ABSTRACT
The performance of Magarose, an agarose-based bead containing a paramagnetic component has been evaluated. The anion exchanger DEAE-Magarose is effective at binding DNA from a crude cell lysate. The plasmid pBluescript was isolated from 1.5 ml Escherichia coli JM109 cell culture, following alkaline lysis yielding 8.2 micrograms high-quality DNA. Under similar binding conditions 21 micrograms of salmon sperm DNA bound to the ion exchangers. The affinity medium oligo-dT Magarose was demonstrated to bind 75 mumol of an oligo-dA probe/g of medium by hybridization. Under similar conditions mRNA could be isolated from a preparation of baby hamster cell total RNA. The magnetic susceptibility of Magarose is very high, facilitating the use of this separation technique for rapid batch chromatographic processes.
Subject(s)
Nucleic Acids/isolation & purification , Animals , Anion Exchange Resins , Chromatography, Ion Exchange , Cricetinae , DNA, Bacterial/isolation & purification , Escherichia coli/chemistry , Magnetics , Male , Oligonucleotides/chemistry , Oligonucleotides/isolation & purification , Plasmids/isolation & purification , RNA, Messenger/isolation & purification , Salmon , Sepharose , Spermatozoa/chemistryABSTRACT
Using partially purified sedoheptulose-1,7-bisphosphatase from spinach (Spinacia oleracea L.) chloroplasts the effects of metabolites on the dithiothreitoland Mg(2+)-activated enzyme were investigated. A screening of most of the intermediates of the Calvin cycle and the photorespiratory pathway showed that physiological concentrations of sedoheptulose-7-phosphate and glycerate specifically inhibited the enzyme by decreasing its maximal velocity. An inhibition by ribulose-1,5-bisphosphate was also found. The inhibitory effect of sedoheptulose-7-phosphate on the enzyme is discussed in terms of allowing a control of sedoheptulose-1,7-bisphosphate hydrolysis by the demand of the product of this reaction. Subsequent studies with partially purified fructose-1,6-bisphosphatase from spinach chloroplasts showed that glycerate also inhibited this enzyme. With isolated chloroplasts, glycerate was found to inhibit CO2 fixation by blocking the stromal fructose-1,6-bisphosphatase. It is therefore possible that the inhibition of the two phosphatases by glycerate is an important regulatory factor for adjusting the activity of the Calvin cycle to the ATP supply by the light reaction.
ABSTRACT
The effect of pH and of Mg(2+) concentration on the light activated form of stromal fructose-1,6-bisphosphatase (FBPase) was studied using the enzyme rapidly extracted from illuminated spinach chloroplasts. The (fructose-1,6-bisphosphate(4-))(Mg(2+)) complex has been identified as the substrate of the enzyme. Therefore, changes of pH and Mg(2+) concentrations have an immediate effect on the activity of FBPase by shifting the pH and Mg(2+) dependent equilibrium concentration of the substrate. In addition, changes of pH and Mg(2+) concentration in the assay medium have a delayed effect on FBPase activity. A correlation of the activities observed using different pH and Mg(2+) concentrations indicates, that the effect is not a consequence of the pH and Mg(2+) concentration as such, but is caused by a shift in the equilibrium concentration of a hypothetical inhibitor fructose-1,6-bisphosphate(3-) (uncomplexed), resulting in a change of the activation state of the enzyme. The interplay between a rapid effect on the concentration of the substrate and a delayed effect on the activation state enables a rigid control of stromal FBPase by stromal Mg(2+) concentrations and pH. Fructose-1,6-bisphosphatase is allosterically inhibited by fructose-6-phosphate in a sigmoidal fashion, allowing a fine control of the enzyme by its product.
