mer: L-Rhamnulose 1-phosphate aldolase has been purified and crystallized from L-rhamnose-induced cultures of Escherichia coli as well as from a strain of the organism constitutive for L-rhamnose utilization. The enzyme is homogeneous by acrylamide gel disc electrophoresis, by electrophoresis on cellulose acetate, and by immunoelectrophoresis and immunodiffusion in agar gel. It is also homogeneous by sedimentation velocity and density gradient centrifugation. Molecular weight determined by density gradient centrifugation and by Sephadex gel thin-layer chromatography is inThe last enzyme in the sequence, L-rhamnulose 1-phosphate L-lactaldehyde lyase (L-rhamnulose-1 -P aldolase), has been purified partially by Sawada and Takagi (1964) from extracts of L-rhamnose-grown E. coli and by Domagk and Heinrich (1965) from suitably induced cells of Lactobacillus plantarum. These studies were done with impure enzymes of relatively low specific activity, which were not investigated extensively. In this paper are described the purification and crystallization of Lrhamnulose-1-P aldolase from a strain of E. coli as well as studies of its catalytic properties and specificity.
Experimental ProcedureMaterials. L-Rhamnulose-1-P was prepared as previously described (Chiu et al., 1966). The procedures used for conversion of ketose 1-phosphates to mixtures of the corresponding epimeric poly01 phosphates by reduction with NaBHl and for isolation and analysis of the products were those of Ginsburg and Mehler (1966). L-(OB 2671). Some of the results presented here were reported in a preliminary communication (Chiu and Feingold, 1967a). $ Career Development grant (1K3-GM-28-296) awardee of @e U. S. Public Health Service. 98 the order of 1.3-1.4 x 106 daltons. Na+, Cs+, " I + ,Rb+, or K+ is required for activity; K. for KCI is 6The enzyme has a sharp pH optimum of 7.5. K, is 0.3 m~ for L-rhamnulose 1-phosphate, 6.0 mM for Llactaldehyde, and 3.0 m~ for dihydroxyacetone phosphate.for the reaction L-rhamnulose 1 -phosphate L-lactaldehyde + dihydroxyacetone phosphate is 8.3 X M. The enzyme is specific for ketose l-phosphates which have the configuration D at C-3 and L at C-4 in the Fischer projection formula. m.
Two lipoteichoic acids, membrane (MLTA) and wall (VVLTA), have been purified from Streptococcus sanguis by Sepharose and Ecteola-cellulose column chromatographies and concanavalin A-conjugated Sepharose affinity column chromatography. The teichoic acids were homogenous as judged by disc gel electrophoresis, column chromatography, and double diffusion tests. Both MLTA and WLTA consisted of glycerol, phosphate, glucose, and fatty acids in the ratios of 0.
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