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Biomedical subjects

M R Kula

Publications and source records attributed to M R Kula.

At least 91 records · Page 5Linked to original sources
PubMed1987

Purification of proteins from cell culture supernatants.

Desired proteins excreted by animal cells usually reach rather low concentrations in the culture supernatant and have to be purified from an excess of serum proteins which are added to the animal cell culture to maintain its viability and/or productivity. Defined media offer among others the advantage of an easier purification procedure. In addition lysis of cells may contribute also to the complexity of the protein mixture encountered. If fetal calf serum or new born calf serum is used in cultivation, bovine serum albumin and globulins will be the most abundant protein in the supernatant. The interaction of albumin especially with hydrophobic proteins represents significant problems for the effective purification of minor constituent. Isolation of a desired protein follows the general scheme: concentration: by precipitation, ultrafiltration, batch adsorption or partition in aqueous phase system; enrichment: by chromatography or partition; high resolution purification: by chromatography and/or immuno adsorption; final concentration and finishing: pyrogen removal, sterilization, formulation. Biochemical engineering aspects of proteins purification are discussed using human interferon-beta produced in a recombinant mouse cell line as an example. The process developed encloses extraction of interferon-beta from the production medium in aqueous two-phase systems and subsequent recovery of interferon-beta from the top phase by high pressure liquid chromatography.

Albumins↗
PubMed1986

Isolation and fractionation of CHO chromosomes in aqueous two phase systems using charged polymers and base specific macroligands.

Chromosomes were isolated in a preparative scale by synchronisation of CHO cells with a double Thymidine block followed by an arrest in the metaphase by addition of Colcemid. Under proper cultivation conditions a mitotic index of 77% total cells could be routinely achieved. Bulk chromosome preparations free of nuclei and other subcellular particles have been obtained by low speed centrifugation followed by a 60 transfer countercurrent distribution using aqueous two phase systems composed of polyethylenglycol and dextran. The partition of CHO chromosomes previously purified in aqueous two phase systems were studied further to develop a protocol for the separation and isolation of individual chromosomes. Partition experiments with chromosomes changing the electrostatic phase potential by addition of charged PEG-derivatives suggest the existence of relatively highly charged chromosome groups. Most promising results with regard to separation were obtained using two PEG-derivatives, which interact specifically with the bases in DNA. For this affinity partitioning a GC- and AT-specific macroligand were employed. Comparing CCD's using each of these ligands information on the GC and AT content of exposed DNA in the chromosomes groups could be derived, demonstrating that specific sequences of DNA are accessible at the surface of metaphase chromosomes.

Adenine↗
PubMed1986

Assay of the glutathione-synthesizing enzymes by high-performance liquid chromatography.

We report a new convenient assay of the activity of gamma-glutamylcysteine synthetase (EC 6.3.2.2) and glutathione synthetase (EC 6.3.2.3) in crude microbial extracts as well as in purified enzyme preparations. The assay is based on the quantitative analysis of the reaction products by high-performance liquid chromatography after derivatization of the thiol group with 5,5'-dithiobis-(2-nitrobenzoic acid) as described by J. Reeve, J. Kuhlenkamp, and N. Kaplowitz [(1980) J. Chromatogr. 194, 424-428]. In addition, the procedure yields information on basal levels of gamma-glutamylcysteine and glutathione in crude microbial extracts. The two enzymes responsible for glutathione biosynthesis can be determined in parallel under the same chromatographic conditions. No prior separation from substrates and by-products is necessary. Product formation is linear with time for at least 30 min between 0.03 and 12 mU for both enzymes. Even in crude extracts 0.2-0.5 nmol of products formed can be detected with certainty. The method was found to be sensitive and highly reproducible.

Candida↗
PubMed1986

Characterization of hydantoinase from Pseudomonas fluorescens strain DSM 84.

