Amino-terminal Amino Acid Sequence Research Articles

Heterologous Overexpression and Characterization of a Flavoprotein-Cytochrome c Complex Fructose Dehydrogenase of Gluconobacter japonicus NBRC3260

A heterotrimeric flavoprotein-cytochrome c complex fructose dehydrogenase (FDH) of Gluconobacter japonicus NBRC3260 catalyzes the oxidation of d-fructose to produce 5-keto-d-fructose and is used for diagnosis and basic research purposes as a direct electron transfer-type bioelectrocatalysis. The fdhSCL genes encoding the FDH complex of G. japonicus NBRC3260 were isolated by a PCR-based gene amplification method with degenerate primers designed from the amino-terminal amino acid sequence of the large subunit and sequenced. Three open reading frames for fdhSCL encoding the small, cytochrome c, and large subunits, respectively, were found and were presumably in a polycistronic transcriptional unit. Heterologous overexpression of fdhSCL was conducted using a broad-host-range plasmid vector, pBBR1MCS-4, carrying a DNA fragment containing the putative promoter region of the membrane-bound alcohol dehydrogenase gene of Gluconobacter oxydans and a G. oxydans strain as the expression host. We also constructed derivatives modified in the translational initiation codon to ATG from TTG, designated (TTG)FDH and (ATG)FDH. Membranes of the cells producing recombinant (TTG)FDH and (ATG)FDH showed approximately 20 times and 100 times higher specific activity than those of G. japonicus NBRC3260, respectively. The cells producing only FdhS and FdhL had no fructose-oxidizing activity, but showed significantly high d-fructose:ferricyanide oxidoreductase activity in the soluble fraction of cell extracts, whereas the cells producing the FDH complex showed activity in the membrane fraction. It is reasonable to conclude that the cytochrome c subunit is responsible not only for membrane anchoring but also for ubiquinone reduction.

Oxygen sensing in plants is mediated by an N-end rule pathway for protein destabilization

The majority of eukaryotic organisms rely on molecular oxygen for respiratory energy production. When the supply of oxygen is compromised, a variety of acclimation responses are activated to reduce the detrimental effects of energy depletion. Various oxygen-sensing mechanisms have been described that are thought to trigger these responses, but they each seem to be kingdom specific and no sensing mechanism has been identified in plants until now. Here we show that one branch of the ubiquitin-dependent N-end rule pathway for protein degradation, which is active in both mammals and plants, functions as an oxygen-sensing mechanism in Arabidopsis thaliana. We identified a conserved amino-terminal amino acid sequence of the ethylene response factor (ERF)-transcription factor RAP2.12 to be dedicated to an oxygen-dependent sequence of post-translational modifications, which ultimately lead to degradation of RAP2.12 under aerobic conditions. When the oxygen concentration is low-as during flooding-RAP2.12 is released from the plasma membrane and accumulates in the nucleus to activate gene expression for hypoxia acclimation. Our discovery of an oxygen-sensing mechanism opens up new possibilities for improving flooding tolerance in crops.

Purification and characterisation of two extracellular acid proteinases from Monascus pilosus

Genus Monascus is one of the most important microorganisms in the fermentation industry in Asia. However, only a little attention has been paid to the proteinases produced by this fungus and their role in the fermentation process. The main objective of this study was to purify and characterise acid proteinases produced by Monascus pilosus. Two acid proteinases (MpiAP1 and MpiAP2) were purified to homogeneity. Both purified enzymes, MpiAP1 and MpiAP2, were monomeric structures with molecular masses of around 43 and 58 kDa, respectively. The former was an acidic non-glycoprotein, whereas the latter was an acidic glycoprotein with 27% carbohydrate content. Although amino-terminal amino acid sequence analysis of both enzymes (MpiAP1 and MpiAP2) of 20 amino acid length showed over 90% similarity, their amino-terminal amino acids were different from each other. Both enzymes were optimally active at 55 °C and at pH 2.5–3.0 against casein or human haemoglobin. The T 1/2 values of MpiAP1 and MpiAP2 were 65 and 70 °C, respectively. Both of the enzymes were completely inhibited by pepstatin A, and markedly by SDS. MoO 3 also showed a partial inhibition of both enzymes. Milk casein and haemoglobin were good substrates for these enzymes. Eleven cleavages were detected using the oxidised insulin B-chain as a peptide for the proteolytic specificity test of MpiAP1, while seven cleavages were detected for MpiAP2.

