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Alfons Lawen

Publications and source records attributed to Alfons Lawen.

15 recordsLinked to original sources
PubMed2005

An improved purification procedure for cyclosporin synthetase.

We have developed expedient and reliable methods to isolate cyclosporin synthetase for in vitro biosynthesis of cyclosporins. We have examined enzyme purification strategies suited to large-scale processing and present a chromatographic sequence that serves as a pilot model for industrial scale preparation of cyclosporin synthetase from cyclosporin producing fungi. A chromatographic sequence consisting of ammonium sulfate precipitation-->gel filtration-->hydrophobic interaction chromatography-->anion exchange chromatography, yielded an electrophoretically homogeneous cyclosporin synthetase preparation (Coomassie G-250 brilliant blue staining). Furthermore, a native polyacrylamide gel electrophoresis system was developed for the isolation of active cyclosporin synthetase enzyme from crude extracts of cyclosporin producing fungi. The environmental factors affecting enzyme stability and the continuity of the in vitro cyclosporin biosynthetic reaction-temperature, pH, and substrate depletion were assessed and manageable conditions have been defined for sustainable cyclosporin biosynthesis with enzyme isolates. Cyclosporin synthetase exhibited an optimal temperature range of 24-29 degrees C and a pH optimum of 7.6. The native enzyme displayed a pI of 5.7, as determined by isoelectric focusing. The industrial implementation of an in vitro biosynthetic approach could potentially prove useful for the production of important therapeutic cyclosporins which occur as only minor fermentation by-products.

Chromatography↗
PubMed2005

Voltage-dependent anion-selective channel 1 (VDAC1)--a mitochondrial protein, rediscovered as a novel enzyme in the plasma membrane.

The eukaryotic porin or voltage-dependent anion-selective channel (VDAC1) is a pore-forming protein discovered twenty five years ago in the mitochondrial outer membrane. Its gene in eukaryotes is known, but its tertiary structure has never been solved. Structure predictions highlight the presence of several amphipathic beta-strands possibly organised in a beta-barrel. VDAC1 has recently been described as being a NADH:ferricyanide reductase in the plasma membrane. There it affects the regulation of cell growth and death. Physiological cell death (apoptosis) has become a major research focus of biomedical research. Regulation of the enzyme will have impacts on cancer and autoimmune diseases (insufficient apoptosis) as well as neurodegenerative diseases (excessive apoptosis). VDAC1 in the plasma membrane establishes a novel level of apoptosis regulation putatively via its redox activity.

Animals↗
PubMed2004

Plasma membrane NADH-oxidoreductase in cells carrying mitochondrial DNA G11778A mutation and in cells devoid of mitochondrial DNA (rho0).

The mammalian plasma membrane (PM) NADH-oxidoreductase (PMOR) system is a multi-enzyme complex located in the plasma membrane of all eukaryotic cells, harboring at least two distinct activities, the plasma membrane NADH-ferricyanide reductase and the NADH-oxidase. To assess the behaviour of the two activities of the PMOR system, we measured the NADH-ferricyanide reductase and NADH-oxidase activities in fibroblast cell lines derived from patients carrying a mitochondrial DNA (mtDNA) G11778A mutation. We also measured the two activities in other cell lines, the HL-60 and HeLa (S3) lines, as well as in rho0 cells (cells devoid of mtDNA) generated from those lines and the fibroblast cells. These rho0 cells consequently lack oxidative phosphorylation and rely on anaerobic glycolysis for their ATP need. We have proposed that in rho0 cells, at least in part, up-regulation of the PMOR is a necessity to maintain the NAD+/NADH ratio, and a pre-requisite for cell growth and viability. We show here that the PM NADH-ferricyanide reductase activity was up-regulated in HL-AV2 (HL-60 rho0) cell lines, but not in the other rho0 and mtDNA mutant lines. The plasma membrane NADH oxidase activity was found to be up-regulated in both HL-AV2 and HeLa rho0 cell lines, but not significantly in the fibroblast rho0 and G11778A lines.

Cell Membrane↗
PubMed2004

Characterization of VDAC1 as a plasma membrane NADH-oxidoreductase.

