LEUCINE AMINOPEPTIDASE AND EXSHEATHING ACTIVITY IN PREPARATIONS FROM ~AE~~~C~~~ C~~T~~T~~ W. P. ROGERS and F. BROOKS* Department
of Entomology,
Waite Agricultural Research Institute, Glen Osmond, South Australia, 5064 (Received 17 January 1978)
Abstr@et-RocERs W. P. and BROOKSF. 1978. Leucine aminopeptidase and exsheathing activity in preparations from Haetnonchus contortus. International Journal for Parasitology 8: 449-452. Exsheathing activity relative to leucine aminopeptidase activity (LAP) was greater in exsheathing fluid of infective juveniles of Huemonchus contortusthan extracts of homogenates of the same organism. In both preparations the biological and enzyme activities were precipitated with acetone 20 v/v and ammonium sulphate, 40% saturation, Broad peaks of exsheathing and LAP activities obtained by sucrose density-~adient centrifu~tion and on Sephadex GlJO overlapped but the peak of biological activity was always found on the low mol. wt. side of the LAP peak. LAP in exsheathing fluid was separated into two sharp peaks in polyacrylamide gradient-pore electrophoresis. In four experiments the major peak gave a mol. wt. within the limits 345,000-354,500. A minor peak was obtained at 1,800,000. Exsheathing activity remained broadly distributed but fell mostly on the low mol. wt. side of the major LAP peak. It is concluded that LAP cannot be the sole agent involved in exsheathment ; a lipase may be necessary to expose the substrate attacked by LAP. INDEX KEY WORDS: ~uemo~chus contortus; exsheathment;
INTRODUCTION
SOME criticism of the proposat that a leucine aminopeptidase (LAP) is involved in the exsheathment of nematodes has been answered. Thus Rogers & Brooks (1978) found LAP in exsheathing fluid of Huemonchus contortus from north America as well as from Australia. Moreover, the enzyme was produced in the presence of 2 x 10--2M-borate which limits bacterial growth in this preparation (Slo~ombe
& Whitlock, 1970). The demonstration that lo-SM, l,lO-phenanthroline inhibited the exsheathment of intact juveniles of H. contortus as well as the action of exsheathing fluid on isolated sheaths (Rogers & Brooks, 1976) also strengthened the view that LAP,
a Zn-protein enzyme, is involved in the process. The failure to show that mammalian LAP attacks isolated sheaths remains as the major objection to the proposal that this enzyme is involved in exsheathment. Though exsheathing fluids of H. contortus and Trichostrongylus colubriformis, both of which contain LAP, show some specificity towards their natural substrates {Rogers, 1965) the specificity of mammaIian LAP cannot be accepted as an explanation for its failure to attack sheaths without further information. -.*Present address: Mrs. F. Brooks, 97, St. Peters Road, Leicester LE2 lDJ, U.K.
449
leucine amino~ptidase.
In this investigation attempts have been made to assess further the role of LAP in exsheathment. This was done by examining: (a) the relative levels of biological and enzyme activity in different preparations, and (b) the effect of purifying LAP in exsheathing fluid on biological activity. EATERIES
AND I+%ETHODS
Exsheathing fluid was prepared as described previousfy (Rogers, 1970); however, solutions were concentrated by using ‘Minicon’ BS cells with a cut-off at a mol. wt. of lS,ooO, or in later work, with ‘Centriflo’ membrane cones with a cut-off at 25,000 (Amicon). Biological activity was measured using isolated sheaths (Rogers &Brooks, 1976). For the highly concentrated preparations of exsheathing fluid needed for separation techniques 6 ml of washed juveniles, ‘packed’ by centrifuging for 5 min at 700 g (103-136 mg dry wt. ml) were stimulated to exsheath (Rogers, 1970) and the exsheathing fluid concentrated to 0.5 ml or 50 ~1 according to the form of separation used, The total protein in these samples varied from 84 to 94 pg. Usually 3 ml of ‘packed’ juveniles were used for preparing homogenates. The juveniles were stimulated to secrete the normal exsheathing enzymes, washed in cold 1 M-NaCl in Tris-HCl at pH 8.3 containing 10-3M-MgZ+ (buffered Mgz+), homogenised, and the extract obtained by centrifuging. Active precipitates were obtained fromexsheathing fluid and tissue extracts by adding 20 v01 of cold acetone, or (NH&Sop to a final concentration of 40% saturation.