Inhibition of a novel sperm gelatinase in prawn sperm by the male reproduction‐related kazal‐type peptidase inhibitor

Previously, we have identified and characterized a male reproduction-related kazal-type peptidase inhibitor (MRPINK) gene from the prawn, Macrobrachium rosenbergii. In the present study, MRPINK was discovered to have an inhibitory effect on the gelatinolytic activity of M. rosenbergii sperm and immunofluorescence analysis revealed it bound specifically onto the base of sperm. The proteolytic activity of sperm extracts to vitelline coat components was also detected to be interfered by MRPINK. Furthermore, a novel gelatinase on sperm was found to be specifically inhibited by MRPINK and was named M. rosenbergii sperm gelatinase (MSG). MSG was then isolated and purified by reversed-phase high performance liquid chromatography combining with gelatinolytic assay. By amino-terminal amino acid sequence analysis and molecular cloning, the primary structure of MSG was determined. The data presented in this study provided evidence that MRPINK has an inhibitory effect on the gelatinolytic activity as well as proteolytic activity of prawn sperm and specifically blocks the activity of MSG.

Proteolysis of purified IgGs by human and bacterial enzymes in vitro and the detection of specific proteolytic fragments of endogenous IgG in rheumatoid synovial fluid

A comparative in vitro survey of physiologically relevant human and microbial proteinases defined a number of enzymes that induced specific hinge domain cleavage in human IgG1. Several of these proteinases have been associated with tumor growth, inflammation, and infection. A majority of the identified proteinases converted IgG to F(ab′) 2, and a consistent feature of their action was a transient accumulation of a single-cleaved intermediate (scIgG). The scIgG resulted from the relatively rapid cleavage of the first hinge domain heavy chain, followed by a slower cleavage of the second chain to separate the Fc domain from F(ab′) 2. Major sites of enzymatic cleavage were identified or confirmed from the mass of the F(ab′) 2 or Fab fragments and/or the amino-terminal amino acid sequence of the Fc for each enzyme including human matrix metalloproteinases (MMPs) 3 and 12, human cathepsin G, human neutrophil elastase (Fab), staphylococcal glutamyl endopeptidase I and streptococcal immunoglobulin-degrading enzyme (IdeS). The cleavage sites in IgG1 by MMP-3, cathepsin G and IdeS were used to guide the synthesis of peptide analogs containing the corresponding carboxy-termini to be used as immunogens in rabbits. Rabbit antibodies were successfully generated that showed selective binding to different human F(ab′) 2s and other hinge-cleavage fragments, but not to intact IgG. In Western blotting studies of synovial fluids from individuals with rheumatoid arthritis, the rabbit antibodies yielded patterns consistent with the presence of endogenous IgG fragments including F(ab′) 2 and the single-cleaved IgG intermediate. The detection in synovial fluid of IgG fragments similar to those observed in the in vitro biochemical studies suggests that proteolysis of IgG may contribute to localized immune dysfunction in inflammatory environments.

Distinguishing Aggrecan Loss from Aggrecan Proteolysis in ADAMTS-4 and ADAMTS-5 Single and Double Deficient Mice

