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PubMed1987

The conformational changes of alpha 2-macroglobulin induced by methylamine or trypsin. Characterization by extrinsic and intrinsic spectroscopic probes.

The conformational changes around the thioester-bond region of human or bovine alpha 2M (alpha 2-macroglobulin) on reaction with methylamine or trypsin were studied with the probe AEDANS [N-(acetylaminoethyl)-8-naphthylamine-1-sulphonic acid], bound to the liberated thiol groups. The binding affected the fluorescence emission and lifetime of the probe in a manner indicating that the thioester-bond region is partially buried in all forms of the inhibitor. In human alpha 2M these effects were greater for the trypsin-treated than for the methylamine-treated inhibitor, which both have undergone similar, major, conformational changes. This difference may thus be due to a close proximity of the thioester region to the bound proteinase. Reaction of trypsin with thiol-labelled methylamine-treated bovine alpha 2M, which retains a near-native conformation and inhibitory activity, indicated that the major conformational change accompanying the binding of proteinases involves transfer of the thioester-bond region to a more polar environment without increasing the exposure of this region at the surface of the protein. Labelling of the transglutaminase cross-linking site of human alpha 2M with dansylcadaverine [N-(5-aminopentyl)-5-dimethylaminonaphthalene-1-sulphonamide] suggested that this site is in moderately hydrophobic surroundings. Reaction of the labelled inhibitor with methylamine or trypsin produced fluorescence changes consistent with further burial of the cross-linking site. These changes were more pronounced for trypsin-treated than for methylamine-treated alpha 2M, presumably an effect of the cleavage of the adjacent 'bait' region. Solvent perturbation of the u.v. absorption and iodide quenching of the tryptophan fluorescence of human alpha 2M showed that one or two tryptophan residues in each alpha 2M monomer are buried on reaction with methylamine or trypsin, with no discernible change in the exposure of tyrosine residues. Together, these results indicate an extensive conformational change of alpha 2M on reaction with amines or proteinases and are consistent with several aspects of a recently proposed model of alpha 2M structure [Feldman, Gonias & Pizzo (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 5700-5704].

Acetylcysteine↗
PubMed1993

Receptor-recognized alpha 2-macroglobulin-methylamine elevates intracellular calcium, inositol phosphates and cyclic AMP in murine peritoneal macrophages.

Human plasma alpha 2-macroglobulin (alpha 2M) is a tetrameric proteinase inhibitor, which undergoes a conformational change upon reaction with either a proteinase or methylamine. As a result, a receptor recognition site is exposed on each subunit of the molecule enabling it to bind to its receptors on macrophages. We have used Fura-2-loaded murine peritoneal macrophages and digital video fluorescence microscopy to examine the effects of receptor binding on second messenger levels. alpha 2M-methylamine caused a rapid 2-4-fold increase in intracellular Ca2+ concentration ([Ca2+]i) within 5 s of binding to receptors. The agonists induced a focal increase in [Ca2+]i that spread out to other areas of the cell. The increase in [Ca2+]i was dependent on the alpha 2M-methylamine concentration and on the extracellular [Ca2+]. Both sinusoidal and transitory oscillations were observed, which varied from cell to cell. Neither alpha 2M nor boiled alpha 2M-methylamine, forms that are not recognized by the receptor, affected [Ca2+]i in peritoneal macrophages under identical conditions of incubation. The alpha 2M-methylamine-induced rise in [Ca2+]i was accompanied by a rapid and transient increase in macrophage inositol phosphates, including inositol tris- and tetrakis-phosphates. Native alpha 2M did not stimulate a rise in inositol phosphates. Finally, binding of alpha 2M-methylamine to macrophages increased cyclic AMP transiently. Thus receptor-recognized alpha-macroglobulins behave as agonists whose receptor binding causes stimulation of signal transduction pathways.

Animals↗
PubMed1995

Mutational analysis of mau genes involved in methylamine metabolism in Paracoccus denitrificans.

