Kinetic analyses of the membrane-bound alkaline phosphatase activity of hymenolepis diminuta (Cestoda: Cyclophyllidea) in relation to development of the tapeworm in the definitive host

The specific activities of the alkaline phosphatase (APase), type I phosphodiesterase and 5'-nucleotidase activities associated with the brush-border plasma membrane of the tapeworm, Hymenolepis diminuta, decrease significantly as the tapeworm grows and matures. Kinetic analyses of the APase activity associated with membrane preparations from whole 6-, 12-, and 18-d-old H diminuta, and individual pieces of 18-d-old H diminuta cut into ten pieces of equal length, failed to demonstrate qualitative changes in the APase activity. Therefore, the decreased specific activities are apparently due to changes in the ratios of enzymatically active to enzymatically inactive membrane proteins (ie, quantitative changes in the membrane proteins) which occur as the tapeworm grows.     

Phosphohydrolase activity of the isolated, brush-border membrane of Hymenolepis diminuta (Cestoda) following sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis

Following electrophoresis of isolated, brush-border membranes of Hymenolepis diminuta on SDS-polyacrylamide gels, three distinct areas of alpha-naphthyl phosphate (NP) hydrolysis were detected; these corresponded to proteins with molecular weights of 106,800, 172,700, and greater than 340,000 Daltons. Hydrolysis of NP was inhibited by adenosine triphosphate, adenosine;5'-monophosphate, p-nitrophenyl-phosphate, glucose-1-phosphate, glucose-6-phosphate, fructose-6-phosphate, fructose-1,6-diphosphate, molybdate, ethylenediaminetetraacetate (EDTA), and ethyleneglycol-bis-(beta-amino-ethyl)-N,N'-tetraacetate (EGTA), but not by fluoride. Inhibition of NP hydrolysis by EDTA was relieved in the presence of Mg++ or Ca++. Heating the isolated, brush-border membrane in the presence of SDS for 5 min at 95 C destroyed all enzymatic activity. These characteristics indicated that the enzyme(s) responsible for NP hydrolysis (following separation of membrane proteins by SDS-polyacrylamide gel electrophoresis) were the same enzymes responsible for the phosphohydrolase activity associated with intact and solubilized, brush-border membrane preparations of H. diminuta.     

Partial characterization of ribonuclease (RNase) activity from the isolated and solubilized brush border of Hymenolepis diminuta

The isolated brush border membrane of Hymenolepis diminuta contained ribonuclease (RNase) activity which was demonstrable using yeast RNA or synthetic homopolymers of adenylic, cytidylic, inosinic, or uridylic acids as substrates. Polyguanylic acid was not hydrolyzed by worm RNase. RNase activity was inhibited by EDTA and divalent cations as well as sulfhydryl blocking and reducing agents. Polyguanylic acid and DNA were also inhibitors of RNase activity; these compounds were not hydrolyzed, but inhibited the hydrolysis of other substrates, possibly by nonproductive substrate binding. Data suggested that RNase (endonuclease) was probably the major enzyme activity in the degradation of long chain polyribonucleotides at the work's surface, while phosphodiesterase (exonuclease) activity did not contribute significantly to the hydrolysis of these compounds.     

Hymenolepis diminuta: interactions of the isolated brush border membrane with proteolytic enzymes

Proteins of the isolated brush border membrane of Hymenolepis diminuta were hydrolyzed in vitro by chymotrypsin, papain, pepsin, subtilopeptidase A (= subtilisin Carlsberg), and trypsin. Neither proteolytic nor amidase activity was demonstrable in the isolated membrane using proteinaceous (casein and hemoglobin) or chromogenic (benzoyl-arginine-p-nitroanilide and succinyl-alanyl-alanyl-propyl-phenylalanine p-nitroanilide) substrates, and the membrane preparation did not inhibit the proteolytic and amidase activities of these enzymes. Thus, the isolated tegumental membrane of H. diminuta is not inherently resistant to the action of proteolytic enzymes, and it does not inhibit proteolytic activity. In control incubations containing only buffer, the alkaline phosphatase activity of the brush border membrane decreased in a time dependent manner, but in the presence of chymotrypsin, subtilopeptidase A, and trypsin, the membrane retained greater alkaline phosphatase activity (pepsin and papain could not be tested for this effect on alkaline phosphatase activity). A similar time dependent decrease in activity was also noted for each of the proteolytic enzymes in control assays, but subtilopeptidase A and papain retained greater activity in the presence of the isolated membrane preparation when these assays were compared to controls.     