Aggrecan loss from mouse cartilage is predominantly because of ADAMTS-5 activity; however, the relative contribution of other proteolytic and nonproteolytic processes to this loss is not clear. This is the first study to compare aggrecan loss with aggrecan processing in mice with single and double deletions of ADAMTS-4 and -5 activity (Deltacat). Cartilage explants harvested from single and double ADAMTS-4 and -5 Deltacat mice were cultured with or without interleukin (IL)-1alpha or retinoic acid and analyzed for (i) the kinetics of (35)S-labeled aggrecan loss, (ii) the pattern of (35)S-labeled aggrecan fragments released into the media and retained in the matrix, (iii) the pattern of total aggrecan fragments released into the media and retained in the matrix, and (iv) specific cleavage sites within the interglobular and chondroitin sulfate-2 domains. The loss of radiolabeled aggrecan from ADAMTS-4/-5 Deltacat cartilage was less than that from ADAMTS-4, ADAMTS-5, or wild-type cartilage under nonstimulated conditions. IL-1alpha and retinoic acid stimulated radiolabeled aggrecan loss from wild-type and ADAMTS-4 Deltacat cartilage, but there was little effect on ADAMTS-5 cartilage. Proteolysis of aggrecan contributed most to its loss in wild-type, ADAMTS-4, and ADAMTS-5 Deltacat cartilage explants. The pattern of proteolytic processing of aggrecan in these cultures was consistent with that occurring in cartilage pathologies. Retinoic acid, but not IL-1alpha, stimulated radiolabeled aggrecan loss from ADAMTS-4/-5 Deltacat cartilage explants. Even though there was a 300% increase in aggrecan loss from ADAMTS-4/-5 Deltacat cartilage stimulated with retinoic acid, the loss was not associated with aggrecanase cleavage but with the release of predominantly intact aggrecan consistent with the phenotype of the ADAMTS-4/-5 Deltacat mouse. Our results show that chondrocytes have additional mechanism for the turnover of aggrecan and that when proteolytic mechanisms are blocked by ablation of aggrecanase activity, nonproteolytic mechanisms compensate to maintain cartilage homeostasis.

Preparation and characterization of a potent, long-lasting recombinant human serum albumin-interferon-α2b fusion protein expressed in Pichia pastoris

A long-lasting recombinant human serum albumin-interferon-α2b fusion protein (rHSA/IFNα2b) was prepared and its structure and biological activities were studied. rHSA/IFNα2b was expressed in methylotrophic yeast Pichia pastoris with HSA’s natural signal peptide and purified by dye affinity chromatography, hydrophobic interaction chromatography, ion exchange chromatography and Sephadex G25. Purity of the prepared rHSA/IFNα2b was greater than 97% analyzed by non-reduced SDS–PAGE and RP-HPLC. Structure and biological activities of the prepared rHSA/IFNα2b were characterized by physical, chemical and biological methods. Its p I was 5.3 and showed a single band on IEF gel. Molecular weight determined by MALDI-TOF was 86004.3 ± 29.2. Amino-terminal and carboxyl-terminal amino acid sequences were identical to predicted sequence. Its specific activity in vitro was 6.3 ± 0.8 × 10 5 IU/mg fusion protein, retaining about 1.4% of that of unmodified rIFNα on a molar basis. After administered in monkeys, significant increases of 2′,5′-oligoadenylate synthetase activity relative to IFN-α were maintained for 14 days in serum and the rHSA/IFNα2b showed more potent biological activity than IFN-α on a molar basis. Therefore, markedly improved in vivo biological activity of rHSA/IFNα2b could exhibit more potent antiviral activity than IFNα2b in future clinical trials.

Identification of a novel, highly variable amino-terminal amino acid sequence element in the nuclear intermediate filament protein lamin B 2 from higher vertebrates

By comparing newly available cDNA sequences of the human intermediate filament protein lamin B 2 with published sequences, we have identified an additional translation initiation codon 60 nucleotides upstream of the previously assumed translation start. In addition, corresponding sequences were identified in the chimpanzee, mouse, rat and bovine genes and cDNAs, respectively. Therefore, we generated antibodies against these potential 20 new amino acids of the human sequence. By immunoblot analysis and immunofluorescence microscopy we show that human lamin B 2 is indeed synthesized as a longer version than previously reported, because it contains these additional 20 amino acids. Notably, the sequence homology to mouse, rat and bovine lamin B 2 is significantly lower in this segment than in that between the second methionine codon and the start of the α-helical rod indicating that the tip of the “head” is engaged in more species-specific functions. Forced expression of the GFP-tagged authentic “long” and the 20 amino acid shorter version of lamin B 2 in human cultured SW-13 cells demonstrated that both the longer and the shorter version are properly integrated into the nuclear lamina, although the shorter version exhibited a tendency to disturb envelope architecture at higher expression levels.

