ABSTRACT
The physical parameters governing adsorption of DNA by various positively charged depth filters and membranes have been assessed. Buffers that reduced or neutralised the depth filter or membrane charge, and those that impeded hydrophobic interactions were shown to affect their operational capacity, demonstrating that DNA was adsorbed by a combination of electrostatic and hydrophobic interactions. The adsorption profile of DNA by a Sartobind Q anion exchange membrane showed immediate breakthrough, irrespective of challenge DNA concentration or flow rate, and in this case adsorption was by electrostatic interactions only. The production-scale removal of DNA from harvest broths containing therapeutic protein by partitioning of cells and debris from protein in sequential centrifugation and filtration steps, and the concentration of DNA in process supernatant were assessed. Centrifugation reduced the quantity of DNA in the process material from 79.8 micrograms ml-1 to 9.3 micrograms ml-1 whereas the concentration of DNA in the supernatant of pre- and post-filtration samples had only marginally reduced DNA content: from 6.3 to 6.0 micrograms ml-1 respectively. DNA was concentrated to 27.3 micrograms ml-1 along with monoclonal antibody in the ultrafiltration step. Similar effects were observed in the harvest step for a second antibody.
Subject(s)
Antibodies, Monoclonal/biosynthesis , DNA/immunology , Filtration/instrumentation , Membranes, Artificial , Adsorption , Animals , Cattle , DNA/isolation & purificationABSTRACT
Acetohydroxyacid synthase (EC 4.1.3.18) has been extracted from leaves of three valine-resistant (Val(r)) tobacco (Nicotiana tabacum) mutants, and compared with the enzyme from the wild-type. The enzyme from all three mutants is appreciably less sensitive to inhibition by leucine and valine than the wild-type. Two of the mutants, Val(r)-1 and Val(r)-6, have very similar enzymes, which under all conditions are inhibited by less than half that found for the wild-type. The other mutant, Val(r)-7, has an enzyme that only displays appreciably different characteristics from the wild-type at high pyruvate or inhibitor concentrations. Enzyme from Val(r)-7 also has a higher apparent Km for pyruvate, threefold greater than the value determined for the wild-type and the other mutants. The sulphonylurea herbicides strongly inhibit the enzyme from all the lines, though the concentrations required for half-maximal inhibition of enzyme from Val(r)-1 and Val(r)-6 are higher than for Val(r)-7 or the wildtype. No evidence has been found for multiple isoforms of acetohydroxyacid synthase, and it is suggested that the valine-resistance of these mutant lines is the result of two different mutations affecting a single enzyme, possibly involving different subunits.
ABSTRACT
Membrane preparations from striatum of pig brain contain endopeptidase activity towards iodoinsulin B-chain. Only 50% of the hydrolysis of insulin B-chain is inhibitable by phosphoramidon, and DEAE-cellulose chromatography can resolve the phosphoramidon-sensitive and -insensitive activities. The former activity (now designated 'endopeptidase-24.11') is responsible for hydrolysis of [D-Ala2,Leu5]enkephalin and is identical with an enzyme in brain that has previously been referred to as 'enkephalinase'. Pig striatal endopeptidase-24.11 has now been purified to homogeneity in a single step by immunoadsorbent chromatography using a monoclonal antibody. The overall purification was 23 000-fold, with a yield of 30%. The brain enzyme appears to be identical with kidney endopeptidase-24.11 in amino acid composition as well as by immunological and kinetic criteria. However, it differs slightly in apparent subunit size (Mr = 87 000), attributable to differences in glycosylation.