450
W. P.
ROGERS
and F. RKOOKS
Calibrations for mol. wt. were carried out using ox liver catalase (Sigma; Samejima & Yang, 1963); yeast alcohol dehydrogenase (Sigma; Armstrong, Coates & Morton, 1963), and rabbit immunoglobulin (IgA antibody to V&o &&rue, Steele, Chaicumpa & Rowley, 1974). A micro modification of the method of ~immelh~h & Peterson (1968) was used for the assay of LAP. Enzyme, 30 )I], was incubated for 2 h at 38°C with 2 1.11 of 0.1-~-tleucinamide in buffered Mgz+ and IO pl of digest was spotted on Whatman 3MM paper with reference spots of leucine and leucinamide. The paper was sprayed with pyridine-acetic acid-water, 1:10:89(by vol) and electrophoresis carried out for 20 min at 500 V and 40 mA. The paper was air dried, dipped in 0+5x ninhydrin (w/v) in 90% ethanol, and dried at 60°C for 30 min. Leucine spots were cut out, eluted with 300 ~1 of 70% ethanol and read at 570 nm (Beckman/Spinco Ultramicro Spectro-Colorimeter) using appropriate blanks from control digests. Activity was measured as leucine released mol/ml/h. The low concentration of the enzyme and the small amounts of exsheathing fluid which could be obtained made it difficult to use more accurate methods depending upon initial rates. Sucrose density-gradient separation was carried out by centrifuging for 20 h at 45,000 rev./min at 2°C in a Beckman/Spinco Model L SW head. A gradient of 5-Z% sucrose (w/v) in buffered Mg*+ was used. Optical densities of 250 ~2 samples were taken at 250 nm: samples at critical points were diluted and read at 280 nm. Though sucrose did not greatly affect the measurement of biological activity it did interfere with the assay of LAP. It was necessary therefore to dialyse the 250 pi samples against buffered Mg 2+ and then to concentrate the samples to the initial volumes. Separation was also carried out by reverse flow elution from a K26-40 (Pharmacia) column containing IO g Sephadex G150 with buffered Mg*+ as eluant. Dextran blue was incorporated in the test solution which was usually 0.5 ml of exsheathing fluid concentrated by precipitation with acetone and/ or (NH&SO,. After the void volume was passed 5 ml samples were taken; each was concentrated to 0.2 ml for examination. Polyacrylamide gradient-pore gels (Pharmacia) were stored in 0.1~M-Tris-boric acid at pH 8.9 containing 5 x IO-~M-M~CI~ for several days at 5°C before use to remove EDTA. Test solutions in buffered MgZ+ containing lO-12% sucrose and tracking dye (bromthymol blue) were loaded onto gels in amounts less than 20 ~1. Electrophoresis was conducted in Trisboric acid Mg2+ buffer for periods up to 24 h at 120V and 60 mA. The temperature of the bridge buffer was kept below 9°C. Extracts from the gel were obtained from 0.5 cm segments. Each segment was homogenized in 06 ml of buffered Mg 2+, centrifuged for 1 min (Beckman/ Spinco Microfuge), and samples taken for measurement of biological and LAP activity. The method of Smith & TABLE
l..l.l’. vo,.. 8.