Proteome Analysis of an Aerobic Hyperthermophilic Crenarchaeon, Aeropyrum pernix K1

We analyzed the proteome of a crenararchaeon, Aeropyrum pernix K1, by using the following four methods: (i) two-dimensional PAGE followed by MALDI-TOF MS, (ii) one-dimensional SDS-PAGE in combination with two-dimensional LC-MS/MS, (iii) multidimensional LC-MS/MS, and (iv) two-dimensional PAGE followed by amino-terminal amino acid sequencing. These methods were found to be complementary to each other, and biases in the data obtained in one method could largely be compensated by the data obtained in the other methods. Consequently a total of 704 proteins were successfully identified, 134 of which were unique to A. pernix K1, and 19 were not described previously in the genomic annotation. We found that the original annotation of the genomic data of this archaeon was not adequate in particular with respect to proteins of 10-20 kDa in size, many of which were described as hypothetical. Furthermore the amino-terminal amino acid sequence analysis indicated that surprisingly the translation of 52% of their genes starts with TTG in contrast to ATG (28%) and GTG (20%). Thus, A. pernix K1 is the first example of an organism in which TTG is the most predominant translational initiation codon.

Purification and properties of citrate synthase from Acetobacter europaeus.

Citrate synthase (EC 4.1.3.7) was purified from the acidophilic bacterium Acetobacter europaeus to electrophoretic homogeneity. The specific activity was 228 units/mg of protein during the exponential ethanol-oxidation growth phase. The enzyme has a molecular mass of 280 kDa and is a hexamer with a subunit size of 46 kDa. The apparent K(m) values were 20 microM for oxaloacetate and 51 microM for acetyl-CoA. Unlike citrate synthase from other Gram-negative bacteria, the activity of the enzyme was inhibited by ATP, slightly enhanced by ADP and not effected by NADH. Acetate caused activation of the enzyme. The pH optimum on the citrate synthase activity in vitro was 8.1. The amino-terminal amino acid sequence of the purified enzyme was ENGKSATISLNGKDVALPVL.

Mouse Inter-Alpha-Trypsin Inhibitor Family Heavy Chain-Related Protein is an Acute Phase Protein Induced by Inflammation

The mouse counterpart of the human inter-alpha-trypsin inhibitor family heavy chain-related protein (IHRP), inter-alpha-trypsin inhibitor family heavy chain-related protein, was partially purified from mouse serum through Western blot analysis using anti-human IHRP antibody. The amino-terminal amino acid sequence was then determined, and the cDNA clone encoding mouse IHRP was isolated and characterized. The mouse IHRP amino-terminal amino acid sequence matched the predicted sequence from the cDNA, and has high homology compared with human and pig IHRP. Furthermore, the whole mouse IHRP amino acid sequence predicted from the cDNA nucleotide sequence showed reasonable homology to that of both human and pig IHRP. Mouse IHRP expression was induced by treatment with typical inflammation-inducing compounds, such as turpentine and lipopolysaccharide.

Cloning and sequencing of an exoglucanase gene from Streptomyces sp. M23, and its expression in Streptomyces lividans TK-24

A gene encoding exoglucanase (CBHII) of Streptomyces sp. M 23 was cloned and sequenced. The cbhII gene consisted of 1359 bp capable of encoding a polypeptide of 453 amino acids with a calculated molecular mass of 45,175 Da. The deduced amino acid sequence showed homology with those of cellulases belonging to family 6 of the glycosyl hydrolases. The cbhII gene was subcloned into the plasmid pSEV1 and expressed in Streptomyces lividans TK-24. The transformed cells were able to secrete the enzyme efficiently in an active form. The CBHII expressed by S. lividans TK-24 was purified to homogeneity by SDS-polyacrylamide gel and characterized. The recombinant CBHII was stable up to 50 degrees C and more than 30% of the original activity remained after heating at 100 degrees C for 10 min. The amino-terminal amino acid sequence of the recombinant CBHII was identified as GPAAPTARVD. These results agreed well with the properties of the authentic CBHII.

