteins has been developed. The method is based on isotopic labeling of alpha-amino groups with 2, 4-dinitrofluorobenzene (DNFB) coupled with electrospray ionization mass spectrometry. Proteins containing covalent cross-link(s) are reductively methylated to convert lysine residues to dimethyl lysine. The methylated protein is partially hydrolyzed and the liberated alpha-amino termini are derivatized with an equimolar mixture of DNFB and [(2)H(3)]DNFB. Dinitrophenyl (DNP)-labeled peptides may be fractionated into mono- and bis-DNP pools by chromatography on phenyl media. The bis-DNP peptides are further separated by reverse-phase HPLC and analyzed by electrospray ionization mass spectrometry. The molecular ions of cross-linked peptides are unambiguously identified as 1:2:1 triplets in the mass spectrum resulting from the binomial distribution of isotopic label in the bis-DNP derivative. Sequence information can be elucidated from the unique product ion patterns which are generated from in-source fragmentation at an elevated cone voltage. Analysis of the disulfide cross-linked peptide (VTCG)(2) was undertaken as a proof of concept and the generality of the method was demonstrated by isolating and sequencing the isopeptide bond of polyubiquitin.     

Effects of pronase on passive ion permeability of the human red blood cell

Summary The effects of pronase fromStreptomyces griseus on sulfate, potassium, sodium, and erythritol permeability of human red blood cells were studied. It was found that the proteolytic enzyme reduces anion permeability, increases cation permeability and has no effect on the nonfacilitated component of the flux of the nonelectrolyte. These findings can be explained on the basis of the fixed charge hypothesis by the assumption that the enzyme exerts its effects by altering the density of positive fixed charges in the membrane.The effects of pronase are qualitatively similar to those of the amino reactive agent, dinitrofluorobenzene (DNFB). Therefore, attempts were made to discover if this similarity is due to alterations of the same membrane sites by the enzyme and the chemical modifier. It was found that the effects of pronase and DNFB were not additive. Hence, the enzyme and the amino reactive agent do not seem to act on two independent and parallel channels. A more detailed analysis of the data suggests that DNFB and pronase affect functionally identical sites.Proteolytic enzymes frequently exhibit some esterase activity. However, the amino-N content of lipid extracts of red cell membranes remained virtually unaltered after exposure of the cells to pronase. This finding indicates that the positive charge of the bulk of the lipid amino groups is not involved in the control of passive ion permeability. The carbohydrate amino groups of the red cell membrane are N-acylated and hence cannot contribute to the positive membrane charge. It seems reasonable to conclude that the effects of pronase on ion permeability are primarily due to alterations of the density of charged protein amino groups in the red cell membrane.     

Permeability of ammonia,methylamine and ethylamine in the cyanobacterium,Synechococcus R-2 (Anacystis nidulans) PCC 7942

Summary Permeabilities of ammonia (NH₃), methylamine (CH₃NH₂) and ethylamine (CH₃CH₂NH₂) in the cyanobacterium (cyanophyte)Synechococcus R-2 (Anacystis nidulans) have been measured. Based on net uptake rates of DCMU (dichlorophenyldimethylurea) treated cells, the permeability of ammonia was 6.44±1.22 m sec^–1 (n=13). The permeabilities of methylamine and ethylamine, based on steady-state¹⁴C labeling were more than ten times that of ammonia (P _methylamine=84.6±9.47 m sec^–1 (76),P _ethylamine=109±11 m sec^–1 (55)). The apparent permeabilities based on net uptake rates of methylamine and ethylamine uptake were significantly lower, but this effect was partially reversible by ammonia, suggesting that net amine fluxes are rate limited by proton fluxes to an upper limit of about 700 nmol m^–2 sec^–1. Increasing concentrations of amines in alkaline conditions partially dissipated the pH gradient across the cell membrane, and this property could be used to calculate the relative permeabilities of different amines. The ratio of ethylamine to methylamine permeabilities was not significantly different from that calculated from the direct measurements of permeabilities; ammonia was much less effective in dissipating the pH gradient across the cell membrane than methylamine or ethylamine. An apparent permeability of ammonia of 5.7±0.9 m sec^–1 could be calculated from the permeability ratio of ammonia to methylamine and the experimentally measured permeability of methylamine. The permeability properties of ammonia and methylamine are very different; this poses problems in the interpretation of experiments where¹⁴C-methylamine is used as an ammonia analogue.