We have recently demonstrated that voltage dependent anion selective channel~1 (porin, isoform 1) can function as a transplasma membrane NADH:ferricyanide-reductase. However, both the specific redox characteristics and the mechanism of electron transport in this enzyme presently remain unclear. Here we demonstrate that the redox capability of porin 1 is specific for ferricyanide as this same enzyme cannot reduce DCIP or cytochrome c in vitro. Furthermore, NADH-dependent ferricyanide reduction associated with VDAC1 is not sensitive to the anion channel inhibitors DIDS and dextran sulfate. However, this activity can be inhibited by thiol chelators, suggesting that at least one of the two cysteine groups present in VDAC1 are critical for electron transfer. We propose a model on how electron transport may occur in VDAC1.

Animals↗
PubMed2003

Novel effects on memory observed following unilateral intracranial administration of okadaic acid, cyclosporin A, FK506 and [MeVal4]CyA.

The involvement of protein phosphatases and peptidyl-prolyl cis/trans isomerases (PPIases) in memory formation in the chick has previously been investigated using a single-trial learning task. In these studies, inhibitory agents were administered bilaterally directly to a critical area of the chick brain. These studies are now extended to investigate whether similar effects are obtained if the drugs are administered unilaterally. All of the effects reported previously following bilateral administration of okadaic acid (OA), cyclosporin A (CyA), FK506 and [MeVal(4)]CyA can be attributed to their action in just one hemisphere. OA, at a concentration known to selectively inhibit PP2A in vitro (0.5 nM) results in permanent memory loss from 30-40 min post-training when injected in the left hemisphere, but has no effect when injected in the right hemisphere. A higher concentration of OA (100 nM), which inhibits both PP2A and PP1 in vitro, has a similar effect in the left hemisphere but causes a transient period of memory loss from 10-20 min post-training when injected in the right hemisphere. CyA (5 nM and 20 nM), which inhibits both PP2B and PPIase activity, causes permanent memory loss from 60 min post-training when injected into the left hemisphere, an effect also observed following administration of FK506 (20 nM), which also inhibits PP2B and PPIase activity, and [MeVal(4)]CyA (5 nM), which inhibits PPIase activity but not PP2B activity. Administration of CyA (20 nM) and FK506, but not [MeVal(4)]CyA, in the right hemisphere leads to a transient period of memory loss from 10-20 min post-training. These results confirm significant roles for phosphatases and PPIases in memory processing but challenge previous conclusions drawn on the basis of bilateral drug administration protocols.

Animals↗
PubMed2003

VDAC1 is a transplasma membrane NADH-ferricyanide reductase.

Porin isoform 1 or VDAC (voltage-dependent anion-selective channel) 1 is the predominant protein in the outer mitochondrial membrane. We demonstrated previously that a plasma membrane NADH-ferricyanide reductase activity becomes up-regulated upon mitochondrial perturbation, and therefore suggested that it functions as a cellular redox sensor. VDAC1 is known to be expressed in the plasma membrane; however, its function there remained a mystery. Here we show that VDAC1, when expressed in the plasma membrane, functions as a NADH-ferricyanide reductase. VDAC1 preparations purified from both plasma membrane and mitochondria fractions exhibit NADH-ferricyanide reductase activity, which can be immunoprecipitated with poly- and monoclonal antibodies directed against VDAC(1). Transfecting cells with pl-VDAC1-GFP, which carries an N-terminal signal peptide, directs VDAC1 to the plasma membrane, as shown by confocal microscopy and FACS analysis, and significantly increases the plasma membrane NADH-ferricyanide reductase activity of the transfected cells. This novel enzymatic activity of the well known VDAC1 molecule may provide an explanation for its role in the plasma membrane. Our data suggest that a major function of VDAC1 in the plasma membrane is that of a NADH(-ferricyanide) reductase that may be involved in the maintenance of cellular redox homeostasis.

Amino Acid Sequence↗
PubMed2003

Apoptosis-an introduction.

Apoptosis has become a major research area in the biomedical sciences. As there are more than 13,000 papers published annually on the topic, it is impossible to keep track on all developments in the area. The individual aspects of molecular control of apoptosis are well reviewed, but more general, introductory recent reviews into the field are lacking. This review aims to give a brief overview of the field, providing an introduction into the literature for students and newcomers; as it is written for the un-initiated, wherever possible, review articles will be cited rather than original papers.

Animals↗
PubMed2003

Non-ribosomal peptide synthetases as technological platforms for the synthesis of highly modified peptide bioeffectors--Cyclosporin synthetase as a complex example.