The hydantoinase (EC 3.5.2.2) from Pseudomonas fluorescens strain DSM 84 was purified either by hydrophobic interaction chromatography on phenyl-Sepharose or by salting out chromatography on Sepharose 4B, gel filtration on Sephacryl S-400, and preparative electrophoresis. Molecular weight values of 230,000 and 60,000 for the native enzyme and each of the four subunits were estimated for the hydantoin hydrolysing activity. The hydantoinase was stable at temperatures up to 40 degrees C but showed an optimal activity at 55 degrees C. The enzyme was markedly inhibited by copper, para-hydroxymercuribenzoate, 8-hydroxyquinoline, and 2,2'-dipyridyl but not by zinc, and poorly by EDTA and o-phenanthroline. The hydantoin-hydrolyzing activity could be reactivated by ferrous ions. Dihydrouracil was the most readily hydrolyzed substrate. The dihydropyrimidinase produced by strain DSM 84 could also hydrolyze 5-substituted hydantions such as isopropylhydantoin (valine derivative) continuously for 10 days in a membrane reactor at a conversion rate of 30%. The only identified end product was N-carbamyl-D-valine.

Amidohydrolases↗
PubMed1985

Improved enzyme screening by automated fast protein liquid chromatography.

The assay for NADH-dependent dehydrogenases in crude extracts is often interfered with non-specific reactions. Therefore a screening for such enzymes is hampered by high blank values. To overcome such problems we chromatographed crude extracts on a fast protein liquid chromatography system during part of an enzyme screening for 2-hydroxyisocaproate dehydrogenases and lactate dehydrogenases. The automated chromatography procedure presented consists of a combination of gel filtration and ion-exchange chromatography. The total time needed to perform one cycle of the two-column purification, including the equilibration and regeneration steps, is about 35 min. The procedure described separates the desired enzyme, 2-hydroxyisocaproate dehydrogenase, totally from any interfering activity such as NADH-oxidase and also from the second enzyme of interest, the lactate dehydrogenase. Besides the elimination of the side reactions the desired enzymes are purified up to 20-fold.

Alcohol Oxidoreductases↗
PubMed1982

Purification and characterization of a nicotinamide adenine dinucleotide-dependent secondary alcohol dehydrogenase from Candida boidinii.

From the yeast Candida boidinii grown on glucose a new secondary alcohol dehydrogenase was purified 426-fold by heat treatment, column chromatography on DEAE-Sephacel, affinity chromatography on Blue Sepharose Cl-6b, and gel filtration on Sephacryl S-300. The purified enzyme was homogeneous as judged by analytical polyacrylamide gel electrophoresis. The molecular weight was found to be 150000 by sedimentation equilibrium as well as by gel filtration. The enzyme appears to be composed of four identical subunits (Mr=38000) as determined by SDS-gel electrophoresis. The enzyme catalyzes the oxidation of isopropanol to acetone in the presence of NAD+ as an electron acceptor. The Km values were found to be 0.099 mM for isopropanol and 0.14 mM for NAD+. Besides isopropanol also other secondary alcohols like butan-2-ol, pentan-2-ol, pentan-3-ol, hexan-2-ol, cyclobutanol, cyclopentanol, and cyclohexanol served as a substrate and were oxidized to the corresponding ketones. Isopropanol seems to be the best substrate for this enzyme which we therefore call isopropanol dehydrogenase. Primary alcohols are not oxidized by the enzyme. The optimum pH for enzymatic activity in the oxidation reaction was found to be 9.0, the optimal temperature is 45 degrees C. The isoelectric point of the isopropanol dehydrogenase was found to be pH 4.9. The enzyme is inactivated by mercaptide-forming reagents and chelating agents, 2-mercaptoethanol is an inhibitor. Zinc ions appear necessary for enzyme production.

Alcohol Oxidoreductases↗
PubMed1981

Reaction kinetics of some important site-specific endonucleases.