Ca2+ inhibition of brush border alkaline phosphatase activity in Hymenolepis diminuta

The brush border membrane of Hymenolepis diminuta contains several Ca2+-dependent enzymes. Following our isolation of a Ca2+-dependent modulator protein we examined the kinetic properties of the brush border marker alkaline phosphatase from fractionated and crude tegument. We show that this enzyme is inhibited by Ca2+ concentrations approaching those in the calcareous corpuscles of H. diminuta.     

Competitive, uncompetitive, and mixed inhibitors of the alkaline phosphatase activity associated with the isolated brush border membrane of the tapeworm Hymenolepis diminuta

Several compounds were tested as inhibitors of the alkaline phosphatase (AlkPase) activity associated with the isolated brush border membrane of the tapeworm, Hymenolepis diminuta. Molybdate, arsenate, arsenite and beta-glycerophosphate (BGP) were competitive inhibitors of the hydrolysis of p-nitrophenyl phosphate, while levamisole and clorsulon were uncompetitive and mixed inhibitors, respectively. Molybdate was also a competitive inhibitor of the hydrolysis of BGP and 5'-adenosine monophosphate, and levamisole was an uncompetitive inhibitor of BGP hydrolysis. The apparent inhibitor constants (Ki') for molybdate and levamisole were virtually identical regardless of the substrate, and these data support the hypothesis that the AlkPase activity is represented by a single membrane-bound enzyme with low substrate specificity. Quinacrine, Hg2+, and ethylenediaminetetraacetate were also potent inhibitors of the AlkPase activity, but the mechanisms by which these latter three inhibitors function were not clear.     

Identification of ADPR-transferase activity in the rat tapeworm, Hymenolepis diminuta

1. The nuclear fraction of the rat tapeworm Hymenolepis diminuta (Cestoda) contains the enzyme adenosine diphosphoribosyl transferase (ADPR-transferase). 2. The enzyme catalyzes the postsynthetic modification of some nuclear proteins by the covalent attachment of the (ADP-ribose) moiety of NAD to such proteins. 3. The reaction is dependent on DNA which contains strand-breaks, and chain lengths equivalent to (ADP-ribose) is estimated. 4. The formation of polynucleotide products was competitively inhibited by 3-acetamidobezamide, with a Km of 125 microM. 5. The catalytic properties of ADPR-transferase in Hymenolepis diminuta are similar to those in T. brucei.     

Localization of cytochrome C oxidase and cytochrome C peroxidase in mitochondria of Hymenolepis diminuta (Cestoda)

The intramitochondrial localization of cytochrome c oxidase and cytochrome c peroxidase in adult Hymenolepis diminuta was investigated. Mitochondria were fractionated into inner membrane, outer membrane, intermembrane space and matrix and the efficacy of fractionation was monitored employing marker enzymes. Cytochrome c oxidase was associated with the mitochondrial inner membrane. Whereas 55% of the cytochrome c peroxidase activity was in the matrix, 32% of the activity was in the intermembrane space fraction. Based upon the distribution of marker enzymes, a dual compartmentalization of cytochrome c peroxidase is apparent in H. diminuta mitochondria.     

