Rule of antibody structure: the primary structure of a human monoclonal IgA1-immunoglobulin (myeloma protein Tro), III. Isolation and characterization of the tryptic peptides of the H-chain (author's transl)]

The reduced and either aminothylated or carboxymethylated H-chain of the monoclonal IgA1 immunoglobulin Tro was digested with trypsin. The tryptic peptides were isolated by gel and ion-exchange chromatography. Because of different methods of alkylation, the cysteine-containing peptides could be obtained in two forms and showed additional overlaps. Sequence studies performed with these fragments elucidated the primary structure of the protein.     

Efficient nitrogen alkylation with carbohydrates

Efficient nitrogen alkylation of various primary and secondary amines, including cyclic, heterocyclic and alkaloid type amines, with a sugar oxetane 3,5-anhydro-1,2-O-cyclohexylidene-alpha-D-xylofuranose is described. As a result, 5-amino-5-deoxy derivatives of xylofuranose were obtained in good yields.     

N-hydroxysuccinimide carbonates and carbamates are useful reactive reagents for coupling ligands to lysines on proteins

Ligands containing amino or hydroxyl groups were converted to their corresponding activated N-hydroxysuccinimidyl carbamate and carbonate by reaction with disuccinimidyl carbonate (DSC). The latter reagents can be used for the group-specific modification of primary amines as an alternative to the widespread usage of N-hydroxysuccinimide esters. Biotin and 2,4-dinitrophenyl (DNP) derivatives were used as examples to demonstrate the approach. Biotin and DNP were each extended by attaching two different spacer arms, carrying either a hydroxyl group or a primary amine as terminal functions. The latter were then activated via their conversion to N-hydroxysuccinimide carbonates and carbamates, respectively. The usefulness of these reagents for protein modification was investigated. The modified proteins obtained exhibited similar stability and activity characteristics compared to those modified with active N-hydroxysuccinimdyl esters. The activation of hydroxy- or amino-terminating compounds with DSC represents a general method that can be applied to any ligand which contains these functional groups for its covalent coupling to amines.     

Polymers of biogenic amines.

Biogenic amines, with a primary amino group, were reacted with glutaraldehyde to form insoluble precipitates. These precipitates had distinctive ultrastructural features upon further reaction with osmic acid. When tested in vitro, they had biological activity and showed evidence that part of this biological activity was due to the large polymer of glutaraldehyde and amine. Experiments with isotope-labelled amines in the production of these precipitates showed that the precipitated polymers were not completely stable and that free amine was liberated from them. Since they were not stable, , they could not be used for the morphological localization of the amines as had been intended, but they may have some use as depot drugs or in the immunization of animals against these amines.     

A convenient one-pot synthesis of asymmetric 1,3,5-triazine-2,4,6-triones and its application towards a novel class of gonadotropin-releasing hormone receptor antagonists

A convenient one-pot synthetic route was developed for the preparation of asymmetric 1,3-dialkyl-1,3,5-triazine-2,4,6-triones from readily available alkyl- or aryl-isocyanates, primary amines and N-chlorocarbonyl isocyanate in excellent yields. Subsequent alkylation with N-protected amino alcohols afforded the desired 1,3,5-triazine-2,4,6-triones in good yields. This methodology was applied to the synthesis of a chemical library acting as antagonists of the hGnRH receptor.     

Oxidation of dimethylamine and trimethylamine in methazotrophic yeasts by microsomal mono-oxygenases sensitive to carbon monoxide

Whole cells of Candida boidinii grown on di- or tri-methylamine as sole nitrogen source readily oxidized both amines. The oxidation was potently inhibited by carbon monoxide. Cell-free extracts required the presence of 20 μM FAD before mono-oxygenase activity with both amines could be demonstrated. NADH was a better electron donor than NADPH. Activity was present in cells grown on secondary and tertiary amines but not on primary amines, and was detected in a number of different yeasts. Enzyme activity could be sedimented at 187 000 x g, and was associated with NADPH-cytochrome c reductase activity. It is thus probably microsomal. Activity was inhibited by cyanide, mercaptoethanol, carbon monoxide and proadifen hydrochloride (SKF 525-A).     

