Calf thymus alkaline phosphatase. II. Interaction with detergents.

1. A number of detergents were used to dissolve calf thymus plasma membranes rich in alkaline phosphatase (orthophosporic-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1) activity. 2. The Stokes' radius (r) of alkaline phosphatase in each detergent was measured by gel filtraton. The size of the solubilized enzyme varied from r = 6.2 nm in sodium cholate to r = 8.3 nm in Berol EMU-043. With N-alkylsulphates, the apparent size increased with alkyl chain length, with r = 6.4 nm (C9) and r = 7.3 nm (C12). Tween 20 failed to solubilise the enzyme. 3. The effect of each detergent on the catalytic activity of alkaline phosphatase was determined. The non-ionic detergents Triton X-100, Nonidet P-40, Berol EMU-043, Tween 20 and the zwitterionic detergent Empigen BB increased V by 10--50% without substantially altering the Km for p-nitrophenylphosphate. The bile salts sodium deoxycholate and sodium cholate decreased V and increased the apparent affinity of the enzyme for nitrophenylphosphate. Inhibition was concentration-dependent up to the critical micellar concentration, above which it remained constant (deoxycholate, 33% cholate, 76%). Alkylsulphates (C8-12) had no significant inhibitory effect during 24 h at 23 degrees C. 4. Exchanging one detergent for another altered alkaline phosphatase activity to a state characteristic for the second detergent, e.g. the activity of cholate-inhibited alkaline phosphatase was restored to normal levels by excess of Triton X-100 and vice versa. The inhibitory effect of deoxycholate and cholate therefore result primarily from interactions between detergent and alkaline phosphate, rather than from selective removal of lipids from the enzyme. 5. Pure lecithin, lysolecithin and an ether-deoxylysolecithin each reactivated cholate-inhibited alkaline phosphatase in a concentration-dependent fashion. Cholesterol had no effect. 6. The half-life (t1/2) of membrane-bound alkaline phosphatase at 55 degrees C was 64 min. With the exception of Berol, solubilisation in non-ionic detergents caused no marked change in this sensitivity. The enzyme became more labile in deoxycholate (t1/2) = 31 min), but less labile in cholate (t1/2 = 99 min). Alkylsulphates, which are strong denaturants, markedly increased the sensitivity of the enzyme to heat-inactivation (C8, t1/2 = 13 min; C9--12, t1/2 less than 2 min). 7. It is concluded that membrane-bound alkaline phosphatase is separated from most if not all of its neighbouring lipid moieties by these detergents, which bind to the solubilised enzyme. The number and character of molecules binding to the enzyme influence its size and shape, its susceptibility to inactivation and its catalytic activity.

Calf thymus alkaline phosphatase. I. Properties of the membrane-bound enzyme.