Many microbial peptide secondary metabolites possess important medicinal properties, of which the immunosuppressant cyclosporin A is an example. The enormous structural and functional diversity of these low-molecular weight peptides is attributable to their mode of biosynthesis. Peptide secondary metabolites are assembled non-ribosomally by multi-functional enzymes, termed non-ribosomal peptide synthetases. These systems consist of a multi-modular arrangement of the functional domains responsible for the catalysis of the partial reactions of peptide assembly. The extensive homology shared among NRPS systems allows for the generalisation of the knowledge garnered from studies of systems of diverse origins. In this review we shall focus the contemporary knowledge of non-ribosomal peptide biosynthesis on the structure and function of the cyclosporin biosynthetic system, with some emphasis on the re-direction of the biosynthetic potential of this system by combinatorial approaches.

Amino Acid Sequence↗
PubMed2003

Involvement of reactive oxygen species in capsaicinoid-induced apoptosis in transformed cells.

Some varieties of sweet pepper accumulate non-pungent isosters of capsaicin, a type of compounds exemplified by capsiate. The only structural difference between capsaicin and capsiate is the link between the vanillyl and the acyl moieties, via an amide bond in the former and via an ester bond in the latter. By flow cytometry analyses we have determined that nor-dihydrocapsiate, a simplified analogue of capsiate, is a pro-oxidant compound that induces apoptosis in the Jurkat tumor cell line. The nuclear DNA fragmentation induced by nor-dihydrocapsiate is preceded by an increase in the production of reactive oxygen species and by a subsequent disruption of mitochondria transmembrane potential. Capsiate-induced apoptosis is initiated at the S phase of the cell cycle and is mediated by a caspase-3-dependent pathway. The accumulation of intracellular reactive oxygen species in capsiate-treated cells is greatly prevented by the presence of ferricyanide, suggesting that capsiates target a cellular redox system distinct from the one involved in the mitochondrial electron-chain transport. Methylation of the phenolic hydroxyl of nor-dihydrocapsiate completely abrogated the ability to induce reactive oxygen species and apoptosis, highlighting the relevance of the presence of a free phenolic hydroxyl for the pro-oxidant properties of capsaicinoids.

Antioxidants↗
PubMed2003

Transplasma membrane electron transport: enzymes involved and biological function.

The notion of transmembrane electron transport is usually associated with mitochondria and chloroplasts. However, since the early 1970s, it has been known that this phenomenon also occurs at the level of the plasma membrane. Ever since, evidence has accumulated for the existence of a plethora of transplasma membrane electron transport enzymes. In this review, we discuss the various enzymes known, their molecular characteristics and their biological functions.

Animals↗
PubMed2003

Photoaffinity labeling of the N-methyltransferase domains of cyclosporin synthetase.

The multifunctional polypeptide cyclosporin synthetase (CySyn) remains one of the most complex nonribosomal peptide synthetase described. In this study we used a highly specific photoaffinity labeling procedure with the natural cofactor S-adenosyl-L-methionine (AdoMet), 14C-isotopically labeled at the Sdelta methyl group to probe the concerted AdoMet-binding interaction of the N-methyltransferase (N-MTase) centers of CySyn. The binding stoichiometry for the enzyme-AdoMet complex was determined to be 1:7, which is in agreement with inferences made from analysis of the complementary DNA sequence of the simA gene encoding the CySyn polypeptide. The photolabeling of the AdoMet-binding sites displayed homotropic negative cooperativity, characterized by a curvilinear Scatchard plot with upward concavity. Although, the process of N-methyl transfer is not a critical event for peptide elongation, the destabilizing homotropic interactions between N-MTase centers that were observed may represent a mechanism whereby the enzyme preserves the proficiency of the substrate-channeling process of cyclosporin peptide assembly over a broad range of cofactor concentrations. Furthermore, we demonstrated the utility of the photolabeling procedure for tracking the enzyme during purification.

Kinetics↗
PubMed2003

New functions of an old protein: the eukaryotic porin or voltage dependent anion selective channel (VDAC).

