The Relationship Between A Phenylalanine-Rich Protein And Glycolate Oxidase In Green Leaves Of Brassica Parachinensis Bailey

Glycolate oxidase (EC 1.1.3.15, GO) is a peroxisomal flavin-dependent enzymein the photorespiratory pathway in plants, which is consisted of identical subunits ofMr 40kD. It was confirmed that GO was bound with a phenylalanine-rich proteinforming a protein complex which was named GO complex. In this paper, therelationship between GO and the phenylalanine-rich protein in GO complex wasstudied.GO complex was separated from green leaves of Brassica parachininensisBailey using 10% acetic acid precipitation, (NH₄)₂SO₄ precipitation, Sephadex G-50molecular sieve chromatograph and DEAE-Cellulose negative ion-exchange chromat-ograph. GO complex exhibited one band of Mr 40kD in SDS-PAGE and GO enzymeactivity was determined in GO complex. Proteins in GO complex migrated to cathodewas detected by SDS-acetate cellulose membrane eletrophoresis. The component ofamino acid in the proteins migrated to cathode in SDS-PAGE was assayed. Thecontent of phenylalanine of proteins in cathode was 12.96%.GC-â… protein and GC-â…¡protein were obtained when SDS-denatured GOcomplex was subjected to Sephadex G-100 gel filtration chromatography. Molecularweight of GC-â… protein and GC-â…¡protein are 67 kD and between 14 kD and 67 kDrespectively. But in SDS-PAGE GC-â… protein exhibited two bands of Mr 40 kD and14 kD or one band of Mr 40 kD, while GC-â…¡protein exhibited one band of Mr 14 kDor no band. This result indicated that the protein of Mr 14kD related closely to GO.GO complex was subjected to dot blot and SDS-PAGE Western blot usingantibody against GO 40 kD subunit and antibody against GO complex respectively,which showed that the GO complex was recognized by antibody against GO 40 kDand GO 40 kD subunit wasn't recognized by antibody against GO complex. The resultsuggested that the phenylalanine-rich protein was in the outside of GO complex andGO was in the inside of GO complex.Crude extract from green leaves of Brassica parachininensis Bailey wassubjected to SDS-PAGE Western blot using antibody against GO complex and there were two main immunity bands of Mr 54 kD and Mr 14 kD. Extract the 54 kD and 14kD protein of Crude extract from green leaves of Brassica parachininensis Bailey,using the antibody against GO complex to SDS-PAGE Western blot assay. There werea set of ladder bands varied from Mr 30 kD to 97 kD, Which showed that the quantityof SDS bound by GO complex was variable.Prepared the antibody against 14kD protein of crude extract from green leavesof Brassica parachininensis Bailey. This antibody was used to dot blot andSDS-PAGE Western blot assay of GO complex., which showed that the antibodyagainst 14 kD protein recognized the GO complex and didn't recognize the 40 kDsubunit of GO. The results indicated that 14kD protein was the phenylalanine-richprotein in GO complex.GO complex was subjected to SDS-acetate cellulose membrane immuno-hybridization using the antibody against GO complex, the antibody against GO 40kD subunit and the antibody against 14kD protein respectively. The results suggestedthat GO migrated to anode and cathode, and the phenylalanine-rich protein migratedto cathode.