A membrane fraction from calf thymocytes was used to investigate molecular and catalytic properties of membrane-bound alkaline phosphatase (ortho-phosphoric-monoester phosphohydrolase EC 3.1.3.1). The principal findings were: 1. Solubilization of membranes with the non-ionic detergent Triton X-100 increases alkaline phosphatase activity by 30-40%. The enzyme activity elutes in a single peak (Stokes' radius = 7.7 nm) after chromatography in Sepharose 6B in the presence of Triton X-100. The activity also sediments as a single component of approx. 6.4 S during centrifugation in sucrose gradients containing Triton X-100. 2. Ion-exchange chromatography and isoelectric focusing in the presence of Triton X-100 indicate substantial charge heterogeneity. Two overlapping bands, a peak at pH 5.92 with a pronounced shoulder at pH 5.29, are apparent by isoelectric focusing. 3. The pH optimum for hydrolysis of p-nitrophenylphosphate (pNPhP) by the undissolved enzyme(s) is 9.57. Half-maximal activity occurs at pH 8.65 and ph 10.45. Triton X-100 has no effect on the pH profile. 4. Catalytic activity is affected by amines, especially analogues of ethanolamine. Diethanolamine exerts a unique stimulatory effect, but does not change the pH dependency. Increasing the concentration of diethanolamine from 0 to 1 M causes a 6-fold increase in Km and a 10-fold increase in the rate of hydrolysis of pNPhP. Glycine is inhibitory. 5. EDTA causes an irreversible loss of activity with t1/2 (1 mM EDTA, pH 8.2, 23 degrees C) = 3.5 h. Optimal activity is achieved in 0.1--1.0 mM Mg2+, although this does not cause the degree of activation reported to occur with the purified enzymes. Other divalent ions are inhibitory. Concentrations required to reduce activity to 50% of control are: Zn2+, 4.0 muM (no added Mg2+) and 30 muM (in the presence of 1 mM Mg2+); Mn2+, 0.25 mM (+/- Mg2+); Ca2+, 20 mM (+/- Mg2+). 6. Monovalent cations have little effect on activity. In the absence of added Mg2+, 50--150 mM Na+ is partially inhibitory, but markedly less so in the presence of 1 mM Mg2+. K+ has no significant effect. 7. Of the substrates tested, pNPhP (Km = 44 muM) was most rapidly hydrolyzed. Other substrates (rate relative to pNPhP) were alpha-naphthylphosphate (0.79), 2'-AMP (0.80), 5'-AMP (0.70), 3'-AMP (0.63), alpha-glycerophosphate (0.47) and glucose 6-phosphate (0.35). Phosphodiesterase activity was less than or equal to 10% of the phosphomonoesterase activity (for pNPhP) as evidenced by the lack of hydrolysis of bis(p-nitrophenyl)-phosphate and cyclic 3',5'-AMP. The ability of these substances to inhibit hydrolysis of pNPhP reflected their capacity as substrates, i.e. the most inhibitory were the most rapidly hydrolyzed.

Identification of a broad-specificity nucleoside transporter with affinity for the sugar moiety in Giardia intestinalis trophozoites.

A broad-specificity nucleoside transporter has been identified in Giardia intestinalis trophozoites, using a rapid sampling assay to measure influx of [3H]deoxycytidine, [3H]adenosine and [3H]guanosine at 0 degrees C. The influx of each labelled nucleoside was inhibited strongly by all common, naturally-occurring nucleosides but only poorly or not at all by nucleobases, indicating that the transporter recognizes structural features on the furanosyl moiety of ribo- and 2'-deoxyribonucleosides. Both 2'- and 5'-deoxyadenosine were potent inhibitors of influx (greater than 95% inhibition at 2 mM), whereas 3'-deoxyadenosine was significantly less effective (approx. 70% inhibition), and 2',3'-dideoxycytidine and cytosine arabinoside were virtually inactive (0-20% inhibition). The data reveal that the 2'- and 5'-hydroxyl groups are not necessary for the recognition of nucleosides by this transporter. However, the 3'-hydroxyl appears to be important. Michaelis-Menten constants (Km) were calculated for the influx at 0 degrees C of deoxycytidine (220 +/- 116 microM) and adenosine (45 +/- 24 microM), with respective Vmax values of 13 +/- 4 and 11 +/- 2 pmol min-1 (10(6) cells)-1. Only 12-26% of [3H]thymidine influx occurred through this transporter, the remainder entering the cells through a thymine/uracil-specific transporter described previously. Thymidine exhibited a Ki of 205 +/- 90 microM against [3H]deoxycytidine influx.

A low-affinity nucleobase transporter in the protozoan parasite Giardia intestinalis.