Rutenberg (1966), with L-leucyl-$-naphthylamide a\ substrate, was used for the histochemical detection, in gels, of ‘leucine aminopeptidase’, and Coomassie Brilliant Blue R250 was used as a stain for proteins (Smith, 1968). Total protein was estimated using a mi~romodification of the method of Lowry. Rosebrough, Farr & Randall (19511. RESULTS The relative biological activity, assessed with isolated sheaths, and LAP activity in I-M-NaCl-extracts of homogenates and exsheathing fluid are shown in Table 1. In all experilnents biologica activity~LAP activity was greater in exsheathing fluid. The addition of 20 vol acetone to exsheathing fluid or to extracts from homogenates caused the precipitation of agents attacking sheaths and leucinamide without appreciable change in relative activity. Ammonium sulphate, at 40% saturation also caused precipitation of both activities. This precipitation was not complete however; about 40% of activity, both biological and enzymic, remained in the supernatant. In two experiments using density-gradient centrifugation leucine aminopeptidase was obtained as a broad peak of mol. wt. somewhat greater than 140,000. Biological activity overlapped the distribution of enzyme but its peak fell below a mol. wt. of 145,000. Fractionation on Sephadex Gl50 gave enzyme activity, and to a lesser extent, biological activity, distributed over broad peaks. Clearer results were obtained when samples were purified by precipitation with acetone and ammonium sulphate before they were placed on the column. In all experiments biological and enzyme activity overlapped, though most biological activity fell in the mol. wt. range of 188,000---107,000 and LAP activity was mostly at higher levels. Gradient-pore gels provided a means for the sharp fractionation of LAP in exsheathing fluid (Fig. I). In the four experiments carried out, peak activity for the agent hydrolysing leucinamide was obtained in the segment 3-3.5 cm (mean value of mol. wt. 350,000) from the origin (cathode). A smaller and more variable peak was obtained in the first segment of mol. wt. about 1,800,OOO. The histochemical detection of ‘LAP’ with Lleucyl-~-naphthylamide as substrate, disclosed several bands, one of which corresponded with the major peak of activity for the hydrolysis of leucin-
l-EXSHEATHMENT ACTIVITY RELATIVE TO LEUCINE AMINOPEPTIDASE ACTIVITY, OF EXTRACTS OF HOMOGENATES OF STIMULATEDJUVENILES,AND OFEXSHEATHINC FLUIDIN TWOSETSOF EXPERIMENTS
_,_--Preparation
--..
__-_
197x
____~
Extract of homogenate Exsheathing fluid .~~~~._~~-~-~-------
Exsheathment time (min)
.~.
110 9.1 65 14.9 1.5 6.7 25 4.0 ---- ---.---~~
Leucine formed (~rno~~rnl~h)
l/f
--_--“x % x >:
10-z 10-q 10-Z 10-2
3.5 4.2 0,3 0.5
Relative activity (biological! enzymic)
1 1.4 86 31
-
Leucine amino~ptidase
I.J.P. VOL.8. 1978
I
I!Il!II/
0°1’ I
I
(al
I
(bl
Fro. 1. The distribution of leucine aminopeptidase with leucinamide as substrate (graph) and of exsheathing activity (above); the distribution of enzyme by histochemical staining is indicated by (a) enzyme(s) from exsheathing fluid, (b) enzyme from pig kidney. Activity from LAP of pig kidney was obtained at mol. wt. of 340,~-380,~O with a minor band at 84,000~88,000. Biological activity was broadly spread and did not coincide with the distribution of true LAP or with the bands located by histochemistry. Most of it fell on the low mol. wt. side of the major LAP peak.