Characterization of reaction conditions providing rapid and specific cysteine alkylation for peptide-based mass spectrometry

Alkylation converts Cys thiols to thioethers and prevents unwanted side reactions, thus facilitating mass spectrometric identification of Cys-containing peptides. Alkylation occurs preferentially at Cys due to its high nucleophilicity, however reactions at other such sites are possible. N-ethylmaleimide (NEM) shows rapid reaction kinetics with Cys and careful definition of reaction conditions results in little reactivity at other sites. Analysis of a protein standard alkylated under differing reaction conditions (pH, NEM concentrations and reaction times) was performed using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) and selected reaction monitoring (SRM) of NEM-modified and unmodified peptide pairs. Mis-alkylation sites at primary and secondary amines were identified and limited to one equivalent of NEM. No evidence for hydroxyl or thioether alkylation was observed. Improved specificity was achieved by restricting the pH below neutral, NEM concentration below 10 mM and/or reaction time to below 5 min. Maximal removal of Cys activity was observed in tissue homogenates at 40 mM NEM within 1 min, dependent upon efficient protein denaturation. SRM assays identified peptide-specific levels of mis-alkylation, indicating that NEM-modified to unmodified ratios did not exceed 10%, with the exception of Cys alkylation that proceeded to 100%, and some Lys residues that resulted in tryptic missed cleavages. High reactivity was observed for His residues considering their relatively low abundance. These data indicate that rapid and specific Cys alkylation is possible with NEM under relatively mild conditions, with more abrasive conditions leading to increased non-specific alkylation without appreciable benefit for MS-based proteomics.     

Synthesis and antifungal activity of six benzylic diamines

Six benzylic diamines were synthesised and examined for antifungal activity. Four of the compounds, KB 2, KB 4, KB 5 and KB 6, reduced radial growth of the oat leaf stripe pathogen Pyrenophora avenae, the largest reduction obtained with 25 μM KB 4, which reduced radial growth by 47%. Surprisingly, these four amines had no effect against infection of barley seedlings with the powdery mildew fungus Erysiphe graminis f.sp. hordei. Instead, two different amines, KB 1 and KB 3, reduced powdery mildew infection on barley. The greatest reduction was obtained with 25 μM KB 3, which reduced mildew infection by 69%. All of the amines which exhibited antifungal or fungicidal properties perturbed polyamine formation as measured by the incorporation of labelled ornithine into polyamines.     

Anti-malarial activity of N(6)-substituted adenosine derivatives. Part I

The synthesis and biological evaluation of novel N(6)-substituted adenosine derivatives is reported. The first series of compounds was obtained using an established procedure for the nucleophilic substitution of a 1-(6-chloro-purin-9-yl)-beta-D-1-deoxy-ribofuranose with various amines. In addition, attachment of two different amino-functionalised spacer arms at the N(6)-position of adenosine enabled derivatisation by an innovative polymer-assisted protocol. Thus, we were able to prepare three series of substituted derivatives that displayed activity versus the multiresistant Plasmodium falciparum strain Dd2 in cell culture experiments.     

A simple conductimetric method as an alternative to the colorimetric p-nitrobenzylpyridine test for the measurement of the reactivity of potentially mutagenic alkylating compounds

A simple method for the measurement of the kinetics of reaction of potentially mutagenic alkyl halides with amines, based on the direct conductimetric monitoring of the quaternary ammonium salt produced in these reactions, is proposed and applied to the alkylation of p-nitrobenzylpyridine (NBP) and triethylamine (TEA) in different solvents. With respect to the classical colorimetric NBP-test, this method has the advantage that the rates can be measured continuously over the entire course of the reactions and the kinetic order and constants can be easily obtained. It is also shown that the previously proposed, NBP modified test', using simultaneously NBP and TEA, gives actually the sum of the rate constants for the reactions of the alkylating reagent with the two amines.     

Purification and properties of methylamine dehydrogenase from Paracoccus denitrificans.