A membrane transporter with general affinity for purine and pyrimidine bases has been identified in Giardia intestinalis trophozoites by measuring cellular influx of [3H]adenine, [3H]guanine and [3H]thymine at 0 degrees C. The base transporter is distinct from the thymine/uracil-specific (type 1) and broad-specificity (type 2) nucleoside transporters of G. intestinalis. Influx of each labelled base was retarded by unlabelled bases, with inhibition in the order: hypoxanthine greater than adenine greater than thymine greater than uracil. The IC50 values for these bases (measured for [3H]adenine influx) were 0.46 mM, 1.15 mM, 1.52 mM and 2.28 mM, respectively. Nucleosides did not inhibit base influx (less than or equal to 15% inhibition at 2 mM, a 400-fold molar excess, at which concentration [3H]nucleoside influx was inhibited by greater than 95%). The Michaelis-Menten constant (Km), calculated for adenine and thymine influx at 0 degrees C, was 1.44 +/- 0.08 mM and 1.61 +/- 0.37 mM, respectively, with corresponding Vmax of 383 +/- 16 and 498 +/- 112 pmol min-1 (10(6) cells)-1. The data demonstrate the existence of a low-affinity, facilitative base transporter with no detectable affinity for nucleosides. The inability of uridine or thymidine to significantly reduce the rate of thymine influx indicates that the previously described thymine/uracil-specific (type 1) thymidine transporter cannot transport thymine, despite its affinity for the base.

Biotinylation: an alternative to radioiodination for the identification of cell surface antigens in immunoprecipitates.

A method has been developed in which the conventional radioiodine label is replaced by non-radioactive biotin in studies involving the immunoprecipitation and analysis of cell surface antigens. The labelling reagent, d-biotinyl-N-hydroxysulfosuccinimide ester (NHSS-biotin), reacts preferentially with lysine residues in polypeptides and possibly also with free amino-groups on carbohydrates and lipids. The reagent can be used as a cell surface label, does not interfere with antigen-antibody interactions and allows labelled molecules to be detected with high sensitivity using streptavidin-peroxidase conjugates. The target antigens of a range of monoclonal antibodies to human cell surface components have been identified using this procedure.

Two genes encoding homologous 70-kDa surface proteins are present within individual trophozoites of the binucleate protozoan parasite Giardia intestinalis.

A 0.52-kb DNA sequence encoding part of a major surface antigen of Giardia intestinalis trophozoites has been amplified by the polymerase chain reaction (PCR) using primers specific for nucleotide sequences that are conserved between two apparently related genes, tsp11 (cloned previously from the Australian G. intestinalis isolate, Ad-1) and tsa417 (cloned from the Afghanistani isolate, WB). Restriction analysis revealed that the DNA amplified from each of seven axenic isolates of G. intestinalis was not homogeneous, even though the template DNA had been purified from cultures that had been established from single trophozoites. Every isolate yielded two PCR products, whose respective cleavage fragments corresponded to tsp11 (HindIII+, PstI-, KpnI-) and to tsa417 (HindIII-, PstI+, KpnI+). This was confirmed by cloning individual amplification products into the plasmid vector, pGEM-7Zf(+). The sequence of one cloned fragment (1-P4), derived from the Ad-1 isolate, but possessing restriction sites characteristic of tsa417, exhibited 98.6% identity over 425 bp with tsa417 and only 63.8% identity with the corresponding region of tsp11. The data indicate that individual trophozoites of all the isolates examined contain a copy of each of these homologous genes.

The interaction of lactose-specific lectins with acid-treated and lactose-substituted sepharose.

The binding of two lectins, one a galactosyl/lactosyl and the other a lactosyl binding protein, to various Sepharose 4B derivatives has been investigated. The adsorbents, lactose-substituted Sepharose, acid-treated Sepharose and acid-treated, lactose-substituted Sepharose, were each tested with regard to their overall binding capacity and for the ability to separate the lectins by differential elution with solutions of galactose and lactose. The binding capacity for both lectins decreased in the order Lac-acid-Sepharose greater than Acid-Sepharose greater than Lac-Sepharose much greater than Untreated Sepharose. The ability of the gels to bind both lectins with a sufficient affinity to allow the proteins to be purified by differential elution decreased in a similar order. Acid-treated, lactose-substituted Sepharose proved the most useful gel and was utilised to isolate each lectin in a pure form.