as 300,000 (Spackman, Smith & Brown, 1955; Himmelhoch & Peterson, 1968) so the figure suggested for the enzyme from the nematode may be too high. However, the mol. wt. of LAP from different sources does vary (DeLange & Smith, 1971). The marked difference in biological activity reiative to LAP activity in exsheathing fluid and homogenates, and lack of coincidence in peaks of activity in fractionating the two functions suggests that (a) LAP is not involved in exsheathment or (b) other factors are involved as well as the enzyme. The inhibition of exsheathing by l,tO-phenanthroline (Rogers & Brooks, 1976) and the presence of LAP in exsheathing fluid of H. contortus of north America (Rogers & Brooks, 1978) support the view that the enzyme is involved in exsheathment so (b) needs further consideration. Exsheathing fluid and hatching fluid of FI. contortus attack each other’s natural substrates and both contain LAP and Iipase(s) (Rogers & Brookes, 1977; Rogers, unpublished). It might be necessary, therefore, for the action of a lipase to precede the action of LAP on sheaths. This proposal, which is presently under examination, could explain the failure of mammalian LAP to attack sheaths and the lack of coincidence of peaks of LAP and exsheathing activity in the fractionation of exsheathing fluid. Ac/cnowledgements--We wish to thank CSIRO McMaster Laboratory for supplying most of the H. contortus used in this work. Support from the Australian Research Grants Committee is gratefully acknowledged. ~~RENCES
amide.
DKXUSSION
The failure to obtain sharp separation of LAP on Sephadex G150 may have been due to varying associations of sub-units and to further associations with other proteins in the preparations. A marked heterogeneity in LAP from pig kidney has been reported by ~immelh~h & Peterson (1968). In previous attempts to fractionate exsheathing fluid on 7.5% polyacrylamide gel (Rogers, 1965) LAP appeared to aggregate and moved only short distances from the origin. It is notable that, in these early experiments, concentrated preparations were difficult to obtain so the enzyme was subjected to electrophoresis on small acrylamide columns from large volumes of Sephadex (Broome, 1963; Rogers, 1964). The mol. wt. of LAP from H. contortus, 350,000, was similar to that obtained from LAP from pig kidney in our experiments using gradient-pore gels. The value for mammalian LAP is generally accepted
451
and exsheathing activity
ARMSTRONGJ. McD., COATESJ. H. & MORTONR. H. 1963. Physiochemical studies on cytochrome b,. Biachemical Journal 86: 136-145. BROOMEJ. 1963. A rapid method of disc electrophoresis. Nature (Land.) 199: 179-180. DELANGER. J. & SMITHE. L. 1971. Leucine aminopep tidase and other N-terminal exopeptidases. In The Enzymes (Edited by BOYERP. D.) Vol. III, pp. 88-l 18. Academic Press, New York. H~MMELH~CH S. R. & PETERK~NE. A. 1968. Preparation of leucine aminopeptidase free of endopeptidase activity. Biochemis&y-7: 2085-2092. LOWRY0. H.. RC@EBROUGH N. J.. FARR A. L. & RAXDALL R. J.’ 1951. Protein m~su~ment with Folin phenol reagent. Jo~n~Z of BioZog~c~~ Chemistry 193: 265-275.
RICERS W. P. 1964. Micromethods for the study of leucine aminopeptidase. Microchemical Journal 8: 194-202.
RCK~ERS W. P. 1965. The role of leucine aminopeptidase in the mouhing of nematode parasites. Catlipararive ~j~che~~~fry and P~ys~~Zu~ 14: 31 l-321. ROGERSW. PI 1970. The fu&tion of leucine aminopeg tidase in exsheathing fluid. Journal qf Pmusitology 56: 138-143.
ROGERSW. P. & BROOKSF. 1976. Zinc as a co-factor for an enzyme involved in exsheathment of Haemonchus contortus.
315-319.
International
Jaurnal
for
Parasitology
6:
452
W. I’. ROGERS and F. BROOKS
ROGERS W. P. & BROOKS F. 1977. The mechanism of hatching of eggs of Haemonrhus contortus. International Journalfor Parasitology 7: 61-65. ROGFRS W. P. & BROOKS F. 1978. Leucine aminopeptidase in exsheathing fluid of north American and Australian Haemonchus cantortrrs. International Journal fbr Parasitology 8: 55-58. SAMEJIMA T. & YANG denatured catalase. 238: 273-277.
J. T. 1963. Reconstitution Journal of Biolugical
of acidChemistry
SLOCOMBE J. 0. D. & WHITLOCK J. H. 1970. The development of a standard method for rapid ecdysis of infective Haemonchus contortus cayugensis larvae. Parasitology 61: 273-277.
