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Isolation and some properties of the digestive amylase of the american lobster (Homarus Americanus)
Comp. Biochem. Physiol., 1972, Vol. 42B, pp. 295 to 302. Pergamon Press. Printed in Great Britain
ISOLATION AND SOME PROPERTIES OF THE DIGESTIVE AMYLASE OF THE AMERICAN LOBSTER (HOM.4RUS
AMERICANUS) M. B. WOJTOWICZ and H. BROCKERHOFF Fisheries Research Board of Canada, Halifax Laboratory, Halifax, Nova Scotia
(Received 14 September 1971) Al~tract--1. Gastric juice of lobster contains an amylase (2 per cent of the total protein) which can be isolated by affinity chromatography on a dextran gel followed by chromatography on a polyacrylamide gel. 2. The specific activity of the enzyme towards starch is 90 (/zmolesmaltose/ min per mg protein), its molecular weight 41,000 and its optimum of activity is at pH 5"2. 3. The purified enzyme is activated by sodium chloride, but does not require added calcium.
INTRODUCTION THE PRESENCEof amylase (alpha-amylase: alpha-l,4-glucan 4-glucanohydrolase, E.C. 3.2.1.1) among the digestive enzymes of crustacea has been recognized for a long time (Hoppe-Seyler, 1876; Wiersma & van der Veen, 1928; Arvy, 1969). Generally, crab enzymes have been investigated (Blandamer & Beechey, 1963; Krishnamoorthy & Reddy, 1968; Nagabhushanam & Sarojini, 1968; Sather, 1969) though there have been qualitative investigations of amylase in the stomach juice of crayfish (Kooiman, 1964; Fingerman et al., 1967) and lobster (Wolvekamp, 1947; Kooiman, 1964) and the digestive tract of spiny lobster (Takahashi et al., 1964). There is a general agreement that the enzymes are alpha-amylases (Vonk, 1960; Blandamer & Beechey, 1966). The isolation of a crustacean amylase has not yet been reported although the enzyme of a crab has been concentrated twenty to forty times (Blandamer & Beechey, 1966) and the amylase of an insect has been isolated recently (Podoler & Applebaum, 1971). In a recent survey on the digestive enzymes of the American lobster we failed to detect a noteworthy amylolytic activity (Brockerhoff et al., 1970). We have since found that appreciable activity could be demonstrated by using soluble starch as a substrate in place of Remazolbrilliant Blue starch. We isolated the enzyme by affinity chromatography on a dextran gel, followed by filtration on a non-retarding gel, and determined some of its characteristics. 295
296
M. B. WOJTOWICZAND H. BROCKERHOFF MATERIALS AND METHODS
Gastric juice of lobster The animals were kept in running sea water at 9-12°C and ted with chopped herring and mackerel. Gastric juice was collected (7-10 days after feeding) by suction with a plastic tube, filtered through glass wool and kept at 2-3°C before analysis, or adjusted to pH 8, dialyzed against water and lyophilized.
Amylase standard Alpha amylase AA (Mann Research Laboratories, New York) from hog pancreas, nominal activity 690 units/rag, measured activity (see below) 460 units/mg.
Assay of activity The procedure of Bernfeld (1955) was used: Incubation of a 1% starch solution at 25°C, reaction of the liberated reducing groups with 3,5-dinitrosalycilic acid; reading of the optical density at 540 #m against a blank prepared at zero time ; calibration with maltose (0"3-3"0/zmoles). All activities are given in international units:/~moles maltose released/rain. Specific activities are units/rag protein. In a few experiments the activity was determined on an insoluble preparation of starch labeled with Remazolbrilliant Blue R (Calbiochem, Los Angeles) according to Rinderknecht et al. (1967), with shaking at 37°C, filtration at the end of the incubation and reading the absorbance at 595 t,m. Different incubation times, up to 2 hr, yielded a flattening curve from which the initial velocity was estimated as the tangent at time 0.
Protein determination Protein was determined by the method of Lowry et al. (1951) using the modifications described by Miller (1959).