A chromosomal fragment containing DNA downstream from mauC was isolated from Paracoccus denitrificans. Sequence analysis of this fragment revealed the presence of four open reading frames, all transcribed in the same direction. The products of the putative genes were found to be highly similar to MauJ, MauG, MauM and MauN of Methylobacterium extorquens AM1. Using these four mau genes, 11 mau genes have been cloned from P. denitrificans to date. The gene order is mauRFBEDACJGMN, which is similar to that in M. extorquens AM1. mauL, present in M. extorquens AM1, seems to be absent in P. denitrificans. MauJ is predicted to be a cytoplasmic protein, and MauG a periplasmic protein. The latter protein contains two putative heme-binding sites, and has some sequence resemblance to the cytochrome c peroxidase from Pseudomonas aeruginosa. MauM is also predicted to be located in the periplasm, but MauN appears to be membrane associated. Both resemble ferredoxin-like proteins and contain four and two motifs, respectively, characteristic for [4Fe-4S] clusters. Inactivation of mauA, mauJ, mauG, mauM and mauN was carried out by introduction of unmarked mutations in the chromosomal copies of these genes. mauA and mauG mutant strains were unable to grow on methylamine. The mauJ mutant strain had an impaired growth rate and showed a lower dye-linked methylamine dehydrogenase (MADH) activity than the parent strain. Mutations in mauM and mauN had no effect on methylamine metabolism. The mauA mutant strain specifically lacked the beta subunit of MADH, but the alpha subunit and amicyanin, the natural electron acceptors of MADH, were still produced. The mauG mutant strain synthesized the alpha and beta subunits of MADH as well as amicyanin. However, no dye-linked MADH activity was found in this mutant strain. In addition, as the wild-type enzyme displays a characteristic fluorescence emission spectrum upon addition of methylamine, this property was lost in the mauG mutant strain. These results clearly show that MauG is essential for the maturation of the beta subunit of MADH, presumably via a step in the biosynthesis of tryptophan tryptophylquinone, the cofactor of MADH. The mau gene cluster mauRFBEDACJGMN was cloned on the broad-host vector pEG400. Transfer of this construct to mutant strains which were unable to grow on methylamine fully restored their ability to grow on this compound. A similar result was achieved for the closely related bacterium Thiosphaera pantotropha, which is unable to utilize methylamine as the sole sources of carbon and energy.

Amino Acid Sequence↗
PubMed1987

Disruption of intracellular processing of epidermal growth factor by methylamine inhibits epidermal growth factor-induced DNA synthesis but not early morphological or transcriptional events.

Upon internalization, epidermal growth factor (EGF) is proteolytically processed from its COOH terminus as it traverses intracellular vesicles and lysosomes. This report describes experiments which were conducted to determine whether lysosomotropic amines such as methylamine, which are known to inhibit degradation of EGF, are able to significantly inhibit the COOH-terminal processing of EGF, and whether disruption of EGF processing would negatively affect EGF-stimulated events such as DNA synthesis and induction of specific mRNA species. The results of these experiments indicated that, whereas methylamine treatment had no effect on EGF binding or internalization, vesicular translocation from endocytic vesicles to lysosomes was halted and processing of EGF was severely inhibited. The stimulation of DNA synthesis beginning 12 h after EGF exposure was also markedly inhibited by methylamine treatment. However, addition of methylamine alone produced a non-specific inhibition of DNA synthesis. The ability of EGF to induce specific transcription of the rat transin gene within 6 h of treatment was also not inhibited by methylamine treatment, but was actually increased in the presence of methylamine. These results suggest that at least some early transcriptionally regulated events induced by EGF do not require vesicular processing of EGF (or its receptor) and that the signal transduced by the binding of EGF to its receptor occurs in, or proximal to, the endocytic vesicles.

Animals↗
PubMed1990

Chemical cross-linking study of complex formation between methylamine dehydrogenase and amicyanin from Paracoccus denitrificans.

Two soluble periplasmic redox proteins from Paracoccus denitrificans, the quinoprotein methylamine dehydrogenase and the copper protein amicyanin, form a weakly associated complex that is critical to their physiological function in electron transport [Gray, K. A., Davidson, V. L., & Knaff, D. B. (1988) J. Biol. Chem. 263, 13987-13990]. The specific interactions between methylamine dehydrogenase and amicyanin have been studied by using the water-soluble cross-linking agent 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC). Treatment of methylamine dehydrogenase alone with EDC caused no intermolecular cross-linking but did cause intramolecular cross-linking of this alpha 2 beta 2 oligomeric enzyme. The primary product that was formed contained one large and one small subunit. Methylamine dehydrogenase and amicyanin were covalently cross-linked in the presence of EDC to form at least two distinct species, which were identified by nondenaturing polyacrylamide gel electrophoresis (PAGE). The formation of these cross-linked species was dependent on ionic strength, and the ionic strength dependence was much greater at pH 6.5 than at pH 7.5. The effects of pH and ionic strength were different for the different cross-linked products. SDS-PAGE and Western blot analysis of these cross-linked species indicated that the primary site of interaction for amicyanin was the large subunit of methylamine dehydrogenase and that this association could be stabilized by hydrophobic interactions. In light of these results a scheme is proposed for the interaction of amicyanin with methylamine dehydrogenase that is consistent with previous data on the physical, kinetic, and redox properties of this complex.

