Table 2.

Dependency of metabolism of [¹⁴C]GA substrates on pH and lysate volumes from E. coli transformed with pMOSElox clone D4

pH	Substrate	Lysate, μl	Products, % of recovered radioactivity
			GA14-ald.	GA14	GA12-ald.	GA12	Compound W	Compound X	Compound Y	Compound Z
7.4	GA12-ald.	700*			0†	82	9	9	0	0
	GA14-ald.	700*	3†	97
5.4	GA12-ald.	0.27			31†	69	0	0	0	0
		1.09			15†	83	0	0	2	0
		4.38			2†	90	2	1	5	0
		17.5			0†	73	5	13	7	2
		35			0†	52	8	32	8	0
		70			0†	27	9	58	3	3
		700*			0†	42	7	51	0	0
	GA12	700*				31†	7	52	10	0
	Compound X	70						95†		5
	GA14-ald.	700*	0†	100

Lysates were prepared as described in Material and Methods using standard lysis buffer or a modified lysis buffer containing 200 mM BisTris⋅HCl (pH 5.4) instead of 200 mM Tris⋅HCl (pH 7.4). All products, except for compound Y and compound Z, were analyzed by GC-MS as their methyl ester trimethylsilyl ether derivatives and identified, where possible, by comparison of their mass spectra with those of published spectra (22). m/z (% relative abundance) for GA₁₂: 368(2.6), 360(0.92), 336(17), 328(8), 308(100), 300(58), 246(43), 240(21); for GA₁₄: 456(4.2), 448(1.2), 424(30), 416(11), 396(25), 388(7), 306(96), 298(65); for compound W (putative 12-hydroxy-GA₁₂): 456(5.4), 448(4.5), 424(56), 416(27), 396(44), 388(25), 334(46), 326(27), 306(100), 239(72); for compound X: 456(5.5), 448(2.8), 424(37), 416(23), 334(21), 326(9), 291(22), 283(15), 245(100), 239(48). 

^*Total volume was 10 times that of the standard assay. 

^†Unmetabolized substrate.