Methylamine dehydrogenase from Paracoccus denitrificans was purified to homogeneity in two steps from the periplasmic fraction of methylamine-grown cells. The enzyme exhibited a pI value of 4.3 and was composed of two 46,700-dalton subunits and two 15,500-dalton subunits. Each small subunit possessed a covalently bound pyrrolo-quinoline quinone prosthetic group. The amino acid compositions of the large and small subunits are very similar to those of other methylamine dehydrogenases which have been isolated from taxonomically different sources. The enzyme was able to catalyze the oxidation of a wide variety of primary aliphatic amines and diamines, but it did not react with secondary, tertiary, or aromatic amines. The enzyme exhibited optimal activity at pH 7.5, with Km values of 12.5 microM for methylamine and 156 microM for phenazine ethosulfate and a Vmax of 16.9 mumol/min per mg of protein. No loss of enzyme activity was observed after incubation for 48 h at pH values ranging from 3.0 to 10.5, and the enzyme was very stable to thermal denaturation. Enzyme activity and immunological detection of each subunit were only observed with cells which had been grown on methylamine as a carbon source.     

Synthesis and biological evaluation of pyrroloiminoquinone derivatives

Synthesis of 10 pyrroloiminoquinone derivatives is presented. The strategy is based around the elaboration of a common intermediate by reaction with primary amines. All the compounds obtained have been subjected to antiproliferative activity with three different cell lines (NCI-H460, HeLa, and HL-60). The capacity of 4 selected compounds to affect the enzymatic activity of the nuclear enzyme DNA topoisomerase II and to form the typical DNA fragmentation which occurs in the apoptotic process is discussed here.     

Glutaraldehyde fixation chemistry: oxygen-consuming reactions

In tissue fixed with glutaraldehyde, dissolved O2 is rapidly consumed by two processes: residual respiration and glutaraldehyde-induced chemical uptake. The nature of the chemistry which consumes O2 during tissue fixation was investigated by studying model reactions of glutaraldehyde with amines and with homogenized tissue suspensions. The addition of glutaraldehyde to solutions of most primary amines and ammonia stimulated rapid O2 consumption. The reaction of glutaraldehyde with primary amines (e.g., 25 mM ethanolamine, glycine, or methylamine) consumed 50% of the dissolved O2 in 15 to 20 s at 37 degrees C. The initial rate of O2 uptake followed second-order kinetics with respect to the primary amine concentration. The total amount of O2 consumed was sufficient to account for the stoichiometric conversion of the primary amines to pyridines. These data are consistent with the synthesis of pyridine derivatives from glutaraldehyde-amine precursors in which the last step is an irreversible oxidation of dihydropyridines to pyridines. The addition of glutaraldehyde to homogenized muscle suspensions, in which respiration was chemically inhibited, significantly increased the rate of O2 uptake. Thus, in tissue O2 is rapidly depleted both by respiration and the chemical demands of the glutaraldehyde-amine reactions during the cross-linking process. Since these experiments were done under conditions commonly used for tissue fixation, hypoxia should be assumed to exist in biological preparations fixed with glutaraldehyde.     

Investigation of the 4-O-alkylamine substituent of non-peptide quinolone GnRH receptor antagonists.

Synthesis and in vitro activity of the enantiomers of quinolone GnRH antagonist (+/-)-1 are reported. Chiral amino alcohols were prepared from the appropriate cyclic D- or L-amino acids by the Amdt-Eistert homologation followed by reduction of the resulting esters. Incorporation of these pharmacophores was achieved via a novel Mitsunobu alkylation of 4-hydroxyquinolones. The key amine pharmacophore for binding to the rat GnRH receptor was most active in the S-configuration. Ring size was not important for potency with 4, 5, 6, and 7-membered ring amines exhibiting similar potency.     

