TABLE 1.

Incorporation of “[¹⁴C]inositol 1-phosphate” into lipid (AIP and AI) under various AIP synthase reaction conditions

The reaction mixture of the AIP synthase reaction is described under “Experimental Procedures.” After incubation at 60 °C for 30 min, radioactivity in the chloroform-soluble materials was counted with the exception that the assay using the P. furiosusmembrane was carried out at 70 °C.

“[14C]Inositol 1-phosphate”	Source of enzyme	Other constituents in the reaction mixturea	Relative incorporation
			%
+b	M. thermautotrophicus membranec	Complete	100
+		−CDP-ArOH	4
+		−Mg2+, Mn2+	2
+		−Mg2+	91
+		−Mn2+	52
+		−Triton X-100	6
+	P. furiosusmembraned	Complete	145
+	None	Complete	1
+	E. coli pET21a-MTH1691e	Complete	281
+	E. coli pET21af	Complete	1
−g	M. thermautotrophicus. membranec	Complete	1

^a Complete mixture other than the enzyme preparation and “[¹⁴C]inositol 1-phosphate” contained 40 nmol of CDP-archaeol, 0.1 m Bicine buffer (pH 8.0), 4 mm MnCl₂, 20 mm MgCl₂, 5 mm 2-mercaptoethanol, and 0.5% (w/v) Triton X-100.

^b [¹⁴C]Inositol 1-phosphate was synthesized from [¹⁴C]glucose 6-phosphate in a complete IP synthase reaction mixture with the supernatant fraction of the M. thermautotrophicus homogenate.

^c The membrane fraction of M. thermautotrophicus homogenates (180 μg of protein/assay).

^d The membrane fraction of P. furiosus homogenates (180 μg of protein/assay).

^e Homogenate of E. coli pET21a-MTH1691 (180 μg of protein/assay).

^f Homogenate of E. coli pET21a (180 μg of protein/assay).

^g The IP synthase reaction mixture was incubated without the addition of the M. thermautotrophicussupernatant fraction. The solution included only [¹⁴C]glucose 6-phosphate as radiolabeled material.