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R D Schmid

Publications and source records attributed to R D Schmid.

At least 73 records · Page 4Linked to original sources
PubMed1997

Possible involvement of nonbilayer lipids in the stimulation of the activity of cytochrome P450SCC (CYP11A1) and its propensity to induce vesicle aggregation.

Phosphatidylcholines with saturated branched fatty acyl chains substituted in the 2-positions of the main chains have a significant stimulating effect on both the side chain cleavage activity of cytochrome P450SCC and the ability of P450SCC to induce a specific vesicle aggregation in lipid-vesicle reconstituted systems. Stimulation increases with increasing content of the branched phosphatidylcholines and is sensitive to its acyl chain structure and composition. Because branched phosphatidylcholines containing 2-alkyl substituted fatty acid chains belong to a new class of phosphatidylcholines forming inverted nonbilayer phases the results indicate that nonbilayer lipids might play an important role in the function of mitochondrial P450SCC.

Cholesterol Side-Chain Cleavage Enzyme↗
PubMed1997

Structural and biochemical properties of glycosylated and deglycosylated glucose oxidase from Penicillium amagasakiense.

Glucose oxidase from Penicillium amagasakiense was purified to homogeneity by ion-exchange chromatography and deglycosylated with endoglycosidase H. On the basis of gas chromatography and sodium dodecyl sulphate/polyacrylamide gel electrophoretic (SDS-PAGE) analyses, the protein-bound high-mannose-type carbohydrate moiety corresponded to 13% of the molecular mass of glycosylated glucose oxidase. A total of six N-glycosylation sites per dimer were determined from the N-acetylglucosamine content. The enzymatically deglycosylated enzyme contained less than 5% of the original carbohydrate moiety. A molecular mass of 130 kDa (gel filtration) and 133 kDa (native PAGE) was determined for the dimer and 67 kDa (SDS-PAGE) for the monomer of the deglycosylated enzyme. The N-terminal sequence, which has not-been published for glucose oxidase from P. amagasakiense to date and which showed less than 50% homology to the N terminus of glucose oxidase from Aspergillus niger, and the amino acid composition were not altered by the deglycosylation. Deglycosylation also did not affect the kinetics of glucose oxidation or the pH and temperature optima. It also did not increase the susceptibility of the enzyme to proteolytic degradation. However, deglycosylated glucose oxidase exhibited decreased pH and thermal stability. The thermal stability of both enzymes was shown to be dependent on the buffer concentration and was enhanced by certain additives, particularly 1 M (NH4)2SO4, which stabilised glucose oxidase 100- to 300-fold at 50 degrees C and pH 7-8, and 2 M KF, which stabilised the enzyme up to 36-fold at 60 degrees C and pH 6. In sodium acetate buffer, changes in pH (4-6) affected the affinity for glucose but had no effect on the Vmax of the reaction. In contrast, in TRIS buffer, pH 8, a 10-fold decrease in Vmax and a 2-fold decrease in K(m) were observed.

Amino Acid Sequence↗
PubMed1996

The folding and activity of the extracellular lipase of Rhizopus oryzae are modulated by a prosequence.

The fungus Rhizopus oryzae synthesizes an extracellular lipase precursor bearing N-terminal pre- and pro-sequences. Our studies in Escherichia coli and using recombinant lipase in vitro indicate that the prosequence of 97 amino acids has at least two functions. First, it modulates the enzyme activity of the lipase so that this enzyme can initially be synthesized in a non-destructive form. Direct synthesis of the mature form of the lipase in the cell has toxic consequences, at least partly because of phospholipase activity that is suppressed in the proprotein. Secondly, it supports folding of the lipase via a pathway influenced by a single cysteine residue at position - 68. Mutational analysis of the prosequence demonstrates not only the key role of this cysteine residue but also the importance of the neighbouring amino acids. In particular, Arg-69 probably enhances the leaving group character of Cys-68. We propose a model in which Cys-68 acts as an intramolecular thiodisulphide reagent, playing a catalytic role in the folding of the enzyme. The prosequence is capable of performing the described functions both in cis and in trans.

Enzyme Activation↗
PubMed1996

Crystal structure of a bacterial lipase from Chromobacterium viscosum ATCC 6918 refined at 1.6 angstroms resolution.

