G van Heeke

Learn More
The gene encoding the Vibrio proteolyticus aminopeptidase was cloned and sequenced and its amino acid sequence was deduced. The gene encodes a 54 kDa protein, larger than the previously reported size of 30 kDa for the purified aminopeptidase. Sequence alignments revealed a 43-45% homology with two other Vibrio sp. extracellular proteinases.
A synthetic gene coding for the inhibitor protein of bovine heart mitochondrial F1 adenosine triphosphatase was designed and cloned in Escherichia coli. Recombinant F1-ATPase inhibitor protein was overproduced in E. coli and secreted to the periplasmic space. Biologically active recombinant F1-ATPase inhibitor protein was recovered from the bacterial cells(More)
In order to obtain large quantities of extremely pure human asparagine synthetase for detailed kinetic and structural studies, its gene was cloned into a 2mu plasmid (pBS24.1GAS) suitable for replication in a Saccharomyces cerevisiae cir0 strain (AB116). In this construct, the transcription of the asparagine synthetase gene is regulated by the alcohol(More)
Site-specific mutagenesis was used to replace the N-terminal cysteine in human asparagine synthetase by an alanine. The mutant enzyme was expressed in the yeast Saccharomyces cerevisiae, and the asparagine synthetase activity was analyzed in vitro. The mutation resulted in the loss of the glutamine-dependent asparagine synthetase activity, while the(More)
Cys-1 mutants of recombinant human asparagine synthetase were constructed and their ability to catalyze the glutamine-dependent nitrogen transfer reaction required for asparagine biosynthesis was determined. In agreement with previous work, altering Cys-1 to either Ala or Ser eliminated the glutamine-dependent activity while only minimally affecting the(More)
Previous studies shows that the replacement of Phe-198 in carbonic anhydrase III to the corresponding Leu residue found in carbonic anhydrase II caused the appearance of isozyme II-like activity (LoGrasso et al. (1991) Biochemistry 30, 8463-8470). Carbonic anhydrase II is more efficient in the catalysis of CO2 hydration by 500-fold and has an apparent pKa(More)
A new expression vector was constructed which allows the overproduction in Escherichia coli of tripartite proteins consisting of human carbonic anhydrase isozyme II (hCAII), a peptide linker containing an enterokinase cleavage site, and a target protein of interest. Carbonic anhydrase is soluble and stable in E. coli and serves as a highly specific(More)
The role of the histidyl residue at position 49 (H49) of the bovine mitochondrial F1-ATPase inhibitor protein (F1I) was examined by site-directed mutagenesis. Six amino acids (Q, E, K, V, L, and I) were substituted for H49 and the activities of the resulting inhibitor proteins were characterized with respect to pH. Each of the six mutations abolished the pH(More)
The replacement His107-->Tyr is a cause of carbonic anhydrase II deficiency syndrome in humans (Venta, P. J., Welty, R. J., Johnson, T. M., Sly, W. S., and Tashian, R. E. (1991) Am. J. Hum. Genet. 49, 1082-1090). We have prepared this mutant of human carbonic anhydrase II by site-directed mutagenesis and expressed it in Escherichia coli. The mutant was too(More)