Disc electrophoresis The purification of the amylase was followed by disc electrophoresis. The Canalco Model 12 apparatus (Canalco, Rockville, Maryland) and the RSD disc standard reagent kit were used, and the basic procedure described for the apparatus was followed. The separating gel of 7% acrylamide, polymerized with ammonium persulfate, was equilibrated and run at pH 9"0-9"5, with 10-150/zg sample protein, for 30-45 min at 5 mA per tube. The gel was fixed with 12% triehloroacetic acid for 30 min and stained with Coomassie Blue,
0.05%. RESULTS
Stability of the enzyme A previous failure to detect significant amylase activity in lyophilized gastric juice (Brockerhoff et al., 1960) led us to suspect the stability of the enzyme. However, the amylase is quite stable. On dialysis of fresh juice (pH 5) against cold water no activity was lost. Adjustment to p H 8 and storage at 4°C overnight, dialysis at p H 8 and lyophilization led to almost quantitative recovery.
Assay of amylase in lobster gastric juice T h e activities of dialyzed and lyophilized juice were measured at p H 5.2 as a function of the time of incubation and of the amount of protein added for assay
DIGESTIVEAMYLASEOF THE AMERICANLOBSTER
297
(Fig. 1). From the figures it can be read that one unit or less of the enzyme will give linear response in the assay for incubation times up to 12 min. & /-~
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0 . 6 mg PROTEIN
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9Mi.
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FIo. 1. Digestion of soluble starch by lobster gastric juice as a function of time (A) and protein concentration (B). One ml 1% starch at pH 5.2, 25°C (Bernfeld, 1955).
Specific activity and quantity of amylase in gastric juice Assays of gastric juice of two to four individual lobsters during the course of 4 months gave specific activities from 1.2 to 1.8 units/mg, an average of 1.5 units/mg. Considering that the specific activity of the pure enzyme is 90, it follows that 1.3-2.0 per cent of the gastric proteins consist of amylase. The activity of the isolated enzyme against soluble starch is 20 per cent of that of the commercial hog pancreatic amylase (sp. act. 460). However, if the lobster enzyme was incubated with Remazolbrilliant Blue starch its activity was only 0.07 per cent of that of the hog enzyme (against this substrate).
Isolation of the amylase; molecular weight; purity Lyophilized gastric juice, 0.25 g in 3 ml 0.1 M ammonium acetate, pH 7, was applied to a 2.5 x 90 em eolunm of Sephadex G-100 and eluted with the same buffer at a temperature of 4°C and a flow rate of 10 ml/hr, and 5-ml fractions were collected (Fig. 2). The amylase is eluted in the region of proteins of an estimated molecular weight around 13,000; this is due to the retarding effect of the carbohydrate matrix of the dextran gel on the enzyme (Gelotte, 1964). The total recovery
298
M.B.
WoJTowlez
AND
H. BROCKERHOFF
of activity was 70 per cent; the middle of the peak (70 per cent of the recovered activity) was collected; a twelvefold purification had been achieved.
A
A /"~\
0
I-(,9 Z w ._1 <
B
I13.. 12)
te
Ve / Vo FIa. 2. Gel filtration of lobster gastric juice proteins on Sephadex G-100 (A) followed by Bio-Gel P-100 (B). Protein (280m/z): , amylase activity: The amylase from the Sephadex column was concentrated to 0.1 vol by ultrafiltration, dialyzed against water, lyophilized and dissolved in buffer, 0.1 M NaC10.05 M Tris, pH 8. It was then applied to a column 2.5 x 90 cm, at 4°C, of Bio-Gel P-100 (Calbiochem, Los Angeles), and eluted with the same buffer (Fig. 2). The effluent was monitored at 280/zm. The column had previously been calibrated (Andrews, 1964) with molecular weight markers (Fig. 3). The amylase was eluted at a V J V o ratio of 1.42 corresponding to a molecular weight of 41,000. A similar determination on Bio-Gel ¢, 0
)4 I-r ¢.9
-e~rum
Albumin Ov0]bumin e~e ~ e
Ld el." ._1 (.)
td -J 0
I 1.2
Chymotrypsinogen~ e ~ I I I L4 1.6 I8 Ve / Vo
FIG. 3. Determination of the molecular weight of lobster amylase on a Bio-Gel P-100 column, calibrated with standard M.W. markers.
FIG. 4. Disc electrophoresis on polyacrylamide gel, pH 9, of crude amylase from a Sephadex G-100 separation (left) and of pure amylase after Bio-Gel P-100 filtration (right) (stain: Coomassie Blue).
