Cell-Bound Thiaminase I ofBacillus thiaminolyticus

Abstract

The distribution of the extracellular enzyme, thiaminase I, was determined for logarithmically growing cultures of Bacillus thiaminolyticus . About 60% of the enzyme is associated with the cells throughout the growth cycle. The remainder of the enzyme is in the culture medium. The release of the cell-bound thiaminase I is examined under a variety of conditions. The rate and extent of release is dependent on the pH and the nature of the incubation solution. The release process appears to be relatively independent of de novo protein synthesis, energy derived from oxidative phosphorylation, or divalent metal ions. The absence of carbon or nitrogen sources has little effect on the release of the enzyme. Cell-bound thiaminase I probably is the immediate precursor for extracellular thiaminase I found in the culture medium. Washed cells continue to release thiaminase I at the expense of cell-bound enzyme. In addition, purified cell-bound thiaminase I is indistinguishable from purified extracellular thiaminase I by a number of physical and kinetic criteria.

Reversible Inactivation of Thiaminase I ofBacillus thiaminolyticusby Its Primary Substrate, Thiamine

“…Reactions mixtures containing 2.1 x 10-I moles of thiaminase I; 50 mMsodium phosphate, pH 5.8; and either 2.0 (0), 4.0 (A), or 6.0 (E) x 10-' moles of thiazole-2-14Cthiamine -hydrochloride are incubated at 37 C. Samples are removed and the reaction stopped by the addition of an equal volume of saturated trichloroacetic acid solution. The samples were then chromatographed and counted as described previously (1). Tz, methylhydroxyethylthiazole.…”

“…tivated thiaminase I regardless of whether the enzyme comes from a crude, partially purified, or homogeneous preparation. They probably do not represent subunits or contaminating proteins because purified thiaminase I migrates as a single, sharp band in SDS-polyacrylamide electrophoresis gels with an apparent molecular weight of 44,000 (1). Attempts to further resolve these two bands by gel filtration or ion exchange chromatography have been unsuccessful.…”

“…Purified, released cell-bound thiaminase I is used in the experiments reported in this communication, except where noted. Techniques for the release and purification of cell-bound thiaminase I have been described previously (1). Thiaminase I activities are assayed by either of two methods.…”

“…In most cases, the spectrophotometric assay of Douthit and Airth (2), as modified by Wittliff and Airth (13), is used. Thiaminase I activity is also determined radiometrically by the method of Agee, et al (1). The presence or absence of thiaminase I activity in the protein bands resolved on Tris-glycine polyacrylamide electrophoresis gels is determined by first cutting the gel in half parallel to its long axis.…”