Acid phosphatase activity in the isolated brush border membrane of the tapeworm, Hymenolepis diminuta: partial characterization and differentiation from the alkaline phosphatase activity

The isolated brush border membrane of the tapeworm, Hymenolepis diminuta, hydrolyzes p-nitrophenyl phosphate over a broad pH range. Acid phosphatase activity (pH optimum at 4.0) is inhibited specifically by sodium dodecyl sulfate (SDS) and NaF, while the alkaline phosphatase activity (pH optimum at 8.8) is inhibited specifically by levamisole, 2-mercaptoethanol, and ethylenediaminetetra-acetate (EDTA). These two phosphatase activities are further differentiated in that (1) there is a rapid decrease in alkaline phosphatase activity when the membrane preparation is incubated at pH 4.0, while there is little loss of acid phosphatase activity, and (2) the alkaline phosphatase activity is solubilized with no loss of activity when the membrane is treated with Triton X-100, while such treatment causes a significant loss of acid phosphatase activity. Both activities are nonspecific and hydrolyze a variety of phosphorylated compounds, but the relative activities of the two phosphatases against these substrates vary significantly.     

Monoamine oxidase in adult Hymenolepis diminuta (Cestoda).

A membrane-bound monoamine oxidase (EC 1.4.3.4) was demonstrated in homogenates of Hymenolepis diminuta. The enzyme oxidized a variety of biologically active amines (in decreasing order: dopamine, adrenaline, noradrenaline, tryptamine, tyramine, octopamine), there was, however, no activity with 5-hydroxytryptamine or benzylamine. No diamine oxidase (EC 1.4.3.6.) could be detected in H. diminuta (using histamine, cadaverine or putrescine as substrates). The monoamine oxidase from H. diminuta was not inhibited by azide, hydroxylamine or semicarbazide, but was inhibited by cupferron, alpha-alpha dipyridyl and iodoacetamide, and by the specific monoamine oxidase inhibitors pargyline, nialamide and iproniazid. Several anthelmintics were also found to be inhibitors of monoamine oxidase. The possible roles of monoamine oxidase in H. diminuta are discussed.     

Coupling of "malic" enzyme and NADPH:NAD transhydrogenase in the energetics of Hymenolepis diminuta (Cestoda)

Acetylpyridine NADP replaced NADP in promoting the Mn2+ ion-requiring mitochondrial "malic" enzyme of Hymenolepis diminuta. Disrupted mitochondria displayed low levels of an apparent oxaloacetate-forming malate dehydrogenase activity when NAD or acetylpyridine NAD served as the coenzyme. Significant malate-dependent reduction of acetylpyridine NAD by H. diminuta mitochondria required Mn2+ ion and NADP, thereby indicating the tandem operation of "malic" enzyme and NADPH:NAD transhydrogenase. Incubation of mitochondrial preparations with oxaloacetate resulted in a non-enzymatic decarboxylation reaction. Coupling of malate oxidation with electron transport via the "malic" enzyme and transhydrogenase was demonstrated by polarographic assessment of mitochondrial reduced pyridine nucleotide oxidase activity.     

Induction of cysteine proteinase in the encapsulation of Hymenolepis diminuta eggs in the American cockroach, Periplaneta americana

Haemocytes play an essential role in defending invertebrates against pathogens and parasites that enter their haemocoel. In the present study, the cockroach, Periplaneta americana was able to encapsulate Hymenolepis diminuta eggs within 24 h after injection. Proteolytic activity of egg capsules was determined by gelatin zymography. A gelatinase-type proteolytic enzyme with molecular weight about 65 kDa was present at the time of capsule formation. Enzyme activity was obviously inhibited by leupeptin but not by ethylenediamine tetraacetic acid (EDTA) or 1,10-phenanthroline or phenylmethanesulfonyl fluoride (PMSF). Hence, we tentatively characterized this enzyme as a cysteine proteinase. The specificity of the cellular immune response in vivo and the increased cysteine proteinase activity coincided with the capsule size and encapsulation process. The possible function of this cysteine proteinase activity during encapsulation of the H. diminuta eggs by P. americana is discussed.     