Azurin↗
PubMed1978

Interactions of methylglyoxal with methylamine.

Ab initio quantum mechanical calculations are used to study the interactions of the aldehydic group of methylglyoxal with the NH2 groups of protein side-chains, using methylamine as a representative molecule. The hydrogen-bonding interaction, C = O...H - N, results in an electronic charge transfer from methylglyoxal to methylamine in both the ground and first excited triplet states. In this latter state a slight possibility is found for the H atom in the hydrogen bond to tunnel from methylglyoxal to methylamine, leading to the possible formation of two free radical fragments. The approach of methylamine to methylglyoxal in the stacked conformation C...N to form a hemiacetal, associated with electron charge transfer from methylamine to methylglyoxal, is energetically unfavourable in vacuum. The concomitant tunnelling of a proton from a proton-donating solvent molecule to the aldehydic oxygen of methylglyoxal is shown to make this approach favourable. The relative stability of the keto and enol forms of methylglyoxal is also investigated, the keto form being found the more stable in vacuum.

Aldehydes↗
PubMed2000

A spectrophotometric method for determining the oxidative deamination of methylamine by the amine oxidases.

Previously published studies on the oxidative deamination of methylamine by the amine oxidases have determined the formation of radioactively labeled formaldehyde from [(14)C]methylamine. The present work describes a coupled spectrophotometric assay, using formaldehyde dehydrogenase, for the continuous determination of the oxidative deamination of methylamine by semicarbazide-sensitive amine oxidase (SSAO) and its potential use for determining methylamine concentrations in plasma. In this assay, the formaldehyde produced by methylamine deamination is further oxidized to formate, with the reduction of NAD(+), by formaldehyde dehydrogenase. The NADH generated is monitored continuously at 340 nm. Interference from the presence of a rotenone-insensitive NADH oxidase activity in crude tissue homogenates and microsomal fractions can be minimized by pretreating samples with Triton X-100 or substituting NAD(+) by APAD(+) in the coupled assay. This relatively inexpensive and reproducible assay procedure avoids the use of radioactively labeled material.

Aldehyde Oxidoreductases↗
PubMed1980

Development of amine oxidase-containing peroxisomes in yeasts during growth on glucose in the presence of methylamine as the sole source of nitrogen.

The metabolism of methylamine as the nitrogen source for growth of the non-methylotrophic yeast Candida utilis and the methylotrophic yeast Hansenula polymorpha was investigated. Growth of both organisms in media with glucose and methylamine was associated with the presence of an amine oxidase in these cells. The enzyme catalyses the oxidation of methylamine by molecular oxygen into ammonia, formaldehyde and hydrogen peroxide and it is considered to be the key enzyme in methylamine metabolism in the organisms studied. In addition to synthesis of amine oxidase, derepression of catalase, formaldehyde and formate dehydrogenase was also observed upon transfer of cells of the two organisms from media containing ammonium ions into media containing methylamine as the nitrogen source. The synthesis of enzymes was paralleled by the development of a number of large microbodies in the cells. Cytochemical staining experiments indicated that the amine oxidase activity was located in the microbodies in both organisms. Catalase-activity was also demonstrated in these organelles, which can therefore be considered as peroxisomes. The present contribution is the first description of a peroxisomal amine oxidase.

Ascomycota↗
PubMed1986

Unusual properties of crocodilian ovomacroglobulin shown in its methylamine treatment and sulfhydryl titration.

The inhibitory activity of chicken and crocodilian ovomacroglobulins against trypsin was measured before and after their incubation with methylamine. The result for crocodilian ovomacroglobulin showed that methylamine treatment destroyed half of its activity, in unique contrast to human alpha 2-macroglobulin and chicken ovomacroglobulin for which methylamine either destroys the inhibitory activity of the former completely or does not affect that of the latter at all. Free sulfhydryl groups of chicken and crocodilian ovomacroglobulins were titrated with 5,5'-dithiobis(2-nitrobenzoic acid) before and after incubation with trypsin. Prior to the incubation with trypsin the chicken and crocodilian proteins respectively had 0 and 1 titratable sulfhydryl per molecule of Mr 720,000. After treatment with trypsin the crocodilian protein had 3.5-4 titratable sulfhydryls, whereas there were no titratable sulfhydryls in the chicken protein. After denaturation of the crocodilian protein in sodium dodecyl sulfate at 100 degrees C the number of titratable sulfhydryls was 4. Chicken ovomacroglobulin again did not have an appreciable number of titratable sulfhydryls under similar denaturing conditions. Incubation of crocodilian protein with [14C]methylamine showed an incorporation of at least 2 mol of methylamine per molecule. The result indicated the presence of three intramolecular thiol ester bonds in crocodilian ovomacroglobulin with differential stability against external perturbations.

