Louis D. Heerze

The use of hydrophobic synthetic glycosides as acceptors in glycosyltransferase assays

A general method is described for the assay of glycosyltransferase activity, which makes use of synthetic glycoside acceptors attached to hydrophobic aglycones. The products formed by incubation of an enzyme with acceptor and radiolabelled sugarnucleotide can then be rapidly (one minute) separated from interfering radioactivity by adsorption on to reverse-phase C-18 cartridges. After aqueous washing, products are easily isolated by elution with methanol. The utility of the method for the assay of β(1–4)galactosyltransferase, α(1–2)fucosyltransferase andN-acetylglucosaminyltransferase I and V is demonstrated.

TREATMENT OF TRAVELLER'S DIARRHEA

This invention relates to treatment of travellers diarrhea, including diarrhea caused by enterotoxigenic E. coli (ETEC), using oligosaccharide compositions which bind E. coli heat-labile toxin (LT) and/or one or more serotypes of enterotoxigenic E. coli organisms. More specifically, the invention concerns neutralization and removal of LT associated with travellers diarrhea. This invention also relates to prevention of ETEC from colonizing the intestinal tract and inducing disease.

Comparison of the lectin-like activity of pertussis toxin with two plant lectins that have differential specificities for α(2-6) and α(2-3)-linked sialic acid

In this report we have compared the lectin-like properties of Pertussis toxin with two plant lectins which are known to possess different specificities towards terminal Neu5Ac Gal linkages on glycoconjugates. The hemagglutinin from elderberry bark (Sambucus nigra) has a binding specificity for terminal Neu5Ac alpha (2-6) Gal sequences and was found to bind a series of glycoconjugates with a similar specificity as Pertussis toxin. The binding specificity of Pertussis toxin was different from that of the leukoagglutinin from the seeds of Maackia amurensis which preferentially binds terminal Neu5Ac alpha (2-3) Gal sequences. These observations confirm the specificity of Pertussis toxin for Neu5Ac alpha (2-6) Gal glycoconjugate sequences.

Synthesis and characterization of a Pertussis toxin-biotin conjugate.

We prepared a Pertussis toxin-biotin conjugate and found its biological properties to be similar to those of native Pertussis toxin with respect to the hemagglutination, Chinese hamster ovary cell, and lymphocyte proliferation assays. Direct binding to Chinese hamster ovary and Jurkat cells was observed using fluorescence microscopy. Pertussis toxin-biotin was also found to possess similar glycoconjugate binding specificities as those of 125I-labeled Pertussis toxin.

Assays for amino acid decarboxylase enzymes using ion-exchange cartridges

A general radiochemical method for estimating the activity of amino acid decarboxylases is reported. This method utilizes ion-exchange cartridges to separate unreacted radiolabeled amino acid substrates from product amines, which can then readily be quantitated by liquid scintillation counting. The assay is simple, rapid, and more sensitive than standard 14CO2 trapping procedures if uniformly labeled amino acid substrates are utilized. Acidic, basic, and aromatic amino acid decarboxylases can be assayed with the appropriate choice of cation or anion exchangers. The utility of the method is demonstrated for aspartate-alpha-decarboxylase, tyrosine decarboxylase, and lysine decarboxylase where kinetic parameters are comparable to values obtained by standard radiochemical 14CO2 trapping assays.