Phloretin inhibition of glucose transport by the tapeworm Hymenolepis diminuta: a kinetic analysis

Phloretin non-competitively inhibits glucose and methionine absorption by Hymenolepis diminuta (Ki = 0.24 and 1.48 mM, respectively). Inhibition of glucose transport by phloretin is reversible. Phloretin and phlorizin binding sites on the surface of H. diminuta are distinct. Phloretin does not inhibit Na+ transport independent of glucose transport in H. diminuta. The data suggest that phloretin inhibits absorptive functions across the surface of H. diminuta via non-specific binding sites associated with the lipid portion of the worm outer membrane.     

Trypsin adsorption by Hymenolepis diminuta (Cestoda)

Significant amounts of radioactivity were associated with Hymenolepis diminuta following incubation in 3H-trypsin. Autoradiography of worms incubated in 3H-trypsin for 30 min demonstrated that all radioactivity was associated with the worm's surface (tegument). The amount of 3H-trypsin adsorbed by the worms was not sufficient to account for the inactivation of this enzyme in the presence of intact worms. Unlabeled trypsin and poly-L-glutamate (but not poly-L-lysine) inhibited adsorption of 3H-trypsin, but were without effect on trypsin inactivation by H. diminuta. Therefore, trypsin was adsorbed by intact H. diminuta, but the process of adsorption apparently did not play any role in inactivation of the enzyme.     

Effects of 5-hydroxytryptamine, cyclic AMP, AMP, and fructose 2,6-bisphosphate on phosphofructokinase activity in Hymenolepis diminuta

1. 5-HT (10(-4) M) had no effect on the activity of phosphofructokinase in Hymenolepis diminuta. Concentrations of ATP above 33 microM inhibited PFK activity; AMP and cyclic AMP relieved this inhibition. 2. Local levels of cyclic AMP may be indirectly modulated by NaF, guanylyl imidophosphate, or 5-HT in the presence of GTP, which stimulates adenylyl cyclase activity x2 in H. diminuta homogenates. 3. Fructose 2,6-bisphosphate (F2BP), a physiological regulator of PFK activity in rat liver, also relieved ATP-induced inhibition of PFK. F2BP was present in supernatants from the worms at about 20 mumol/g wet wt. 4. 5-HT may cause an increase in the rate of glycolysis in H. diminuta by elevating either cyclic AMP and/or AMP levels; these nucleotides can in turn increase PFK activity.     

A comparison of phlorizin and phloretin adsorption by the tapeworm Hymenolepis diminuta

Phloretin and phlorizin adsorb to the tegument surface of Hymenolepis diminuta, with KDs of 2.39 mM and 14.7 microM, respectively, and Vmaxs of 1446 and 12.54 nmoles/g tissue per 2 min, respectively. Phloretin adsorption is not inhibited by phlorizin or glucose. Glucose partially inhibits phlorizin adsorption. Phlorizin, but not phloretin, adsorption to isolated tegument brush border membrane preparations is partially inhibited by N-ethylmaleimide. No indications of phlorizin hydrolysis to phloretin during incubation with H. diminuta were obtained. The data are supportive of spacially separate and distinct binding sites for phloretin and phlorizin in the tegument brush border.     

Glycosyl transferase activity in the brush border membrane of Hymenolepis diminuta

The isolated brush border membrane of Hymenolepis diminuta incorporates radiolabeled glucose when incubated in the presence of uridine diphospho(UDP)-D-(6-3H)glucose. The pH optimum for incorporation was 7.0 to 7.2 regardless of the buffer used. Transferase activity was maximal in 200 mM Tris buffer; 100 mM phosphate buffer inhibited significantly the incorporation of radiolabeled glucose, whereas 50 mM Tris-maleate and 100 mM PIPES resulted in moderate inhibition of activity. Incorporation of labeled glucose was not inhibited by low concentrations (0.01%) of Triton X-100, but activity was inhibited 50% by 0.25% Triton X-100. Addition of divalent cations to the brush border membrane preparation did not activate transferase activity, but addition of chelating agents (i.e., EDTA or EGTA) inhibited transferase activity nearly 90%. Incorporation of labeled glucose was inhibited by UDP, guanosine diphosphate (GDP), UDP- and GDP-activated monosaccharides, and monosaccharides, indicating that the transferase activity lacked substrate specificity.     

