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Preliminary studies on nucleotide sugars in T. brucei subgroup extracts
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T H I R T E E N T H SEMINAR ON TRYPANOSOMIASIS
P r e l i m i n a r y s t u d i e s on n u c l e o t i d e s u g a r s in 7". b r u c e i s u b g r o u p e x t r a c t s
O. K. A. I T A Z I AND A. R. N J O G U
East African Trypanosomiasis Research Organization, Tororo, Uganda Following the discovery in this laboratory, that the 4S antigens were glycoproteins, experiments were initiated to search for sugar intermediates in the antigen synthesis. These intermediates are designated as sugar nucleotides. Trichloroacetic acid (TCA) soluble trypanosome extracts have been analysed by paper and ion-exchange chromatography. 3 components have been resolved by paper chromatography and sometimes up to 5 peaks could be isolated by ion exchange chromatography. The latter method gave a better resolution of the extract than paper chromatography and it was, therefore, the technique mainly used for resolving sugar nucleotides in trypanosome extracts. One of the peaks from the Dowex column was found to contain uridine diphosphate glucose (UDPG). This substance is a key intermediate in the synthesis of other sugar nucleotides (see reaction scheme below) : U D P galactose epimerase U D P glucose
U D P galactose
NAD ~ U D P galactose is converted into other sugar nucleotides that are added separately to the growing polysaccharide unit of the glycoprotein. The presence of U D P G has been confirmed by isolating it and enzymically determining it as in the reaction below. NAD + NADH U D P glucose ~' ~ U D P glucuronic acid U D P G Dehydrogenase The change in absorbance at 340 nm. was monitored using a unicam SP 500 spectrophotometer. From the reaction curve it was possible to calculate the amount of U D P glucose in the trypanosome extract. The other nucleotides have yet to be identified.
Trypanocidal substances in human serum
FRANK HAWKING
Clinical Research Centre, Northwick Park, Harrow, Middlesex It has long been known that human serum is trypanocidal for non-pathogenic trypanosomes, e.g.T, brucei. I have investigated this further at this Centre. Preliminary work has shown that really there are two substances involved. These are closely similar and may be 2 modifications of an original substance, but they differ in their properties and can be obtained separate from one another. Substance A is active upon trypanosomes in vitro and substance B is active upon trypanosomes in mice. The amounts present in the blood of normal individuals varies greatly; perhaps it is genetically controlled. I n particular, many individuals have practically no substance A, although considerable amounts of substance B. When human serum is injected into a rat or mice, substance A is destroyed or suppressed, but substance B remains intact. The 2 substances are also distributed slightly differently during column fractionation. I n collaboration with Dr. David Ramsden, we have found that the substances are macroglobulins of approximately 820,000 molecular weight, but they travel with the alpha --2 macroglobulins and are not IgM or immunoglobulins, as was incorrectly assumed by Miss Aaronovitch at last year's seminar. When trypanosomes are exposed to these substances and then studied by the electron microscope in collaboration with Dr. Dourmashkin, we have found that the first effect is the formation of large clear spaces in the cytoplasm of the trypanosome (presumably distended with fluid) while the external membranes remain intact for sometime.
T H I R T E E N T H SEMINAR ON TRYPANOSOMIASIS
P r e l i m i n a r y s t u d i e s on n u c l e o t i d e s u g a r s in 7". b r u c e i s u b g r o u p e x t r a c t s
O. K. A. I T A Z I AND A. R. N J O G U
East African Trypanosomiasis Research Organization, Tororo, Uganda Following the discovery in this laboratory, that the 4S antigens were glycoproteins, experiments were initiated to search for sugar intermediates in the antigen synthesis. These intermediates are designated as sugar nucleotides. Trichloroacetic acid (TCA) soluble trypanosome extracts have been analysed by paper and ion-exchange chromatography. 3 components have been resolved by paper chromatography and sometimes up to 5 peaks could be isolated by ion exchange chromatography. The latter method gave a better resolution of the extract than paper chromatography and it was, therefore, the technique mainly used for resolving sugar nucleotides in trypanosome extracts. One of the peaks from the Dowex column was found to contain uridine diphosphate glucose (UDPG). This substance is a key intermediate in the synthesis of other sugar nucleotides (see reaction scheme below) : U D P galactose epimerase U D P glucose
U D P galactose
NAD ~ U D P galactose is converted into other sugar nucleotides that are added separately to the growing polysaccharide unit of the glycoprotein. The presence of U D P G has been confirmed by isolating it and enzymically determining it as in the reaction below. NAD + NADH U D P glucose ~' ~ U D P glucuronic acid U D P G Dehydrogenase The change in absorbance at 340 nm. was monitored using a unicam SP 500 spectrophotometer. From the reaction curve it was possible to calculate the amount of U D P glucose in the trypanosome extract. The other nucleotides have yet to be identified.
Trypanocidal substances in human serum
FRANK HAWKING
Clinical Research Centre, Northwick Park, Harrow, Middlesex It has long been known that human serum is trypanocidal for non-pathogenic trypanosomes, e.g.T, brucei. I have investigated this further at this Centre. Preliminary work has shown that really there are two substances involved. These are closely similar and may be 2 modifications of an original substance, but they differ in their properties and can be obtained separate from one another. Substance A is active upon trypanosomes in vitro and substance B is active upon trypanosomes in mice. The amounts present in the blood of normal individuals varies greatly; perhaps it is genetically controlled. I n particular, many individuals have practically no substance A, although considerable amounts of substance B. When human serum is injected into a rat or mice, substance A is destroyed or suppressed, but substance B remains intact. The 2 substances are also distributed slightly differently during column fractionation. I n collaboration with Dr. David Ramsden, we have found that the substances are macroglobulins of approximately 820,000 molecular weight, but they travel with the alpha --2 macroglobulins and are not IgM or immunoglobulins, as was incorrectly assumed by Miss Aaronovitch at last year's seminar. When trypanosomes are exposed to these substances and then studied by the electron microscope in collaboration with Dr. Dourmashkin, we have found that the first effect is the formation of large clear spaces in the cytoplasm of the trypanosome (presumably distended with fluid) while the external membranes remain intact for sometime.