Assays for total and antigen-specific polymeric IgA in serum based on binding to secretory component.

Binding assays with secretory component (SC) were used to detect polymeric IgA antibody to E. coli lipopolysaccharide and to estimate total polymeric IgA in sera from 14 patients with alcoholic liver disease and eight normal controls. Radioiodinated human SC was shown to bind to polymeric IgA and IgM but not to monomeric IgA, secretory IgA or IgG. Serum aliquots (0.5 ml) were totally depleted of IgM using 2 ml anti-IgM affinity columns and the effluent sera were titrated in microtitre plates coated with lipopolysaccharide, the binding of polymeric IgA being detected by adding 10 ng radiolabelled SC. Total polymeric IgA was measured via its capacity to inhibit the binding of 5 ng labelled SC to IgM coated wells, quantitation being achieved by comparison with the inhibition produced by purified polymeric IgA. Total lipopolysaccharide-specific IgA antibody was detected by ELISA in sera from both patients and controls, 1185 +/- 793 and 56 +/- 19 U/100 microliters (mean +/- SD), respectively; but polymeric IgA antibody was detected only in patients' sera (131 +/- 214 U/100 microliters). The concentration of total polymeric IgA was higher in patients' sera than in control sera (488 +/- 333 and less than 120 micrograms/ml respectively).

Characteristics of thymidine transport in Giardia intestinalis trophozoites.

The transport of thymidine by the protozoan parasite Giardia intestinalis was examined at 0 degrees C. This temperature prevented attachment of the cells to vessel walls, so that a rapid sampling technique could be used. Thymidine influx (distinguished from gross uptake) was readily measurable at 0 degrees C and was specific and saturable. The transporter appears to be a facilitative carrier, exhibiting a high affinity for thymidine (Km = 50 microM). Thymine and uracil were the most effective inhibitors (Ki = 30 microM and 45 microM, respectively), followed by thymidine, deoxyuridine and uridine (Ki = 64-96 microM). Cytosine, cytidine and deoxycytidine were not inhibitory, even at high concentrations. The data indicate that the oxygen at position 4 of the pyrimidine ring is essential for recognition by the transporter, whereas the 5-methyl group of thymine is unimportant. The furanose ring appears not to be recognized, since D-ribose was non-inhibitory and uridine and deoxyuridine were equally inhibitory but less so than uracil and thymine. This carrier probably mediates the transport of uracil, as well as uridine and thymidine, although influx of the base remains to be measured.

A new locus (vsp417-4) belonging to the tsa417-like subfamily of variant-specific surface protein genes in Giardia intestinalis.

A new variant-specific surface protein gene locus (vsp417-4) of Giardia intestinalis is described. Vsp417-4 represents the fourth member of a gene subfamily that is based on a previously described gene, tsa417 ( =vsp417-1). The new locus was detected by characterising DNA amplified in polymerase chain reactions from the 3' ends of divergent homologues (vsp417-4(A-I), vsp417-4(A-II)) found respectively in isolates belonging to the genetic Assemblage A/Group I ('A-I') and Assemblage A/Group II ('A-II') subtypes of G. intestinalis. The complete vsp417-4(A-I) gene was isolated on a 6.2-kb HindIII fragment by screening a genomic DNA library prepared from a type A-I isolate, Ad-1/C7. The deduced polypeptide (VSP417-4(A-I); 709 amino acids, Mr 72662) has properties characterising it as a Giardia variant-specific surface protein, namely a high cysteine content (11.85 mol%), 29 copies of the four amino-acid 'CXXC' motif, and conserved N-terminal signal peptide and C-terminal hydrophobic (membrane-spanning) segments--the latter terminating with the invariant, hydrophilic motif '-CRGKA'. An extended polyadenylation signal sequence (CTTAGRTAGTAAAY), which appears to be a characteristic feature of VSP genes in Giardia, is situated immediately beyond the stop codon. VSP417-4(A-I) shares 87% sequence identity with VSP417-4(A-II) over its C-terminal 235 amino acids, but only 57-58% identity with VSP417-1, VSP417-2 and VSP417-3 which are encoded by other vsp417 family genes identified in these genotypes. Southern hybridisations, using probes derived from the 5' segment of vsp417-4(A-I), indicated the presence of at least five to six closely related loci in both type A-I and type A-II isolates.