The crystal structure of a lipase from the bacterium Chromobacterium viscosum ATCC 6918 (CVL) has been determined by isomorphous replacement and refined at 1.6 angstroms resolution to an R-factor of 17.8%. The lipase has the overall topology of an alpha/beta type protein, which was also found for previously determined lipase structures. The catalytic triad of the active center consists of the residues Ser87, Asp263 and His285. These residues are not exposed to the solvent, but a narrow channel connects them with the molecular surface. This conformation is very similar to the previously reported closed conformation of Pseudomonas glumae lipase (PGL), but superposition of the two lipase structures reveals several conformational differences. r.m.s. deviations greater than 2 angstroms are found for the C alpha-atoms of the polypeptide chains from His15 to Asp28, from Leu49 to Ser54 and from Lys128 to Gln158. Compared to the PGL structure in the CVL structure, three alpha-helical fragments are shorter, one beta-strand is longer and an additional antiparallel beta-sheet is found. In contrast to PGL, CVL displays an oxyanion hole, which is stabilized by the amide nitrogen atoms of Leu17 and Gln88, and a cis-peptide bond between Gln291 and Leu292. CVL contains a Ca2+, like the PGL, which is coordinated by four oxygen atoms from the protein and two water molecules.

Amino Acid Sequence↗
PubMed1996

Thermoalkalophilic lipase of Bacillus thermocatenulatus. I. molecular cloning, nucleotide sequence, purification and some properties.

An expression library was generated by partial Sau3A digestion of genomic DNA from the thermophile Bacillus thermocatenulatus and cloning of DNA fragments in pUC18 in Escherichia coli DH5alpha. Screening for lipase activity identified a 4.5 kb insert in pUC18 which directed the production of lipase in E. coli DH5alpha. A subclone with a 2.2 kb insert was sequenced. The lipase gene codes for a mature lipase of 388 amino acid residues, corresponding to a molecular weight of 43 kDa. As in other Bacillus lipases, an Ala replaces the first Gly in the conserved pentapeptide Gly-X-Ser-X-Gly found in most lipases. The region upstream of the lipase gene contains a Bacillus promoter which directs the expression of lipase in E. coli DH5alpha. The expressed lipase was isolated and purified 312-fold to homogeneity. N-terminal sequencing of the purified lipase revealed a correct cleavage of the preprotein in E. coli DH5alpha. Maximum activity was found at pH 8.0-9.0 with tributyrin and olive oil as substrates and at 60-70 degrees C with p-NPP and olive oil as substrates. The lipase showed high stability at pH 9.0-11.0 and towards various detergents and organic solvents.

Amino Acid Sequence↗
PubMed1996

Alpha-branched 1,2-diacyl phosphatidylcholines as effectors of activity of cytochrome P450SCC (CYP11A1). Modeling the structure of the fatty acyl chain region of cardiolipin.

Cardiolipin has been shown to be the most effective activator of cholesterol side chain cleavage activity of cytochrome P450SCC, and evidence has been provided for a lipid effector site on the enzyme. Results suggested the headgroup of cardiolipin as major determinant of lipid interaction with P450SCC (Lambeth, J. D. (1981) J. Biol. Chem. 256, 4757-4762). The role of unsaturation is contradictory and open to question (Igarashi, Y. and Kimura, T. (1986) Biochemistry 25, 6461-6466). We synthesized phosphatidylcholines with fully saturated branched fatty acyl chains substituted in the 2-positions of the main chains and studied the influence of these lipids on the activity and other properties of P450SCC in vesicle-reconstituted systems. These saturated branched lipids, with regard to the fatty acyl moiety in molecular shape similar to cardiolipin but with the headgroup of phosphatidylcholines retained, showed a stimulatory efficiency higher than any other phospholipid and at least comparable to cardiolipin. Activation is sensitive to the acyl chain structure and composition. Results suggest that the shape of the molecule at least partially plays an important role in the process of stimulation of the activity of P450SCC. Because binding of cholesterol was increased by the branched lipids monitored optically by the fraction of P450SCC in the high spin form, it was concluded that these lipids, like cardiolipin and other lipids, exert their effects by regulating the binding of cholesterol to P450SCC. These data suggest that polymorphic lipids such as branched phosphatidylcholines and cardiolipin might influence P450SCC function by maintenance of the membrane curvature at a value optimal for activity.

