Lysyl oxidase sequencing

For positive controls, it is extremely important to have a tissue culture cell line with an abundant amount of target nucleic acid. Varieties of tissue culture cell lines are available from American Tissue Culture Center, Rockville, MD. Rapidly growing tissue culture cell lines transfected with the target nucleic acid could be successfully used for positive controls. For example, formalin-fixed paraffin-embedded foreskin fibroblast tissue culture cell-line FS4 can be used as a positive control for human lysyl oxidase mRNA detection and c-W-ras transformed RS-485 cell line for normal ras message detection. Thin sections (4-5 im) of paraffin-embedded positive control cell lines could be placed simultaneously near the side of the experimental human or animal tissue. Similarly, one can also use freshly cut animal or human tissue (with the target sequences) preserved, sectioned, and mounted under standard laboratory conditions. For negative controls, choose a cell line (or a tissue) that completely lacks the target nucleic acid see Note 12). 

Trackman, P. C., Pratt, A. M., Wolanski, A., Tang, S.-S., Offer, G. D., Troxler, R. F., and Kagan, H. M., 1990, Cloning of rat lysyl oxidase cDNA complete codons and predicted aminoacid sequences. Biochemistry 29 4863ii4870. 

Structure of the active center. The active centers of this dimeric enzyme are so well embedded into its protein structure that they are inaccessible to the solvent. The two centers are situated approximately 30 A apart from each other but connected by /3-strands. The active center consists of a type 2 copper center and a cofactor. Sequence comparisons have established that the residues His 8, His 246, and His 357 coordinate the copper ions in both yeast and plants (e.g., lentil seeds) [120,122]. The participating cofactor is typical for amine oxidases, diamine oxidases, and lysyl oxidases but has not yet been found in any other protein - 2,4,5-trihydroxy-phenylalanine quinone [123, 124] (also known as TOPA-quinone, TPQ or 6-hydroxy-DOPA quinone), an internal cofactor which is created by post-translational modification of the tyrosine in position 387 [120]. The consensus sequence of the amino acids neighboring the TOPA cofactor are conserved in all known amine oxidases - Asn-TOPA-Asp/Glu [113,120, 123,125-127]. The positions of the histidine ligands relative to TOPA quinone are conserved in all known amine oxidases as well. The chain lengths of the amine oxidase monomers vary according to the organism of origin 692 residues in yeast [128], 762 in bovine serum amine oxidase [128,129] and 569 in the enzyme from lentil seeds [120,130]. 

A phylogenetic relationship may be assumed for amine oxidase and diamine oxidase which shows distinct sequence homologies in their C-terminal regions [128]. Lysyl oxidase is not related to the other amine oxidases as it does not share any homology in structure or amino acid sequence [128] with the other amine oxidases. Non-copper enzymes with similar structures or sequences have not yet been found for any of the three enzymes. 

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