Carboxymethyl-substituted bifunctional chelators: preparation of aryl isothiocyanate derivatives of 3-(carboxymethyl)-3-azapentanedioic acid, 3,12-bis(carboxymethyl)-6,9-dioxa-3,12-diazatetradecanedioic++ + acid, and 1,4,7,10-tetraazacyclododecane-N,N',N",N'-tetraacetic acid for use as protein labels

3-(Carboxymethyl)-3-azapentanedioic acid (NTA), 3,12-bis(carboxymethyl)-6,9-dioxa-3,12-diazatetradecanedioic acid (EGTA), and 1,4,7,10-tetraazacyclododecane-N,N',N",N'-tetraacetic acid (DOTA) structures having a 4-nitrophenyl substituent attached via an alkyl spacer to the methylene carbon atom of one carboxymethyl arm of the chelator were obtained by alkylation of 4-nitrophenylalanine with bromoacetic acid (NTA), by reductive alkylation of 1,8-diamino-3,6-dioxaoctane with (4-nitrophenyl)-pyruvic acid followed by alkylation with bromoacetic acid (EGTA), and by alkylation of the trimethyl ester of 1,4,7,10-tetraazacyclododecane-N,N',N"-triacetic acid with the methyl ester of alpha-bromo-4-(4-nitrophenyl)pentanoic acid and subsequent saponification (DOTA). The nitrophenyl-substituted chelators were converted to the corresponding amines by hydrogenation then reacted with thiophosgene to give the protein-reactive aryl isothiocyanate derivatives.     

Pulmonary surfactant apoprotein A structure and modulation of surfactant secretion by rat alveolar type II cells

The pulmonary surfactant apoprotein with a reduced denatured molecular mass of 26-38 kDa (PSP-A) has recently been identified as an inhibitor of surfactant phospholipid secretion by isolated rat alveolar type II cells. We have investigated some of the structural determinants of PSP-A that are relevant to the inhibitory process. The PSP-A was isolated from rats given an intratracheal instillation of silica. The yield of PSP-A from silica-treated animals was 20-40-fold higher than that obtained from untreated animals. Reduction of PSP-A with 2-mercaptoethanol caused a reversible loss of biological activity that was restored by mild oxidation. Alkylation of the protein with excess iodoacetamide also led to inactivation, although titration with 5,5'-dithiobis-(2-nitrobenzoic acid) indicated that the protein initially contained no free sulfhydryl moieties. Neither alkylation nor reduction plus alkylation completely prevented the formation of oligomers as determined by gel permeation analysis. The apparent molecular mass of PSP-A at 4 degrees C in low ionic strength buffers was 1.6 megadaltons, and at 37 degrees C in normal ionic strength buffers was greater than 1.5 megadaltons. Removal of the oligosaccharide moiety with endoglycosidase F also had no effect upon biological activity. Five distinct monoclonal antibodies recognizing peptides epitopes on PSP-A were produced. All monoclonal antibodies exhibited similar affinity for PSP-A and recognized the delipidated and deglycosylated form. Four monoclonal antibodies reacted with epitopes on PSP-A that altered its function as an inhibitor. One monoclonal antibody was clearly ineffective at altering the activity of PSP-A. These results demonstrate that: 1) disulfide bonds are required for the activity of PSP-A, 2) disruption of disulfides does not prevent the formation of oligomeric forms of PSP-A, 3) the oligosaccharide moiety is not essential for biological activity, and 4) monoclonal antibodies can be used to map the epitopes responsible for biological activity.     

A combinatorial peptoid library for the identification of novel MSH and GRP/bombesin receptor ligands

A tripeptoid library was synthesized using 69 different primary amines in initially 69 individual reactions by the mix and split approach. The resulting library consisted of 328,509 (69(3)) single compounds, divided in 69 subpools each containing 4,761 entities. The 69 subpools were tested in two binding assays, one for alpha-MSH (alpha-melanotropin) and one for GRP (gastrin-releasing peptide)/bombesin. The sublibraries with the highest affinity to the MSH receptor (i.e. melanocortin type 1 or MC1 receptor) and, respectively, the GRP-preferring bombesin receptor were identified by an iterative process. Individual tripeptoids with good binding activity were resynthesized, analyzed and their dissociation constants and biological activity determined. The KD of the most potent MC1 receptor ligand was 1.58 mumol/l and that of the GRP-preferring bombesin receptor 3.40 mumol/l. Extension of this latter tripeptoid structure whose KD value increased to 280 nmol/l. A similar increase in activity was not observed with the most potent MSH tripeptoid ligand when extended by one residue, but a compound suitable for radioiodination and lacking the N-terminal amino group had a slightly higher binding activity than the tripeptoids (KD approximately 850 nmol/l). These results demonstrate that testing a peptoid library containing 328,509 single compounds led to the successful identification of new ligands for both the MC1 receptor as well as the GRP-preferring bombesin receptor.     