CHARACTERIZATION OF DECORIN OF THE HUMAN YELLOW LIGAMENT

Decorin, a component of the low-molecular-weight proteoglycans (PG) of yellow Iigaments was purified, and the structures of the core protein and the glycosaminoglycan (GAG) chains were analyzed. The crude PG fraction extracted under dissociative conditions was subjected to DEAE-Sephacel ion-exchange chromatography, gel-filtration on Sepharose CL-4B, and Octyl-Sepharose CL-4B chromatography, and decorin was obtained. The amino-terminal amino acid sequence of decorin was found to be identical to those of human bone PG-II and human fetal membrane PG-II. The analyses of the GAG chains using two-dimensional electrophoresis on cellulose acetate membranes, affinity high-performance liquid chromatography (HPLC) with a hydroxyapatite column and gel-filtration HPLC after chondroitinase digestion, showed the presence of dermatan sulfate (DS) chains and chondroitin sulfate (CS). Further, it was suggested that CS located in the reducing termini and DS in the non-reducing termini in the hybrid chain. Hirosaki Med. J. 56: 45-54, 2005 ~~ g~~~0~~~0h~~m~~~ ~~0~~.~.*~GM~G, ~.~0~~~57~-~M~G~ ~, ~0~ 7 -3l J\~:BetU!f I) -!t -2 J ~ 0 fJ (GAG) mt~ji0t~m~~~fJT Gt.::. 7 -3l~)\~ 0N*jJiM7 -2 J ~~2:71J~J: e: l-~PG-II:B etUe: ~ij~ij~PG-II0~hC :-3G)t.::. GAGM~ji0-t)vD-;Z 7-t~-~ij~=jzjI;~%r*J} ), /\-1 ~0:::f:~7/\-3l-1 ~fJ ~~fJT~ ~J:, GAGM~ji~J:JijI;*j/iM{WH::~ 0 -1T ~1Jj1t~ (CS), ~FJijI;*j/iM{J!lH::~)v~ -3l ~~1rE~ (DS) i6~~tt9~/\-1 :1' I) 'Y ~~M 1) ~b*tk~~~ff~~*H1. *4~~fiJ~ 2) %iW*~~~ff~~-j::1 t;~~fiJ~

DNA-Binding Protein Involved in the Regulation of Chitinase Production inStreptomyces lividans

A protein that binds specifically to the promoter region of chiA was purified from the cell lysate of Streptomyces lividans by using an affinity purification method. Determining the amino-terminal amino acid sequence of the purified protein led to cloning of a gene (cpb1) encoding the chitinase promoter-binding protein, Cpb1. The deduced amino acid sequence of Cpb1 showed significant similarity to the sequences of a group of hypothetical proteins of S. coelicolor that have been revealed by the genome project, and the amino-terminal region of Cpb1 showed similarity to the DNA-binding domains of several transcription factors. The Cpb1 proteins expressed in S. lividans or Escherichia coli showed specific binding activity to the chiA promoter. The disruption of cpb1 resulted in partial relief of the glucose repression of chitinase production, indicating that cpb1 took part in the regulation of chitinase expression in S. lividans.

Major outer membrane proteins in moderately halophilic eubacteria of genera Chromohalobacter and Halomonas.

Outer and inner membrane fractions of Chromohalobacter marismortui and Halomonas elongata were isolated by differential detergent solubilization, and profiles of membrane proteins, especially major outer membrane proteins, were analyzed. These type strains possessed one extremely abundant outer membrane protein which showed similarity in amino-terminal amino acid sequence with the outer membrane porin proteins in other Gram-negative bacteria. Three halophilic eubacterial strains isolated from saline environments were also characterized. Strains 160 and 43 were found to be Chromohalobacter spp. and strain 40 to be a Halomonas sp. by sequence analysis of their 16 S ribosomal RNA genes. Extremely abundant porin proteins with an apparent molecular mass of 49 kDa were found in Chromohalobacter sp.160 and Halomonas sp. 40, but no major outer membrane protein was detected in Chromohalobacter sp. 43, suggesting strain 43 was most likely a naturally defective porin mutant. Porin proteins from Chromohalobacter spp. and Halomonas spp. showed the same migration on SDS-polyacrylamide gel electrophoresis with or without heat-treatment, indicating that these porin proteins did not form a SDS-resistant trimeric structure, which was detected in most of the Gram-negative bacterial porin proteins.

Characterisation of proteoglycans and their catabolic products in tendon and explant cultures of tendon.

