Galactosyltransferase purified from rat milk is distinct from the human and bovine enzyme.

N-Acetylglucosaminide beta 1, 4-galactosyltransferase (EC 2.4.1.22) was purified from rat milk by affinity chromatography on N-acetylglucosamine-Sepharose and alpha-lactalbumin-Sepharose columns. The purified enzyme migrated as three polypeptides of relative molecular weight 59,000, 54,000, and 27,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Antiserum raised against the 54K rat protein immunoprecipitated all three polypeptides, suggesting that they share common antigenic sites. The human milk galactosyltransferase, purified under similar conditions, was electrophoretically homogeneous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with relative molecular weight 54K, and was not immunoprecipitated by the antiserum to the 54K rat milk protein. In addition, Michaelis constants for the enzyme from rat and human milk differed. The apparent Michaelis constant for N-acetylglucosamine and uridine 5'-diphosphate-galactose were 4.8 and .3 mM, respectively, for the rat enzyme, and 1.8 and .028 mM, respectively, for the human enzyme.

Energization of the transport systems for arabinose and comparison with galactose transport in Escherichia coli.

1. Strains of Escherichia coli were obtained containing either the AraE or the AraF transport system for arabinose. AraE+,AraF- strains effected energized accumulation and displayed an arabinose-evoked alkaline pH change indicative of arabinose-H+ symport. In contrast, AraE-,AraF+ strains accumulated arabinose but did not display H+ symport. 2. The ability of different sugars and their derivatives to elicit sugar-H+ symport in AraE+ strains was examined. Only L-arabinose and D-fucose were good substrates, and arabinose was the only inducer. 3. Membrane vesicles prepared from an AraE+,AraF+ strain accumulated the sugar, energized most efficiently by the respiratory substrates ascorbate + phenazine methosulphate. Addition of arabinose or fucose to an anaerobic suspension of membrane vesicles caused an alkaline pH change indicative or sugar-H+ symport on the membrane-bound transport system. 4. Kinetic studies and the effects of arsenate and uncoupling agents in intact cells and membrane vesicles gave further evidence that AraE is a low-affinity membrane-bound sugar-H+ symport system and that AraF is a binding-protein-dependent high-affinity system that does not require a transmembrane protonmotive force for energization. 5. The interpretation of these results is that arabinose transport into E. coli is energized by an electrochemical gradient of protons (AraE system) or by phosphate bond energy (AraF system). 6. In batch cultures the rates of growth and carbon cell yields on arabinose were lower in AraE-,AraF+ strains than in AraE+,AraF- or AraE+,AraF+ strains. The AraF system was more susceptible to catabolite repression than was the AraE system. 7. The properties of the two transport systems for arabinose are compared with those of the genetically and biochemically distinct transport systems for galactose, GalP and MglP. It appears that AraE is analogous to GalP, and AraF to MglP.

Proton-linked D-xylose transport in Escherichia coli.

The addition of xylose to energy-depleted cells of Escherichia coli elicited an alkaline pH change which failed to appear in the presence of uncoupling agents. Accumulation of [14C]xylose by energy-replete cells was also inhibited by uncoupling agents, but not by fluoride or arsenate. Subcellular vesicles of E. coli accumulated [14C]xylose provided that ascorbate plus phenazine methosulfate were present for respiration, and this accumulation was inhibited by uncoupling agents or valinomycin. Therefore, the transport of xylose into E. coli appears to be energized by a proton-motive force, rather than by a phosphotransferase or directly energized mechanism. Its specificity for xylose as inducer and substrate and the genetic location of a xylose-H+ transport-negative mutation near mtl showed that the xylose-H+ system is distinct from other proton-linked sugar transport systems of E. coli.