I.J.P. vol..x. 197X
SMITH I. 196X. Disc electrophoresis in polyacrylamide gels. In Chromatography and Electrophoresk (Edited by SMITH I.) vol. II. pp. 365-388. Heinemann Medical Books, Bath SMITH E. E. & RUTENB~RC A. M. 1966. Starch-gel electrophoresis of human enzymes which hydrolyse Lleucyl-p-naphthylamide. Science 152: 1256. SPACKMAN D. H., SMITH E. L. & BROWN D. M. 1955. Leucine aminopeptidase. IV. Isolation and properties of the enzyme from swine kidney. Jorwnal of’ Biological Chemistry 212: 255-269. STEELE E. J., CHAICUMPA W. &ROWLEY D. 1971. Isolation and biological properties of three classes of rabbit antibody to Vihrio cholerea. Journal of Infectioxr Diseases 130: 93-103.
of Entomology,
Waite Agricultural Research Institute, Glen Osmond, South Australia, 5064 (Received 17 January 1978)
Abstr@et-RocERs W. P. and BROOKSF. 1978. Leucine aminopeptidase and exsheathing activity in preparations from Haetnonchus contortus. International Journal for Parasitology 8: 449-452. Exsheathing activity relative to leucine aminopeptidase activity (LAP) was greater in exsheathing fluid of infective juveniles of Huemonchus contortusthan extracts of homogenates of the same organism. In both preparations the biological and enzyme activities were precipitated with acetone 20 v/v and ammonium sulphate, 40% saturation, Broad peaks of exsheathing and LAP activities obtained by sucrose density-~adient centrifu~tion and on Sephadex GlJO overlapped but the peak of biological activity was always found on the low mol. wt. side of the LAP peak. LAP in exsheathing fluid was separated into two sharp peaks in polyacrylamide gradient-pore electrophoresis. In four experiments the major peak gave a mol. wt. within the limits 345,000-354,500. A minor peak was obtained at 1,800,000. Exsheathing activity remained broadly distributed but fell mostly on the low mol. wt. side of the major LAP peak. It is concluded that LAP cannot be the sole agent involved in exsheathment ; a lipase may be necessary to expose the substrate attacked by LAP. INDEX KEY WORDS: ~uemo~chus contortus; exsheathment;
INTRODUCTION
SOME criticism of the proposat that a leucine aminopeptidase (LAP) is involved in the exsheathment of nematodes has been answered. Thus Rogers & Brooks (1978) found LAP in exsheathing fluid of Huemonchus contortus from north America as well as from Australia. Moreover, the enzyme was produced in the presence of 2 x 10--2M-borate which limits bacterial growth in this preparation (Slo~ombe
& Whitlock, 1970). The demonstration that lo-SM, l,lO-phenanthroline inhibited the exsheathment of intact juveniles of H. contortus as well as the action of exsheathing fluid on isolated sheaths (Rogers & Brooks, 1976) also strengthened the view that LAP,
a Zn-protein enzyme, is involved in the process. The failure to show that mammalian LAP attacks isolated sheaths remains as the major objection to the proposal that this enzyme is involved in exsheathment. Though exsheathing fluids of H. contortus and Trichostrongylus colubriformis, both of which contain LAP, show some specificity towards their natural substrates {Rogers, 1965) the specificity of mammaIian LAP cannot be accepted as an explanation for its failure to attack sheaths without further information. -.*Present address: Mrs. F. Brooks, 97, St. Peters Road, Leicester LE2 lDJ, U.K.
449
leucine amino~ptidase.