Alligators and Crocodiles↗
PubMed1994

[14C]Methylamine accumulation in cultured human skin fibroblasts--a biochemical test for lysosomal storage and lysosomal diseases.

Incorporation of the lysosomotropic amine [14C]methylamine by fibroblasts cultured from patients with lysosomal storage diseases and from controls was used to estimate the size of the lysosomal compartment. All cell lines from patients with infantile and juvenile forms of mucopolysaccharidoses, mucolipidoses and oligosacharidoses showed markedly increased radioactivity compared with the normal range of controls. In cells from patients with sphingolipidoses and adult forms of storage diseases, however, methylamine accumulation was not significantly increased. Experimentally induced lysosomal storage by enzyme inhibitors (leupeptin, suramin) also caused increased methylamine accumulation. When the lysosomal pH was determined with fluorescein isothiocyanate-dextran, it was in the range of normal controls (pH 4.7-5.0) in patients cells. Thus, [14C]methylamine accumulation should depend on the volume rather than differences in acidity of the lysosomal compartment and be a measure of its eventual pathological enlargement. We conclude that the determination of [14C]methylamine accumulation in fibroblasts provides a valuable tool in the screening for a variety of lysosomal storage disorders.

Cells, Cultured↗
PubMed1995

Reversed electron transfer through the bc1 complex enables a cytochrome c oxidase mutant (delta aa3/cbb3) of Paracoccus denitrificans to grow on methylamine.

In Paracoccus denitrificans four classes of redox proteins are involved in the electron transfer from methylamine to oxygen:methylamine dehydrogenase (MADH), amicyanin, cytochrome c and cytochrome c oxidase. MADH and its electron acceptor amicyanin are indispensable for growth on methylamine. At least three different cytochromes c and two types of cytochrome c oxidase, cytochromes aa3 and cbb3, have previously been proposed to participate in the electron transfer pathways from methylamine to oxygen. In this study, participation of both cytochrome c oxidases and of the quinol oxidase (cytochrome bb3) has indeed been confirmed by analysis of a series of oxidase mutants. Interestingly, a P. denitrificans cytochrome c oxidase mutant (delta aa3/cbb3) retains the capacity to oxidise methylamine. It is demonstrated that the oxidation of the cytochrome c pool in this mutant does not proceed via an alternative cytochrome c oxidase, but rather via an 'uphill' electron transfer through the bc1 complex to ubiquinone, driven by the membrane potential. The subsequent oxidation of ubiquinol proceeds via the only remaining terminal oxidase, the bb3-type quinol oxidase.

Culture Media↗
PubMed1985

Dissociation by methylamine of insulin release from glucose-induced electrical activity in isolated mouse islets of Langerhans.

The effect of methylamine on electrical activity and simultaneously measured insulin release was investigated in single perifused islets of normal mice. Methylamine, (2 mmol/L or 6 mmol/L) failed to affect beta-cell input resistance and only caused a modest and transient inhibition of electrical activity of islets exposed to 11.1 mmol/L glucose. Methylamine (2 mmol/L) inhibited insulin release evoked by a five-minute rise in glucose concentration from 5.6 to 22.2 mmol/L, even when the glucose-induced electrical activity remained unaltered. Methylamine, at 2 or 5 mmol/L, partially inhibited insulin release but failed to affect the continuous electrical activity in islets exposed throughout to 22.2 mmol/L glucose. At 10 mmol/L, methylamine reduced both insulin release and electrical activity. These data reinforce the idea that the glucose-induced changes in beta-cell membrane potential represent an early event in the process of stimulus-secretion coupling and can be dissociated from the subsequent process of insulin release.

Animals↗
PubMed1983

Structural and physical changes in lysosomes from isolated rat hepatocytes treated with methylamine.