Substrate adhesion of rat hepatocytes: a comparison of laminin and fibronectin as attachment proteins

In previous studies rat hepatocytes have been shown to adhere to substrates composed of collagen or fibronectin. In the present communication, the basement membrane protein laminin is reported to mediated the attachment and spreading of hepatocytes. The cell attachment-mediating activity of laminin was compared with that of fibronectin. The activity of fibronectin was heat sensitive, whereas laminin retained its activity after boiling. On the other hand, reduction and alkylation or periodate oxidation of the proteins affected only the cell attachment activity of laminin. Preincubation of cells with soluble fibronectin inhibited initial cell attachment to fibronectin but not to laminin substrates, and, reversely, soluble laminin selectively inhibited cell attachment to laminin. These results suggest that attachment of cells to substrates of the two proteins involves different cellular receptors recognizing distinct and nonidentical structures in the proteins.     

Sulfate Conjugation of Dopamine in Rat Brain: Regional Distribution of Activity and Evidence for Neuronal Localization

Brain tissue contains at least two forms of phenolsulfotransferase that are involved in the sulfate conjugation of biogenic amines and their metabolites. Two apparent Km values were obtained for p-nitrophenol at pH 7.4 (0.6 microM and 0.3 mM) but only one enzyme had the capacity to conjugate dopamine (Km = 130 microM). Dopamine sulfotransferase activity was found to vary 17-fold in different brain regions, with the highest levels in diencephalon, hippocampus, and striatum. To determine the cellular localization of the enzymes, phenolsulfotransferase activity was measured in striatum following selective destruction of striatal neurons by stereotaxic injection of 2 micrograms kainic acid. Fourteen days after injection the catecholamine sulfotransferase activity in the lesioned striatum was reduced to approximately 40-50% of that in the control contralateral striatum. There was a statistically significant correlation between the ratio of lesioned to control activity for the sulfotransferase and the neuronal marker enzymes glutamate decarboxylase and neuron-specific enolase. p-Nitrophenol sulfotransferase activity was also decreased in the lesioned striatum. These results suggest that PST activity is present within the kainic acid-sensitive neurons of the striatum. The regional variation in activity, together with the results of the kainic acid studies, suggest that sulfate conjugation of biogenic amines and their metabolites in brain may take place within specific types of neurons.     

Inhibition of platelet aggregation by primary amines. Evidence for a possible role of membrane-associated calcium

The aggregation of human blood platelets by thrombin, adenosine diphosphate, wheat germ agglutinin or ristocetin was inhibited by primary amines. In general, thrombin-induced platelet aggregation was strongly affected by the amines while the effect was weak on cell aggregation by ristocetin. Usually, the diamines were stronger inhibitors of aggregation than the monoamines with cadaverine as the strongest and ethylamine as the weakest inhibitor. At concentration where platelet aggregation was inhibited, the amines neither displaced serotonin from serotinin-loaded platelets nor caused lysis of human red cells. The lectin activity of wheat germ agglutinin on human red cells was not affected by the amines indicating that the amines probably acted on platelets and not on the agglutinin. The clotting activity of thrombin on fibrinogen was partially inhibited by the amines while its esterolytic activity remained unaltered. The inhibitory action of the amines on platelet aggregation could be overcome with small amounts of calcium while other divalent cations tested had little effect. It is suggested that the amines affect platelet aggregation by interfering with the actions of membrane-associated calcium.