In this investigation attempts have been made to assess further the role of LAP in exsheathment. This was done by examining: (a) the relative levels of biological and enzyme activity in different preparations, and (b) the effect of purifying LAP in exsheathing fluid on biological activity. EATERIES
AND I+%ETHODS
Exsheathing fluid was prepared as described previousfy (Rogers, 1970); however, solutions were concentrated by using ‘Minicon’ BS cells with a cut-off at a mol. wt. of lS,ooO, or in later work, with ‘Centriflo’ membrane cones with a cut-off at 25,000 (Amicon). Biological activity was measured using isolated sheaths (Rogers &Brooks, 1976). For the highly concentrated preparations of exsheathing fluid needed for separation techniques 6 ml of washed juveniles, ‘packed’ by centrifuging for 5 min at 700 g (103-136 mg dry wt. ml) were stimulated to exsheath (Rogers, 1970) and the exsheathing fluid concentrated to 0.5 ml or 50 ~1 according to the form of separation used, The total protein in these samples varied from 84 to 94 pg. Usually 3 ml of ‘packed’ juveniles were used for preparing homogenates. The juveniles were stimulated to secrete the normal exsheathing enzymes, washed in cold 1 M-NaCl in Tris-HCl at pH 8.3 containing 10-3M-MgZ+ (buffered Mgz+), homogenised, and the extract obtained by centrifuging. Active precipitates were obtained fromexsheathing fluid and tissue extracts by adding 20 v01 of cold acetone, or (NH&Sop to a final concentration of 40% saturation.
450
W. P.
ROGERS
and F. RKOOKS
Calibrations for mol. wt. were carried out using ox liver catalase (Sigma; Samejima & Yang, 1963); yeast alcohol dehydrogenase (Sigma; Armstrong, Coates & Morton, 1963), and rabbit immunoglobulin (IgA antibody to V&o &&rue, Steele, Chaicumpa & Rowley, 1974). A micro modification of the method of ~immelh~h & Peterson (1968) was used for the assay of LAP. Enzyme, 30 )I], was incubated for 2 h at 38°C with 2 1.11 of 0.1-~-tleucinamide in buffered Mgz+ and IO pl of digest was spotted on Whatman 3MM paper with reference spots of leucine and leucinamide. The paper was sprayed with pyridine-acetic acid-water, 1:10:89(by vol) and electrophoresis carried out for 20 min at 500 V and 40 mA. The paper was air dried, dipped in 0+5x ninhydrin (w/v) in 90% ethanol, and dried at 60°C for 30 min. Leucine spots were cut out, eluted with 300 ~1 of 70% ethanol and read at 570 nm (Beckman/Spinco Ultramicro Spectro-Colorimeter) using appropriate blanks from control digests. Activity was measured as leucine released mol/ml/h. The low concentration of the enzyme and the small amounts of exsheathing fluid which could be obtained made it difficult to use more accurate methods depending upon initial rates. Sucrose density-gradient separation was carried out by centrifuging for 20 h at 45,000 rev./min at 2°C in a Beckman/Spinco Model L SW head. A gradient of 5-Z% sucrose (w/v) in buffered Mg*+ was used. Optical densities of 250 ~2 samples were taken at 250 nm: samples at critical points were diluted and read at 280 nm. Though sucrose did not greatly affect the measurement of biological activity it did interfere with the assay of LAP. It was necessary therefore to dialyse the 250 pi samples against buffered Mg 2+ and then to concentrate the samples to the initial volumes. Separation was also carried out by reverse flow elution from a K26-40 (Pharmacia) column containing IO g Sephadex G150 with buffered Mg*+ as eluant. Dextran blue was incorporated in the test solution which was usually 0.5 ml of exsheathing fluid concentrated by precipitation with acetone and/ or (NH&SO,. After the void volume was passed 5 ml samples were taken; each was concentrated to 0.2 ml for examination. Polyacrylamide gradient-pore gels (Pharmacia) were stored in 0.1~M-Tris-boric acid at pH 8.9 containing 5 x IO-~M-M~CI~ for several days at 5°C before use to remove EDTA. Test solutions in buffered MgZ+ containing lO-12% sucrose and tracking dye (bromthymol blue) were loaded onto gels in amounts less than 20 ~1. Electrophoresis was conducted in Trisboric acid Mg2+ buffer for periods up to 24 h at 120V and 60 mA. The temperature of the bridge buffer was kept below 9°C. Extracts from the gel were obtained from 0.5 cm segments. Each segment was homogenized in 06 ml of buffered Mg 2+, centrifuged for 1 min (Beckman/ Spinco Microfuge), and samples taken for measurement of biological and LAP activity. The method of Smith & TABLE
l..l.l’. vo,.. 8.
