5N1-Labeled hypoxanthine and 1,3-15N-labeled uracil were synthesized chemically and used to prepare labeled yeast tRNAPhe biosynthetically. Maps (500 MHz) of 15N chemical shift vs. proton chemical shift were obtained, for each ring NH group, by means of INDOR (difference heterodecoupling) and also by means of a proton-observe two-dimensional method involving coherences of forbidden resonances of the NH system. Resonances of GC11, T54-m1A58, GU4, and A psi 31 were confirmed, assigned, or reassigned. psi 39 was found to be in anti conformation, not syn as previously stated. Almost all the uracil NH group resonances could be separated, but most of the GC resonances are too close even in two dimensions to be separately resolved with the observed 20-Hz 15N line width.

Phosphofructokinase (PFruK) from the slime mold Dictyostelium discoideum has been purified to homogeneity over 15000-fold with a 29% yield. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis of the final preparation revealed a single band of 95 kDa. The native molecular mass was determined by gel filtration to be 382 kDa, indicating that the enzyme is a homotetramer. An antibody raised in rabbits against the 95-kDa band immunoprecipitated PFruK activity while it did not react with the enzyme from yeast and mammalian cells. The apparent PI was 6.8 and the pH optimum was 7.6. The enzyme had an activation energy (EJ of 29

We demonstrate a fairly general method for identification of NMR absorption lines of macromolecules extracted from microorganisms, based on nuclear Overhauser effects (NOE). Several

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