Acylation↗
PubMed1996

Purification and some properties of a novel microbial lactate oxidase.

Geotrichum candidum was found to produce a lactate oxidase. The enzyme was purified by gel filtration and ion-exchange chromatography. The purified lactate oxidase showed a molecular mass of 50 kDa under denaturing and about 400 kDa under non-denaturing conditions. Transmission electron microscopy analysis confirmed an octameric structure. FMN was found to be a cofactor for this enzyme. Polarographic studies confirmed an oxygen uptake by the lactate oxidase. The enzyme showed specificity towards the L isomer of lactate and did not oxidise pyruvate, fumarate, succinate, maleate and ascorbate. It was stable at alkaline pH and also for 15 min at 45 degrees C. The addition of glycerol and dextran 500,000 to the enzyme sample enhanced storage stability.

Amino Acid Sequence↗
PubMed1996

Development and evaluation of a dipstick immunoassay format for the determination of atrazine residues on-site.

On the basis of a semi-quantitative dipstick immunoassay (IA) for atrazine with visual detection (Giersch, T., J. Agric. Food Chem., 1993, 41, 1006), a quantitative format suitable as a field assay for the analysis of pesticide residues in water and liquid food samples on-site is described. For antibody immobilization, different membranes and immobilization techniques were investigated. The measuring range for atrazine was 0.3-10 micrograms l-1 using reflectance detection. The total assay time was about 25 min with dipsticks previously coated with antibody. Atrazine-spiked water and liquid food samples were selected for assay evaluation. The samples could be measured directly without the need for any prior enrichment or clean-up steps. A satisfactory agreement was found between the results of the dipstick IA and HPLC or GC measurements of both the original and spiked samples.

Atrazine↗
PubMed1996

Analysis of the catalytic mechanism of a fungal lipase using computer-aided design and structural mutants.

Both an active enzyme conformation and stabilization of tetrahedral transition states are essential for the catalysis of ester bond hydrolysis by lipases. X-ray structural data and results from site-directed mutagenesis experiments with proteases have been used as a basis for predictions of amino acid residues likely to have key functions in lipases. The gene encoding a lipase from Rhizopus oryzae was cloned and expressed in Escherichia coli. Site-directed mutagenesis of this gene was used to test the validity of computer-aided predictions of the functional roles of specific amino acids in this enzyme. Examination of the kinetic constants of the Rhizopus oryzae lipase variants allowed us to identify amino acid residues which are directly involved in the catalytic reaction or which stabilize the active geometry of the enzyme. The combination of these results with molecular mechanics simulations, based on a homology-derived structural model, provided new information about structure-function relationships. The interpretation of the data is consistent with results obtained with other hydrolases, such as proteases.

Amino Acid Sequence↗
PubMed1995

On-line monitoring of glucose, glutamate and glutamine during mammalian cell cultivations.

Amperometric biosensors (based on rhodinised carbon electrodes) for glucose, glutamine and glutamate were constructed. The sensors were incorporated into a three cell parallel FIA system and used to monitor the three analytes on-line during two mammalian cell perfusion cultures. All measurements were made simultaneously from undiluted media sample. Use of the FIA system enabled easy and rapid exchange of the sensors, during cultivation. The inclusion of a calibration step, regularly for all sensors, helped to maintain the accuracy of all measurements. Comparison with off-line measurements indicated that all three biosensors operated successfully, providing accurate information.

Biosensing Techniques↗
PubMed1995

The design of enzyme sensors based on the enzyme structure.

The size of some of the reported electron mediators for glucose oxidase compared with the available space to penetrate the active site, implies that electrons have to move along the protein structure. Theoretical and experimental evidence predicts that it is possible to have electron transfer at the required rate used in biosensors (200 to 1840 electrons s-1) for the distances in glucose oxidase (20 to 25 A). Use of the program "Pathways" (together with the knowledge of the enzyme structure) allowed us to find an electron pathway within the enzyme. This pathway has a maximum electron coupling between the active site and the surface of the enzyme. The pathway reaches the surface near functional groups that can be used for oriented immobilization of the enzyme. Experimental confirmation of this particular pathway has been attempted but it is still elusive.