Rutenberg (1966), with L-leucyl-$-naphthylamide a\ substrate, was used for the histochemical detection, in gels, of ‘leucine aminopeptidase’, and Coomassie Brilliant Blue R250 was used as a stain for proteins (Smith, 1968). Total protein was estimated using a mi~romodification of the method of Lowry. Rosebrough, Farr & Randall (19511. RESULTS The relative biological activity, assessed with isolated sheaths, and LAP activity in I-M-NaCl-extracts of homogenates and exsheathing fluid are shown in Table 1. In all experilnents biologica activity~LAP activity was greater in exsheathing fluid. The addition of 20 vol acetone to exsheathing fluid or to extracts from homogenates caused the precipitation of agents attacking sheaths and leucinamide without appreciable change in relative activity. Ammonium sulphate, at 40% saturation also caused precipitation of both activities. This precipitation was not complete however; about 40% of activity, both biological and enzymic, remained in the supernatant. In two experiments using density-gradient centrifugation leucine aminopeptidase was obtained as a broad peak of mol. wt. somewhat greater than 140,000. Biological activity overlapped the distribution of enzyme but its peak fell below a mol. wt. of 145,000. Fractionation on Sephadex Gl50 gave enzyme activity, and to a lesser extent, biological activity, distributed over broad peaks. Clearer results were obtained when samples were purified by precipitation with acetone and ammonium sulphate before they were placed on the column. In all experiments biological and enzyme activity overlapped, though most biological activity fell in the mol. wt. range of 188,000---107,000 and LAP activity was mostly at higher levels. Gradient-pore gels provided a means for the sharp fractionation of LAP in exsheathing fluid (Fig. I). In the four experiments carried out, peak activity for the agent hydrolysing leucinamide was obtained in the segment 3-3.5 cm (mean value of mol. wt. 350,000) from the origin (cathode). A smaller and more variable peak was obtained in the first segment of mol. wt. about 1,800,OOO. The histochemical detection of ‘LAP’ with Lleucyl-~-naphthylamide as substrate, disclosed several bands, one of which corresponded with the major peak of activity for the hydrolysis of leucin-
l-EXSHEATHMENT ACTIVITY RELATIVE TO LEUCINE AMINOPEPTIDASE ACTIVITY, OF EXTRACTS OF HOMOGENATES OF STIMULATEDJUVENILES,AND OFEXSHEATHINC FLUIDIN TWOSETSOF EXPERIMENTS
_,_--Preparation
--..
__-_
197x
____~
Extract of homogenate Exsheathing fluid .~~~~._~~-~-~-------
Exsheathment time (min)
.~.
110 9.1 65 14.9 1.5 6.7 25 4.0 ---- ---.---~~
Leucine formed (~rno~~rnl~h)
l/f
--_--“x % x >:
10-z 10-q 10-Z 10-2
3.5 4.2 0,3 0.5
Relative activity (biological! enzymic)
1 1.4 86 31
-
Leucine amino~ptidase
I.J.P. VOL.8. 1978
I
I!Il!II/
0°1’ I
I
(al
I
(bl
Fro. 1. The distribution of leucine aminopeptidase with leucinamide as substrate (graph) and of exsheathing activity (above); the distribution of enzyme by histochemical staining is indicated by (a) enzyme(s) from exsheathing fluid, (b) enzyme from pig kidney. Activity from LAP of pig kidney was obtained at mol. wt. of 340,~-380,~O with a minor band at 84,000~88,000. Biological activity was broadly spread and did not coincide with the distribution of true LAP or with the bands located by histochemistry. Most of it fell on the low mol. wt. side of the major LAP peak.