A gene encoding a 69-kilodalton major surface protein of Giardia intestinalis trophozoites.

A gene encoding a 68.5-kDa trophozoite surface protein (TSP11) of the Australian Giardia intestinalis (syn. G. lamblia) isolate, Ad-1, has been cloned from a genomic expression library screened with an antiserum specific for 3 major surface antigens. Sequence analysis of two overlapping genomic fragments identified a single open reading frame that contained no introns and predicted a cysteine-rich, 667-residue polypeptide with features common to other trophozoite surface proteins. These include the presence of 27 copies of the 4-amino acid Cys-X-X-Cys motif, an N-terminal signal sequence and a highly conserved, hydrophobic C-terminal segment. Transcripts from the tsp11 gene were detected as a single band on Northern blots using total RNA extracted from Ad-1 trophozoites. Primer extension analysis indicated that the mRNA has a 5' untranslated region of only 5 nt, similar to the very short (1-6 nt) leader sequences reported for other Giardia mRNAs. A large portion of the promoter distal segment of tsp11 has homology with tsa417, a gene encoding a 72.5-kDa trophozoite surface antigen of the Afghanistan-derived G. intestinalis isolate, WB [13].

Identification of the HA-2 agglutinin in the haemolymph of the ascidian Botrylloides leachii as the factor promoting adhesion of sheep erythrocytes to mouse macrophages.

The HA-2 agglutinin purified from B. leachii haemolymph is shown to mediate the binding of sheep erythrocytes to mouse macrophages. This agglutinin appears to be wholly responsible for the adhesive activity of haemolymph, since upon chromatography of the latter through Sephadex G-200, the adhesive activity for both sheep and guinea pig erythrocytes was confined to those fractions containing the HA-2 agglutinin. The HA-1 agglutinin recovered in earlier fractions was unable to promote the adhesion of guinea pig erythrocytes to macrophages. Adsorption experiments indicated that this was due to a lack of ligand sites on the macrophage surface. These data support earlier conclusions that the HA-1 and HA-2 agglutinins have distinct binding specificities.

Giardia intestinalis: detection of major genotypes by restriction analysis of gene amplification products.

The polymerase chain reaction (PCR) has been used to amplify a 0.52 kb segment of Giardia intestinalis DNA, using primers specific for nucleotide sequences conserved within two genes (tsp11 and tsa417) that encode homologous, cysteine-rich trophozoite surface proteins. Using products amplified from axenic isolates belonging to genetic groups I and II (defined on the basis of allozyme electrophoresis data), restriction endonuclease analysis revealed both tsp11-like and tsa417-like fragments within all samples. The study also identified among the amplification products of group II organisms an additional fragment, containing a novel PstI site, that is not detected in the reaction products of group I isolates. The recovery of three distinct PCR products from each group II isolate was verified by cloning the fragments into the plasmid vector pGEM-7. Fragments containing the new PstI site possess the ClaI site common to both tsp11 and tsa417-like fragments, but they lack the HindIII site which characterizes tsp11-like fragments and also lack the PstI and KpnI sites which characterize tsa417-like fragments. Spot-blot analyses using cloned fragments of all three types as probes showed strong homologous hybridization but weak heterologous hybridization, indicating that each type differs substantially in nucleotide sequence from the others. Because the samples of Giardia DNA used in the PCR were purified from cultures that had been established from single trophozoites, the data indicate that individual trophozoites belonging to genetic group II possess three homologous genes defined by these related fragments. The presence of a PstI site in the amplified segment of the newly-discovered third gene of group II organisms provides a simple diagnostic means of differentiating group I and II isolates.