Tendons are collagenous tissues made of mainly Type I collagen and it has been shown that the major proteoglycans of tendons are decorin and versican. Little is still known about the catabolism of these proteoglycans in tendon. Therefore, the aim of the study was to characterise the proteoglycans including their catabolic products present in uncultured bovine tendon and in the explant cultures of tendon. In this study, the proteoglycans were extracted from the tensile region of deep flexor tendon and isolated by ion-exchange chromatography and after deglycosylation analysed by SDS-polyacrylamide electrophoresis, Western blotting and amino-terminal amino acid sequence analysis. Based on amino acid sequence analysis, approximately 80% of the total proteoglycan core proteins in fresh tendon was decorin. Other species that were detected were biglycan and the large proteoglycans versican (splice variants V(0) and/or V(1)) and aggrecan. Approximately 35% of decorin present in the matrix showed carboxyl-terminal proteolytic processing at a number of specific sites. The analysis of small proteoglycans lost to the medium of tendon explants showed the presence of biglycan and decorin with the intact core protein as well as decorin fragments that contained the amino terminus of the core protein. In addition, two core protein peptides of decorin starting at residues K(171) and D(180) were observed in the matrix and one core protein with an amino-terminal sequence commencing at G(189) was isolated from the culture medium. The majority of the large proteoglycans present in the matrix of tendon were degraded and did not contain the G1 globular domain. Furthermore the aggrecan catabolites present in fresh tendon and lost to the medium of explants were derived from aggrecanase cleavage of the core protein at residues E(373)-A(374), E(1480)-G(1481) and E(1771)-A(1772). The analysis of versican catabolites (splice variants V(0) and/or V(1)) also showed evidence of degradation of the core protein by aggrecanase within the GAG-beta subdomain, as well as cleavage by other proteinase(s) within the GAG-alpha and GAG-beta subdomains of versican (variants V(0) and/or V(2)). Degradation products from the amino terminal region of type XII collagen were also detected in the matrix and medium of tendon explants. This work suggests a prominent role for aggrecanase enzymes in the degradation of aggrecan and to a lesser extent versican. Other unidentified proteinases are also involved in the degradation of versican and small leucine-rich proteoglycans.

Characterisation of proteoglycans and their catabolic products in tendon and explant cultures of tendon

Tendons are collagenous tissues made of mainly Type I collagen and it has been shown that the major proteoglycans of tendons are decorin and versican. Little is still known about the catabolism of these proteoglycans in tendon. Therefore, the aim of the study was to characterise the proteoglycans including their catabolic products present in uncultured bovine tendon and in the explant cultures of tendon. In this study, the proteoglycans were extracted from the tensile region of deep flexor tendon and isolated by ion-exchange chromatography and after deglycosylation analysed by SDS-polyacrylamide electrophoresis, Western blotting and amino-terminal amino acid sequence analysis. Based on amino acid sequence analysis, ∼80% of the total proteoglycan core proteins in fresh tendon was decorin. Other species that were detected were biglycan and the large proteoglycans versican (splice variants V0 and/or V1) and aggrecan. Approximately 35% of decorin present in the matrix showed carboxyl-terminal proteolytic processing at a number of specific sites. The analysis of small proteoglycans lost to the medium of tendon explants showed the presence of biglycan and decorin with the intact core protein as well as decorin fragments that contained the amino terminus of the core protein. In addition, two core protein peptides of decorin starting at residues K171 and D180 were observed in the matrix and one core protein with an amino-terminal sequence commencing at G189 was isolated from the culture medium. The majority of the large proteoglycans present in the matrix of tendon were degraded and did not contain the G1 globular domain. Furthermore the aggrecan catabolites present in fresh tendon and lost to the medium of explants were derived from aggrecanase cleavage of the core protein at residues E373–A374, E1480–G1481 and E1771–A1772. The analysis of versican catabolites (splice variants V0 and/or V1) also showed evidence of degradation of the core protein by aggrecanase within the GAG-β subdomain, as well as cleavage by other proteinase(s) within the GAG-α and GAG-β subdomains of versican (variants V0 and/or V2). Degradation products from the amino terminal region of type XII collagen were also detected in the matrix and medium of tendon explants. This work suggests a prominent role for aggrecanase enzymes in the degradation of aggrecan and to a lesser extent versican. Other unidentified proteinases are also involved in the degradation of versican and small leucine-rich proteoglycans.

A conspicuous nickel protein in microbial mats that oxidize methane anaerobically.