as 300,000 (Spackman, Smith & Brown, 1955; Himmelhoch & Peterson, 1968) so the figure suggested for the enzyme from the nematode may be too high. However, the mol. wt. of LAP from different sources does vary (DeLange & Smith, 1971). The marked difference in biological activity reiative to LAP activity in exsheathing fluid and homogenates, and lack of coincidence in peaks of activity in fractionating the two functions suggests that (a) LAP is not involved in exsheathment or (b) other factors are involved as well as the enzyme. The inhibition of exsheathing by l,tO-phenanthroline (Rogers & Brooks, 1976) and the presence of LAP in exsheathing fluid of H. contortus of north America (Rogers & Brooks, 1978) support the view that the enzyme is involved in exsheathment so (b) needs further consideration. Exsheathing fluid and hatching fluid of FI. contortus attack each other’s natural substrates and both contain LAP and Iipase(s) (Rogers & Brookes, 1977; Rogers, unpublished). It might be necessary, therefore, for the action of a lipase to precede the action of LAP on sheaths. This proposal, which is presently under examination, could explain the failure of mammalian LAP to attack sheaths and the lack of coincidence of peaks of LAP and exsheathing activity in the fractionation of exsheathing fluid. Ac/cnowledgements--We wish to thank CSIRO McMaster Laboratory for supplying most of the H. contortus used in this work. Support from the Australian Research Grants Committee is gratefully acknowledged. ~~RENCES
amide.
DKXUSSION
The failure to obtain sharp separation of LAP on Sephadex G150 may have been due to varying associations of sub-units and to further associations with other proteins in the preparations. A marked heterogeneity in LAP from pig kidney has been reported by ~immelh~h & Peterson (1968). In previous attempts to fractionate exsheathing fluid on 7.5% polyacrylamide gel (Rogers, 1965) LAP appeared to aggregate and moved only short distances from the origin. It is notable that, in these early experiments, concentrated preparations were difficult to obtain so the enzyme was subjected to electrophoresis on small acrylamide columns from large volumes of Sephadex (Broome, 1963; Rogers, 1964). The mol. wt. of LAP from H. contortus, 350,000, was similar to that obtained from LAP from pig kidney in our experiments using gradient-pore gels. The value for mammalian LAP is generally accepted
451
and exsheathing activity
ARMSTRONGJ. McD., COATESJ. H. & MORTONR. H. 1963. Physiochemical studies on cytochrome b,. Biachemical Journal 86: 136-145. BROOMEJ. 1963. A rapid method of disc electrophoresis. Nature (Land.) 199: 179-180. DELANGER. J. & SMITHE. L. 1971. Leucine aminopep tidase and other N-terminal exopeptidases. In The Enzymes (Edited by BOYERP. D.) Vol. III, pp. 88-l 18. Academic Press, New York. H~MMELH~CH S. R. & PETERK~NE. A. 1968. Preparation of leucine aminopeptidase free of endopeptidase activity. Biochemis&y-7: 2085-2092. LOWRY0. H.. RC@EBROUGH N. J.. FARR A. L. & RAXDALL R. J.’ 1951. Protein m~su~ment with Folin phenol reagent. Jo~n~Z of BioZog~c~~ Chemistry 193: 265-275.
RICERS W. P. 1964. Micromethods for the study of leucine aminopeptidase. Microchemical Journal 8: 194-202.
RCK~ERS W. P. 1965. The role of leucine aminopeptidase in the mouhing of nematode parasites. Catlipararive ~j~che~~~fry and P~ys~~Zu~ 14: 31 l-321. ROGERSW. PI 1970. The fu&tion of leucine aminopeg tidase in exsheathing fluid. Journal qf Pmusitology 56: 138-143.
ROGERSW. P. & BROOKSF. 1976. Zinc as a co-factor for an enzyme involved in exsheathment of Haemonchus contortus.
315-319.
International
Jaurnal
for
Parasitology
6:
452
W. I’. ROGERS and F. BROOKS
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