Mitochondrial porin or VDAC (Voltage Dependent Anion selective Channels) was identified for the first time in 1976, on the basis of the evolutionary similarity between the gram negative and mitochondrial outer membranes. Since this achievement VDAC has been extensively investigated: its functional features have been sharply defined upon reconstitution in artificial membranes and its sequence has been determined in many genomes. Unfortunately the tertiary structure has not yet been solved, mainly because it proved to be very difficult to get suitable crystals. Despite this established knowledge, in the last few years this protein has attracted renewed interest. There are two main reasons for this interest: the discovery, in most eukaryotes, of a family of genes encoding VDAC isoforms and the claims of VDAC involvement in the intrinsic pathway of apoptosis and in particular in the mechanism of cytochrome c release from mitochondria. We can affirm that nowadays the eukaryotic porin (or VDAC) is studied in a more general cellular contest, looking at the interactions and integration with other molecules, since VDAC is in a crucial position in the cell, forming the main interface between the mitochondrial and the cellular metabolisms. In this minireview we will briefly focus our attention onto the following topics: 1) recent advances about the structure of VDAC; 2) the VDAC-related multigene families; 3) the presence, targeting and function of VDAC in various cell membranes.

Animals↗
PubMed2002

Mapping and molecular modeling of S-adenosyl-L-methionine binding sites in N-methyltransferase domains of the multifunctional polypeptide cyclosporin synthetase.

We employed a highly specific photoaffinity labeling procedure, using (14)C-labeled S-adenosyl-l-methionine (AdoMet) to define the chemical structure of the AdoMet binding centers on cyclosporin synthetase (CySyn). Tryptic digestion of CySyn photolabeled with either [methyl-(14)C]AdoMet or [carboxyl-(14)C]AdoMet yielded the sequence H(2)N-Asn-Asp-Gly-Leu-Glu-Ser-Tyr-Val-Gly-Ile-Glu-Pro-Ser-Arg-COOH (residues 10644-10657), situated within the N-methyltransferase domain of module 8 of CySyn. Radiosequencing detected Glu(10654) and Pro(10655) as the major sites of derivatization. [carboxyl-(14)C]AdoMet in addition labeled Tyr(10650). Chymotryptic digestion generated the radiolabeled peptide H(2)N-Ile-Gly-Leu-Glu-Pro-Ser-Gln-Ser-Ala-Val-Gln-Phe-COOH, corresponding to amino acids 2125-2136 of the N-methyltransferase domain of module 2. The radiolabeled amino acids were identified as Glu(2128) and Pro(2129), which are equivalent in position and function to the modified residues identified with tryptic digestions in module 8. Homology modeling of the N-methyltransferase domains indicates that these regions conserve the consensus topology of the AdoMet binding fold and consensus cofactor interactions seen in structurally characterized AdoMet-dependent methyltransferases. The modified sequence regions correspond to the motif II consensus sequence element, which is involved in directly complexing the adenine and ribose components of AdoMet. We conclude that the AdoMet binding to nonribosomal peptide synthetase N-methyltransferase domains obeys the consensus cofactor interactions seen among most structurally characterized low molecular weight AdoMet-dependent methyltransferases.

Amino Acid Sequence↗
PubMed2002

Cyclosporin A, FK506 and rapamycin produce multiple, temporally distinct, effects on memory following single-trial, passive avoidance training in the chick.

Few studies have used a pharmaco-behavioural methodology to directly investigate roles for the calcium-dependent protein phosphatase calcineurin (CaN) in memory formation, due partly to the absence of specific inhibitory agents. A number of drugs with different inhibitory profiles were used to examine this issue in groups of chicks trained on a single-trial, passive-avoidance task. Bilateral intracranial administration of the immunosuppressants FK506 and cyclosporin A (CyA) led to two temporally distinct effects, distinguished by the concentration of drug required and the effective time of administration relative to training. In addition to inhibiting CaN, CyA and FK506 inhibit distinct classes of peptidyl prolyl-cis/trans-isomerases (PPIases). Other agents known to inhibit these enzymes, including the Map kinase inhibitor Rapamycin, also induced memory deficits in a complex, dose- and time-of-administration-dependent, manner. The data fail to conclusively implicate CaN in memory formation, but are consistent with proposals that a phosphatase cascade may participate in an early stage of information storage. PPIases may be required at a later stage to catalyse the folding of new or translocated proteins, the synthesis of which is required for formation of long-term memory, although other possible explanations for the data remain to be investigated.

Animals↗