Incubation of isolated rat hepatocytes with 10 mM methylamine resulted in an inhibition of endogenous protein degradation and a microscopically visible enlargement of the lysosomes. Lysosomes from methylamine-treated cells exhibited increased buoyancy in metrizamide gradients and increased fragility as measured by the release of acid phosphatase activity in vitro, despite the fact that no methylamine remained in the gradient-isolated organelles. When methylamine was extracted from intact cells, the inhibition of protein degradation was immediately relieved, whereas the lysosomal enlargement (and to a certain extent also the increased fragility) persisted for some time. The methylamine-induced osmotic swelling of the lysosomes would thus seem to involve not merely a passive stretching of the lysosomal membrane, but rather some structural change (e.g., an increased amount of membrane material) which is relatively slowly reversible, but without effect on lysosomal function.

Acid Phosphatase↗
PubMed1985

The enzymatic systems involved in the mammalian metabolism of methylamine.

The metabolism of methylamine has been investigated in the rat in order to elucidate the role of monoamine oxidase (MAO; EC 1.4.3.4) and intestinal bacteria in the metabolism of the compound. In a series of experiments in which short- and long-acting inhibitors of MAO were administered either alone or in combination prior to methyl-[14C]amine hydrochloride injection, the excretion of radioactivity in the expired air and the urine was examined to indirectly assess the role of MAO in the metabolism of methylamine. The data presented provide indirect evidence to demonstrate that the effect of iproniazid, an inhibitor of methylamine oxidation, is mediated through enzyme systems separate from MAO systems which have been invoked as major contributors to metabolism of methylamine by other investigators. The bacterial oxidation of methylamine in the intestine plays a minor role in the overall metabolism of the compound.

Animals↗
PubMed1994

The effects of pH and cations on the spectral and kinetic properties of methylamine dehydrogenase from Thiobacillus versutus.

The catalytic parameters of Thiobacillus versutus methylamine dehydrogenase (MADH) with the physiological substrates methylamine and amicyanin show a pH profile that is quite different from the one found in commonly used assays with artificial electron acceptors. The optimum at pH 7.5, observed for kcat in the latter case, is absent with amicyanin as the reoxidizing substrate. With amicyanin kcat scarcely depends on pH; the same is true for the maximal rate of reduction of MADH by methylamine (kred). Conversely, both the specificity constant (kcat/Km) of MADH for amicyanin and the apparent second-order rate constant for the reduction of MADH by methylamine (kassocapp) increase very sharply with pH. MADH has a high- and a low-affinity binding site for monovalent cations. Cation binding to the high-affinity site, which only binds the larger cations (Cs+, Rb+, and NH4+), is accompanied by a red shift in the absorbance spectrum, whereas cation binding to the low-affinity site, which, less specifically, favors binding of the smaller cations, leads to a bleaching of the visible spectrum with a concomitant increase in the near-UV. Cation binding to either site strongly affects the reactivity of MADH. The reduction of MADH by methylamine is inhibited by monovalent cations, whereas the oxidation of reduced MADH by amicyanin is strongly stimulated. For the former reaction it was established that cations affect only kassocapp, not kred. Some speculations about the molecular basis for the effects of pH and cations are presented.

Bacterial Proteins↗
PubMed1997

Mechanism-based inactivation of a yeast methylamine oxidase mutant: implications for the functional role of the consensus sequence surrounding topaquinone.

The copper-containing yeast methylamine oxidase E406N mutant has an altered consensus sequence surrounding the topaquinone cofactor (residue 405). The mutation has no effect on the final yield of the active-site topaquinone cofactor during biogenesis but causes the enzyme to be inactivated by substrate methylamine [Cai, D., and Klinman, J. P. (1994) Biochemistry 33, 7674-7653]. In this study we show that the inactivation leads to the formation of a covalent adduct, which has a UV/vis spectrum very similar to that of a product Schiff base, an intermediate of topaquinone-catalyzed amine oxidation reactions. The kinetic isotope effects on the second-order rate constant for the inactivation and catalytic turnover are identical, indicating that the two processes share a common intermediate that follows C_H bond cleavage. Resonance Raman spectroscopy provides direct evidence for the accumulation of a neutral product Schiff base species. Removal of excess methylamine leads to recovery of both activity and the native absorption spectrum for E406N, indicating that the cofactor in the inactivated enzyme is chemically competent for hydrolysis. The rate of the reactivation is slow, however; the shortest half-life of the inhibited E406N at 25 degrees C is 5.9 min at pH 6.15. pH effect experiments show that the inactivation and reactivation steps are controlled by a single ionizable group with a pKa of 6.9-7.1; under basic conditions, when this residue is deprotonated, the inactivation is the fastest and the half-life of the inhibited enzyme is the longest. On the basis of the available crystal structures of copper amine oxidases, we propose that a histidine residue in the dimer interface is responsible for the observed ionization. In the wild-type enzyme this histidine is kept protonated by virtue of Glu at position 406. Unlike methylamine, the larger substrates ethylamine and benzylamine give normal turnover with E406N. Disruption of structure at the subunit interface in E406N may allow a rotation of the relatively small topa-product Schiff base complex (formed from methylamine) away from the active-site base to a conformation that is incompetent toward hydrolysis.