Reaction kinetics of the site-specific endonucleases BamHI, BgIII, C1aI, EcoRI, HpaII, PstI, SaII, SmaI, and XorII were investigated employing some frequently used substrates. Six of these enzymes could be analyzed under steady-state conditions. Kinetic data were obtained from progress curves applying an integrated Michaelis-Menten equation. KM ranged from 4 x 10(-9) M to 4 x 10(-11) M. Activities also spanned two orders of magnitude. In the case of C1aI the analysis of the pre-steady-state kinetics ("burst reaction") allowed the assessment of several rate constants. The rate-limiting step is the very slow dissociation of the enzyme-product complex (0.22 min(-1)). This complex is formed from the enzyme-bound nicked intermediate at a rate of 1.7 min(-1). The introduction of the first cut is again faster by a factor of about 6. SmaI and XorII resembled C1aI in their kinetics. The burst reaction can be used for the easy and unambiguous determination of molar concentrations of site-specific endonucleases in any preparation, which is free of non-specific DNases.

DNA Restriction Enzymes↗
PubMed1981

Technical aspects of extractive enzyme purification.

We see the advantages of extraction processes for large-scale enzyme isolation and purification in the high capacity of the method, savings in process time and energy, high activity yields and the possibilities for continuous processing at all stages. Commercially available equipment can be used for separation of aqueous two-phase systems with minor modifications. It is hoped that, through using such technology, intracellular enzymes will become available in large quantities and at lower cost.

Biochemistry↗
PubMed1980

Studies on an enzyme, S-formylglutathione hydrolase, of the dissimilatory pathway of methanol in Candida boidinii.

In Candida boidinii, S-formylglutathione formed by reaction of the glutathione-dependent formaldehyde dehydrogenase is hydrolyzed to formate and glutathione by a special enzyme, S-formylglutathione hydrolase which is induced in C. boidinii along with the other enzymes of the dissimilatory pathway during growth on CH3OH. The S-formylglutathione hydrolase was purified to apparent homogeneity and a specific activity of 1390 U/mg. The molecular weight of the native enzyme was determined as 61 000 by gel filtration and 64 000 by sedimentation-diffusion equilibrium. It is composed of two nonidentical polypeptide chains of 35 000 and 25 000 daltons. The Km-value of S-formylglutathione was found to be 0.21 mM. Glutathione is a competitive inhibitor with a Ki vaue of 18.5 mM. The enzyme is very specific for S-formylglutatione, S-acetylglutathione gave 1.3%, respectively. Other glutathione derivatives of hydroxyacids tested were not split by the S-formylglutatione hydrolase.

Candida↗
PubMed1980

Binding of non-substrate nucleotides to a restriction endonuclease: a model for the interaction of bam HI with its recognition sequence.

The kinetic constants of the site-specific endonuclease Bam HI for various substrates were determined and binding of non-substrate nucleotides to the enzyme was studied. Agarose gel assays in combination with an integrated Michaelis-Menten equation were used for the evaluation of data. The turnover number was 2.2 min-1 at 37 degrees C with pJC80 DNA as the substrate. It depends on the conformation and base composition of the substrate. Michaelis constants also depend on substrate conformation. Non-substrate polynucleotides were found to inhibit Bam competitively with KI ranging from 10(-6) to > 10(-3) M depending on base composition, base pairing, and helix conformation. Dinucleotides showed sequence-specific, competitive inhibition with KIs ranging from 10(-5) to > 10(-3) M. Mononucleotides and -nucleosides acted noncompetitively. Binding was influenced by the extent of phosphorylation, but not by the nature of the base. KIs varied between 10(-3) and 10(-2) M. The results are discussed with respect to the recognition requirements of Bam HI.

Bacteriophage lambda↗
PubMed1980

Physical and kinetic properties of the site specific endonuclease Bam HI from Bacillus amylolique-faciens.

The site specific endonuclease Bam HI which is composed of subunits of a molecular weight of 22 000 [1] can aggregate to complexes of a molecular weight of 360 000. It is an acidic protein with an isoelectric point at pH 5.3. Optimal activity is reached at 13 mM MgCl2. A very simple method is presented to determine kinetic constants of restriction enzymes directly from agarose gel photographs without any further equipment applying the integrated Michaelis Menten equation. With pJC 80 DNA as a substrate KM was found to be 3.6 10(-10) M. The method can be used to redefine the unit activity of site specific endonucleases unambigously.

Bacillus↗