Comparison of the levels of intra-specific genetic variation within Giardia muris and Giardia intestinalis.

The extent of intra-specific genetic variation between isolates of Giardia muris was assessed by allozyme electrophoresis. Additionally, the levels of allozymic variation detected within G. muris were compared with those observed between members of the two major assemblages of the morphologically distinct species Giardia intestinalis. Four isolates of G. muris were analysed. Three (Ad-120, -150, -151) were isolated from mice in Australia, while the fourth (R-T) was isolated from a golden hamster in North America. The 11 isolates of G. intestinalis (Ad-1, -12, -2, -62, representing genetic Groups I and II of Assemblage A and BAH-12, BRIS/87/HEPU/694, Ad-19, -22, -28, -45, -52, representing genetic Groups III and IV of Assemblage B) were from humans in Australia. Intra-specific genetic variation was detected between G. muris isolates at four of the 23 enzyme loci examined. Similar levels of variation were found within the genetic groups that comprise Assemblages A and B of G. intestinalis. These levels of intra-specific variation are similar to those observed within other morphologically-distinct species of protozoan parasites. We suggest that the magnitude of the genetic differences detected within G. muris provides an indication of the range of genetic variation within other species of Giardia and that this can be used as a model to delineate morphologically similar but genetically distinct (cryptic) species within this genus.

Activation of complement by tetrathyridia of Mesocestoides corti: enhancement by antibodies from infected mice and lack of effect on parasite viability.

Tetrathyridia of the cestode Mesocestoides corti were isolated from the peritoneal cavity of infected mice. The parasites activated guinea pig and mouse complement (C) in vitro by both the classical and alternative pathways as shown by quantitative C fixation and crossed immunoelectrophoresis. The ability of tetrathyridia to activate mouse C was enhanced by preincubating the parasites in serum obtained from mice infected with M. corti. Antibodies of the IgG1 class, an immunoglobulin found in profoundly increased amounts in mice infected with M. corti, as well as IgM and IgG2 antibodies, bound to cultured tetrathyridia and facilitated deposition of the third component of C (C3) from dilute mouse serum, presumably via classical pathway activation. The results demonstrate that mouse IgG1 antibodies do not prevent the activation of C by the tetrathyridia or by C-fixing antibodies of other classes which become attached to the tetrathyridia. The activation of C in vitro by tetrathyridia did not affect their ability to grow in mice, even though C3-derived polypeptides could be detected by immunofluorescence on the surface of the parasites.

Heligmosomoides polygyrus: retarded development and stunting of larvae by antibodies specific for excretory/secretory antigens.

The migration of young adult-stage Heligmosomoides polygyrus from beneath the muscularis mucosa to the lumen of the intestine was monitored to compare the rate of development and maturation of larvae in normal and previously infected mice. The development of surviving larvae was significantly retarded in mice that had experienced one or more previous infections and the adult worms arising from a challenge infection were stunted and appeared anaemic. Identical effects were observed with worms recovered from mice that had been injected with immune mouse serum at the time of challenge, and the magnitude of these effects was related to the amount of serum given. Larval maturation was also retarded in mice immunized with larval excretory/secretory (ES) antigens, even though the antibody response was poor due to the very small (submicrogram) amounts of antigen available for injection. In contrast, larvae developed at a normal rate in mice that had been hyperimmunized with killed exsheathed larvae. These mice had serum antibody titres against both "internal" and cuticular antigens similar to those of highly immune (4x-infected) mice, but they had no detectable antibody against ES antigens. The results indicate that the growth and development in vivo of H. polygyrus larvae are retarded by antibodies specific for larval ES antigens. Stunting is permanent, with female worms being affected more severely than males and egg output per worm correspondingly reduced.