The phospholipases of pathogenic and non-pathogenic Trypanosoma species.

Four species of trypanosome were examined for phospholipase activities using 1-[3H]palmitoyl-2-acyl-sn-glycero-3-phosphocholine and 1-acyl-2[14C]linoleoyl-sn-glycero-3-phosphocholine as substrates. The major activity in each species is a phospholipase A1 (EC 3.1.1.32) which does not require calcium. The most effective of the detergents tested for activation of the enzyme from each species, and the Ph optima, are as follows: Trypanosoma brucei, 0.125% Triton X-100 at pH 6.0-8.5; T. congolense, 0.5 mM linoleate at pH 6.0; T. theileri, 0.1% Triton X-100 at pH 6.75; T. lewisi, 0.2 mM sodium dodecyl sulfate at pH 5.2. The specific activity of the enzyme from a pathogenic species, T. brucei, is very high (145 nmol/min/mg/protein) and could contribute to the tissue damage characteristically caused by this parasite. The level in T. lewisi, a non-pathogenic species, is relatively low (1 nmol/min/mg). The levels in T. theileri (31 nmol/min/mg) and T. congolense (10 nmol/min/mg are intermediate. These results are compatible with the hypothesis that phospholipases contribute to the pathogenicity of trypanosomes.

The heterogeneous distribution of beta-glucuronidase in purified Golgi membranes.

An improved procedure for the preparation of purified Golgi membranes by upward flotation is described. The mode of binding of membrane fragments to an affinity adsorbent specific for beta-glucuronidase shows that this enzyme has a heterogeneous distribution in Golgi membranes.

Accumulation of phospholipase A1 in tissue fluid of rabbits infected with Trypanosoma brucei.

Samples of tissue fluid were obtained from plastic cages implanted subcutaneously in rabbits. A phospholipase A1 similar to that found in Trypanosoma brucei appeared in the tissue fluid about seven days after infection with this parasite, increasing with parasite burden, and reaching levels of more than 5 nmol phosphatidylcholine hydrolysed per min per ml tissue fluid. The large amount of phospholipase A1 found free in the tissue fluid appeared to be of trypanosomal origin and was either secreted by living parasites or released from dying organisms. Phospholipase A1 was detectable in blood plasma from the infected rabbits, but at a level considerably lower than in the tissue fluid. An inhibitor of the phospholipase was present in the plasma after the first two week of infection which may be partly responsible for this lower level.

The phospholipase A1 of Trypanosoma brucei does not release myristate from the variant surface glycoprotein.

[3H]Myristoyl-labeled variant surface glycoprotein (VSG) has been isolated from Trypanosoma brucei by reverse phase high performance liquid chromatography and used as substrate for the conversion by trypanosomal enzymes of membrane-form VSG to soluble VSG. Conversion is detected by the release of myristoyl-containing lipids. The major lipolytic enzyme of T. brucei, phospholipase A1, is effective for the hydrolysis of myristoyl esters of p-nitrophenol, in a colorimetric assay. However, the phospholipase is unable to cleave the myristoyl ester linkage of VSG. The phospholipase can be separated from the myristoyl-releasing activity of trypanosome homogenate by centrifugation, affinity chromatography, and anion-exchange chromatography. Elution profiles on anion-exchange high performance liquid chromatography also indicate that the phospholipase is inactive against VSG. A small amount of myristoyl-releasing activity associated with the purified phospholipase is probably due to contamination with a phosphodiesterase which releases myristoyl-containing diglyceride from VSG.

Lysophospholipase 1 in Trypanosoma brucei.

Protein fractions from Trypanosoma brucei brucei showed lysophospholipase 1 activity (E.E.3.1.1.5), against the substrate 1-acyl-sn-glycero-3-phosphocholine, and also phospholipase A1 activity (E.C.3.1.1.4) by hydrolysis of the 1-acyl bond of 1,2-diacyl-sn-glycero-3-phosphocholine. Both enzyme activities were eluted together and showed a 12-fold purification following Sephacryl S-200 column chromatography. A final 96-fold increase in activity was obtained by electrophoresis on nondenaturing polyacrylamide gels to yield a band containing both enzymic activities. Phospholipase A1 showed maximum activity between pH 6.0--8.5 and lysophospholipase 1 had a pH optimum of 8.5. Both activities were found mainly in the soluble fraction of disrupted trypanosomes and were similarly inhibited by N-ethylmaleimide and p-chloromercuribenzoic acid. Although Triton X-100 stimulated phospholipase A1 activity, it inhibited lysophospholipase 1 activity. The Km value for the lysophospholipase 1 was found to be 0.15 mM. It was not possible to resolve separate activities for lysophospholipase 1 and phospholipase A1 and the ratio of the two activities was approximately 1 : 10 for a variety of preparations and treatments. It is probable that a single enzyme displays both activities.

Isolation of phospholipase A1 from Trypanosoma brucei.

Phospholipase A1 from Trypanosoma brucei brucei has been purified 380-fold by column chromatography on phosphatidylcholine-Sepharose affinity columns followed by DEAE-cellulose anion-exchange chromatography and Sephacryl S-200 molecular exclusion chromatography. Octyl-Agarose hydrophobic column chromatography can be substituted for the PC-Sepharose column. The molecular weight of trypanosomal PLase A1 was found to be 26,000 by SDS-polyacrylamide gel electrophoresis.