Animals↗
PubMed1995

Crystal structure of NADH oxidase from Thermus thermophilus.

The crystal structures of the flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) containing isoforms of NADH oxidase from Thermus thermophilus have been determined by isomorphous and molecular replacement and refined to 2.3 A and 1.6 A resolution with R-values of 18.5% and 18.6% respectively. The structure of the homodimeric enzyme consists of a central 4-stranded antiparallel beta-sheet covered by helices, a more flexible domain formed by two helices, and a C-terminal excursion connecting the subunits. The active sites are located in a deep cleft between the subunits. The binding site of the flavin cofactor lacks the common nucleotide binding fold and is different from the FMN binding site found in flavodoxins.

Binding Sites↗
PubMed1994

Improved properties of bovine erythrocyte acetylcholinesterase, isolated by papain cleavage.

A simple and rapid procedure involving papain cleavage of the membrane anchor was used to isolate membrane-bound acetylcholinesterase from bovine erythrocytes. The solubilized enzyme was purified 930-fold by ion exchange chromatography and gel filtration. The properties of the papain-cleaved acetylcholinesterase were compared with those of a commercial acetylcholinesterase, solubilized from the erythrocyte membranes by detergents. Cleavage of the membrane anchor eliminated dimer aggregation, caused a pH shift in thermal stability and resulted in increased stability in organic solvents. Bovine serum albumin, used as stabilizer of the commercial enzyme preparation, increased the thermal stability but concomitantly decreased the activity of acetylcholinesterase at pH 6-8. The improved stability of the cleaved acetylcholinesterase, especially in organic solvents, may enhance the biosensor performance of the enzyme.

Acetylcholinesterase↗
PubMed1994

Screening, purification and properties of a thermophilic lipase from Bacillus thermocatenulatus.

By screening of 15 thermophilic Bacillus strains, five strains exhibiting lipase activity were found. Among these the strain Bacillus thermocatenulatus (DSM 730) produced the highest lipase activity. The lipase proved to be inducible and extracellular and was purified 67-fold to homogenous state by hexane extraction, methanol precipitation and ion-exchange chromatography on Q-Sepharose. The molecular weight of the lipase determined by SDS-PAGE is 16 kDa. However, the lipase forms very large aggregates (> 750 kDa) as observed after native PAGE, which makes handling of the lipase very difficult. The lipase binds almost irreversibly on different chromatography matrices, e.g., Amberlite and Serolite, and is very stable in the immobilised form. The N-terminal sequence consists of 53% apolar amino acids and shows no significant homology towards other known lipase sequences. Maximum activity was found at pH 7.5-8.0 and 60-70 degrees C with pNPP and olive oil as substrates.

Amino Acid Sequence↗
PubMed1994

Properties of unusual phospholipids, II: Synthesis, NMR studies and monolayer investigations of diacylglycerophosphocholines containing alpha-branched acyl chains.

Diacylglycerophosphocholines, containing various alpha-branched fatty acids, were synthesized and purified to homogeneity. The influence of the alpha-branched acyl chains on the monolayer packing properties of these phospholipids was studied by the Langmuir-Blodgett film balance technique. NMR investigations were used for conformational studies, including the determination of rotamer populations about the dihedral angles (theta 2, theta 4 and alpha 1) in the glycerol region of the diacylglycerophosphocholines.

Fatty Acids↗
PubMed1994

Properties of unusual phospholipids: I. Synthesis, monolayer investigations and calorimetry of diacylglycerophosphocholines containing monoacetylenic acyl chains.

Isomeric diacylglycerophosphocholines containing various octadecynoic acids (4-, 6-, 9-, 12-, 14- and 17-octadecynoic acid) were synthesized and purified to homogeneity. Their behaviour in monolayers, when studied by the Langmuir-Blodgett film balance technique, revealed systematic relationships between structure and packing properties. The thermotropic phase behaviour of these novel phospholipids, as measured by differential scanning calorimetry, depended in a systematic fashion on the position of the triple bond: the gel-to-liquid-crystalline phase transition temperature (Tm) passed through a minimum of -3.4 degrees C for a triple bond in position 9.

Calorimetry, Differential Scanning↗