Anaerobic oxidation of methane (AOM) in marine sediments is an important microbial process in the global carbon cycle and in control of greenhouse gas emission. The responsible organisms supposedly reverse the reactions of methanogenesis, but cultures providing biochemical proof of this have not been isolated. Here we searched for AOM-associated cell components in microbial mats from anoxic methane seeps in the Black Sea. These mats catalyse AOM rather than carry out methanogenesis. We extracted a prominent nickel compound displaying the same absorption spectrum as the nickel cofactor F430 of methyl-coenzyme M reductase, the terminal enzyme of methanogenesis; however, the nickel compound exhibited a higher molecular mass than F430. The apparent variant of F(430) was part of an abundant protein that was purified from the mat and that consists of three different subunits. Determined amino-terminal amino acid sequences matched a gene locus cloned from the mat. Sequence analyses revealed similarities to methyl-coenzyme M reductase from methanogenic archaea. The abundance of the nickel protein (7% of extracted proteins) in the mat suggests an important role in AOM.

Generation and characterization of recombinant unmodified and phosphorylatable murine IFN-alpha1 in the methylotropic yeast Pichia pastoris.

In order to generate reagents to study the murine type I interferon (IFN) system, recombinant murine IFN-alpha1 (rMuIFN-alpha1) protein was expressed in the methylotropic yeast Pichia pastoris. rMuIFN-alpha1 with a phosphate acceptor site engineered at the C-terminus (rMuIFN-alpha1P) to enable radiolabeling by gamma(32)P-ATP and cAMP-dependent protein kinase was also generated. Proteins of 20, 25 (MuIFN-alpha1) and 25.5 (MuIFN-alpha1P), kDa were detected in the yeast growth medium, had type I IFN activity, and were recognized by antimurine L929 cell IFN antibodies. The MuIFN-alpha1 proteins produced in P. pastoris were a mixture of glycosylated and unglycosylated forms, with sugars of approximately 5 kDa added via N-linked glycosylation. The recombinant proteins were highly purified using a single RP-HPLC elution step, and their authenticity was confirmed by amino-terminal amino acid sequencing. The MuIFN-alpha1 and MuIFN-alpha1P protein preparations had specific antiviral activities of 1.3 x 10(7) and 4.7 x 10(6) IU/mg protein, respectively. MuIFN-alpha1P could be radiolabeled to a high specific radioactivity (0.6-2 x 10(8) cpm/microg protein) with gamma(32)P-ATP and cAMP-dependent protein kinase without significantly altering its biologic activity or electrophoretic properties. Binding experiments on COS-7 cells transiently transfected with MuIFNAR-2 and IFNAR-2 demonstrated specific and dose-dependent binding of gamma(32)P-ATP-MuIFN-alpha1P to cell surface type I IFN receptors.

Oxygen-insensitive nitroreductases NfsA and NfsB of Escherichia coli function under anaerobic conditions as lawsone-dependent Azo reductases.

Quinones can function as redox mediators in the unspecific anaerobic reduction of azo compounds by various bacterial species. These quinones are enzymatically reduced by the bacteria and the resulting hydroquinones then reduce in a purely chemical redox reaction the azo compounds outside of the cells. Recently, it has been demonstrated that the addition of lawsone (2-hydroxy-1,4-naphthoquinone) to anaerobically incubated cells of Escherichia coli resulted in a pronounced increase in the reduction rates of different sulfonated and polymeric azo compounds. In the present study it was attempted to identify the enzyme system(s) responsible for the reduction of lawsone by E. coli and thus for the lawsone-dependent anaerobic azo reductase activity. An NADH-dependent lawsone reductase activity was found in the cytosolic fraction of the cells. The enzyme was purified by column chromatography and the amino-terminal amino acid sequence of the protein was determined. The sequence obtained was identical to the sequence of an oxygen-insensitive nitroreductase (NfsB) described earlier from this organism. Subsequent biochemical tests with the purified lawsone reductase activity confirmed that the lawsone reductase activity detected was identical with NfsB. In addition it was proven that also a second oxygen-insensitive nitroreductase of E. coli (NfsA) is able to reduce lawsone and thus to function under adequate conditions as quinone-dependent azo reductase.