Consensus Sequence↗
PubMed1992

Binding of transforming growth factor-beta 1 to methylamine-modified alpha 2-macroglobulin and to binary and ternary alpha 2-macroglobulin-proteinase complexes.

The binding of 125I-labelled transforming growth factor-beta 1 (TGF-beta 1) to human alpha 2-macroglobulin (alpha 2M) was studied by native PAGE and autoradiography. TGF-beta 1 bound preferentially to alpha 2M-methylamine and minimally, if at all, to native alpha 2M. Preparations of alpha 2M-proteinase complex were generated by incubating a standard concentration of alpha 2M (0.4 microM) with different concentrations of trypsin, chymotrypsin or neutrophil elastase (0.04-2.0 microM). The 125I-TGF-beta 1-binding activity depended on the initial ratio of active proteinase to alpha 2M, or r value, used to form the alpha 2M-proteinase complex. With all three proteinases, r values of 2 or greater yielded preparations with unchanged or decreased TGF-beta 1-binding activity relative to native alpha 2M. By contrast, r values near 1 yielded preparations with significantly increased TGF-beta 1-binding activity. The results of [3H]thymidine-incorporation studies performed in mouse keratinocytes were consistent with the 125I-TGF-beta-binding experiments. alpha 2M-trypsin and alpha 2M-chymotrypsin prepared at an r value of 1.0 counteracted the activity of TGF-beta 1, whereas the equivalent complexes prepared at an r value of 3.0 had no effect. As determined by SDS/PAGE, 125I-TGF-beta 1 binding to alpha 2M-methylamine was at least 80% non-covalent. Reaction of alpha 2M-methylamine with iodoacetamide or 5,5'-dithiobis-(2-nitrobenzoic acid) decreased the percentage of covalent binding but had no effect on total binding. Neuraminidase treatment had no effect on the binding of 125I-TGF-beta 1 to alpha 2M-methylamine. Cleavage of the 'bait regions' in alpha 2M-methylamine by prolonged treatment with trypsin also had no effect. These studies suggest that TGF-beta 1 binding to alpha 2M is enhanced by conformational change in the proteinase inhibitor resulting from reaction with proteinase or amine. If both proteinase-binding sites in a single alpha 2M molecule are occupied, TGF-beta 1-binding activity is decreased or perhaps eliminated.

Animals↗
PubMed1996

Methylamine decreases trafficking and packaging of newly synthesized phosphatidylcholine in lamellar bodies in alveolar type II cells.

Lung lamellar bodies, the storage organelles for lung surfactant phosphatidylcholine (PC), maintain an acidic pH that can be increased with weak bases. This study investigates the effect of a weak base, methylamine, on the pH in lamellar bodies and on the trafficking and packaging of newly synthesized PC in lamellar bodies. Methylamine increased the pH of isolated lung lamellar bodies and of lamellar bodies in intact cells. Metabolic labelling of isolated type II cells with [methyl-3H]choline showed that although methylamine (2.5-10 mM) did not alter the labelling of cellular or microsomal PC and disaturated PC, it decreased the labelling of the PC and disaturated PC in lamellar bodies. The packaging of PC in lamellar bodies (the specific activities ratio between the PC in lamellar bodies and the microsomal PC) also decreased in a time- and concentration-dependent manner. The cellular synthesis of PC or its packaging into lamellar bodies was unaltered by brefeldin A, suggesting that the Golgi was not involved in PC packaging. Although methylamine also increased surfactant secretion, the inhibition of PC packaging in lamellar bodies seems unrelated to the secretagogue effect, (1) on the basis of metabolic consequences of increased secretion and (2) because ATP, another secretagogue, did not inhibit PC packaging. Methylamine seems to inhibit PC packaging by inhibiting trafficking of PC to lipid-rich light subcellular fractions. Together our results suggest that the trafficking of surfactant PC into lamellar bodies might be sensitive to changes in the pH of lamellar bodies.

Acridine Orange↗