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General Understanding
The classical model of enzyme chemistry defines the movement of a substrate through the matrix (often cytoplasm) until it reaches the proper target to be altered and released to find the next enzyme in a pathway.  This process can be seen as inefficient due not only to concentration limitations of the enzyme but also diffusion and compartmental limitations of the cell.  Testing of cellular metabolism found that classical models of enzymes chemistry did not match that of invivo experimentation.  This led scientist to believe that proteins found in a give pathway were held together by weak interactions  forming a metabolic complex or metabolon, to increase product yield by decreasing diffusion and compartmental limitations.  This research isolated and cloned Glucose-6-Phosphate Dehydorgenase (G6PDH) from soybean, the first enzyme in the Pentose Phosphate Pathway (PPP), as the first step to isolate the entire pathway for invitro testing of a metabolon.
 
MOLECULAR BIOLOGY:  The Cloning and Isolation of Glucose-6-Phosphate Dehydrogenase (G6PDH).
 

Glucose-6-Phosphate Dehydorgenase (G6PDH) is the first enzyme of the oxidative limb of the Pentose Phosphate Pathway (PPP) which supplies NADPH for the reductive synthesis as well as ribose-5-phosphate, a precursor for nucleotide synthesis.  Recent evidence indicates that the PPP in soybean nodules function as a metabolic complex or metabolon.  Cloning the soybean gene was useful to begin investigating this interaction.  G6PDH has been previously cloned from E.coli, cyanobacteria, rat, yeast, and potato and these sequences were used to design PCR primers.  PCR reactions were attempted with total genomic DNA and total RNA from soybean leaf, nodule and callus culture.  RNA extraction from potato leaf was used as a positive control.  Total RNAextracted from soybean callus culture produced a clear, reproducible PCR product that was consistent with the positive control.  PolyA+ selected mRNA from soybean callus tissue was then used to construct a cDNA library in lambda ZapII (Stratgene, Lajolla CA).  PCR was used to follow soybean G6PDH during the library construction with samples of the first strand, second strand, size fractioned cDNA, and finally a portion of the finished lambda phage library was used as template DNA.  In each of these samples it was possible to detect a soybean G6PDH PCR product.  Further experiments sought to isolate and sequence the soybean G6PDH from this newly constructed library.