The alpha macroglobulins of rat serum.

Cortex of rat kidney was homogenized and fractions enriched in plasma membrane, endoplasmic reticulum or brush border were prepared by several techniques of differential centrifugation. The identity and homogeneity of the membrane fragments were investigated by assaying marker enzymes and by transmission and scanning electron microscopy. Kallikrein was present in both plasma-membrane- and endoplasmic-reticulum-enriched fractions isolated by two fractionation procedures. Kallikrein was highly concentrated in a plasma-membrane fraction but was absent from the brush-border membrane of proximal tubular cells. Cells of transplanted renal tumours of the rat, originating from the proximal tubule, had no kallikrein activity. Kininase activity, angiotensin I-converting enzyme (kininase II) and angiotensinase were found in a plasma-membrane-enriched fraction and especially in the fraction containing isolated brush border. It is suggested that after renal kallikrein is synthesized on endoplasmic reticulum, it is subsequently reoriented to a surface membrane for activation and release. Renal kallikrein may enter the tubular filtrate distal to the proximal tubules. The brush-border membrane of proximal tubule is the major site of inactivation of kinins and angiotensin II..     

Renal cortical brush-border and basolateral membranes: Cholesterol and phospholipid Composition and relative turnover

Summary A new procedure for the rapid isolation of renal cortical brush-border and basolateral membranes from the same homogenate is described. Brush-border membranes isolated using Mg²⁺-EGTA precipitation were enriched 18-fold for leucine aminopeptidase and had a recovery of 32.5%. Basolateral membrane fractions were isolated using a discontinuous sucrose gradient and showed an enrichment of 10.7-fold and recovery of 12.8% using (Na⁺, K⁺)-ATPase as a marker enzyme. Lipid analysis using two-dimensional TLC separation of phospholipids and gas liquid chromatography for cholesterol showed marked differences in the lipid composition of the brush-border and basolateral membranes. The brush-border membrane had increased sphingomyelin, phosphatidylserine, ethanolamine plasmalogens, and an increased cholesterol-to-phospholipid and sphingomyelin-to-phosphatidylcholine ratio compared to the basolateral membrane. The relative turnover of total membrane and individual phospholipid species using a double isotope ratio method was carried out. Phospholipids were labeled with either phosphorus 32 and 33 or acetate (³H, 1-¹⁴C). The relative turnover of phospholipid species and cholesterol differed strikingly. Phosphatidylcholine showed a high turnover, phosphatidylethanolamine and phosphatidylinositol had intermediate values and sphingomyelin, phosphatidylserine and cholesterol had low relative turnover rates. The order of phospholipid class relative turnover was independent of the labeled precursor used. The brush-border membrane had a significantly reduced relative turnover rate for total membrane phospholipids, sphingomyelin and cholesterol compared to the basolateral membrane. These data show marked differences in the lipid composition and relative turnover rates of the phospholipid species of the brush-border and basolateral membranes. They provide a biochemical basis for the recently reported differences in brush-border and basolateral membrane fluidity and suggest independent cellular regulation of brush-border and basolateral membrane lipids.     

Concurrent infections of the trematode Echinostoma caproni and the tapeworms Hymenolepis diminuta and Hymenolepis microstoma in mice

Superimposing the intestinal tapeworm Hymenolepis diminuta on an established infection with the trematode Echinostoma caproni or simultaneous infection of mice with H. diminuta and Hymenolepis microstoma caused destrobilation and expulsion of H. diminuta, whereas establishment and growth of H. microstoma under the same infection regimes were not affected. In contrast, simultaneous superimposition of H. diminuta and H. microstoma on an established E. caproni infection caused destrobilation and expulsion of both H. diminuta and H. microstoma.