DIGESTIVE AMYLASE OF T H E AMERICAN LOBSTER
299
P-200 (a gel with larger pores) gave an identical value. Since the Bio-Gels are polyacrylamides and free of carbohydrates they will not interact with amylase, and the determined molecular weight will be near to the true one. The compounds of low molecular weight that contaminated the amylase after the Sephadex filtration were now widely separated from the enzyme (Fig. 2A and B). The absorbent material that appeared in the void volume may consist of denaturated (unfolded) protein. The yield of activity in the collected middle of the amylase peak was 50 per cent, so that the overall yield, from gastric juice, was around 25 per cent. By taking wider fractions the yield of pure enzyme could undoubtedly be increased. The overall purification from the gastric proteins (about 1 unit/mg) was approximately 100-fold. Figure 4 shows disc gel electrophoretic patterns (pH 9) of a crude amylase (Fig. 4, left) from a Sephadex G-100 separation and of the final preparation (Fig. 4, right).
Optimal pH of activity The enzyme was assayed in the following buffers: Na-acetate, 0.02 M, pH 4.05-5; Na-Tris-maleate, 0.02 M, pH 5.2-8.0; Na-phosphate, 0.02 M, pH 6.0-7.0. Activities measured at the same pH but with different buffers were similar. The optimal pH was found to be around 5.2 (Fig. 5).
/\
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o
x,.
J
W
>
/
F-
/
\
\
_J hi
fll~
o
I 4
I 5
I 6
I 7
8
pH FIG. 5. pH activity profile of lobster amylase. The pH profile against the insoluble Remazolbrilliant Blue starch showed a peak around pH 5, rather inprecise because of the very low activity of the enzyme against this substrate.
Activation by sodium chloride The enzyme was dialyzed exhaustively against water until the molarity measured by conductivity (if assumed to be due to NaCI) was reduced to 10-4. This preparation was strongly activated by NaC1, to a maximum of almost fifteen times at the optimal concentration of 0.05-0.1 M (Fig. 6).
300
M . B . WOJTOWlCZ AND H. BROCKERHOFF
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LOG (. NcICI) FIG. 6. Activation of lobster amylase by sodium chloride.
Influence of calcium ion Semi-purified enzyme from the Sephadex filtration was used for this experiment. It was dissolved in 0.01 M sodium ethylenediaminetetra-aeetate (EDTA) and dialyzed against the 500-fold volume of 0.01 M EDTA. This process was repeated three times. The influence of calcium was tested by the addition of calcium acetate in concentrations of 0.025-0.075 M; these have been reported by Blandamer & Beechey (1964) to activate the amylase of a crab. No increase of activity could be observed. The addition of 0.05 M NaC1 to the same preparation restored the activity of the enzyme, but the simultaneous addition of calcium had again no activating effect. In fact, at 0.05 M concentration, calcium acetate inhibited the enzyme by 50 per cent. DISCUSSION The isolation of an amylase by consecutive gel filtration on a retarding and non-retarding gel was recently described by Frati & Caputo (1970) for the proteins from mouse submaxillary gland; a pure enzyme was obtained by those authors. The facility with which amylase can be isolated from the very complex protein mixture of lobster gastric juice underlines the general applicability and simplicity of this method. The amylase of the lobster has many features in common with previously described amylases of crustacea (Vonk, 1960; Arvy, 1969) and amylases in general (Fischer & Stein, 1960). Its pH optimum is acidic, pH 5.2; similar optima have been reported for crayfish stomach juice (Wiersma & van der Veen, 1928; Wolvekamp, 1947) and spiny lobster stomach extract (Takahashi et al., 1964), although pH 7 for hepatopancreas (Blandamer & Beechey, 1964), and pH 8 for midgut gland of crabs (Sather, 1969) have also been given. The enzyme is activated
DIGESTIVE AMYLASE OF THE AMERICAN LOBSTER
301
by chloride ion like all amylases (Fischer & Stein, 1960) but activation by calcium cannot be demonstrated, presumably because the enzyme binds its obligatory calcium so tightly that it cannot be removed by dialysis against EDTA (Fischer & Stein, 1960). The molecular weight of the lobster amylase, 41,000, is in the expected region; that of the amylase of hog pancreas, e.g. is 45,000 (Fischer & Stein, 1960). The enzyme appears to be rather resistant towards attack by the proteinases of the gastric juice, lobster trypsin and the low molecular weight proteinases (Brockerhoff et al., 1970). There is a general consensus that the amylases of invertebrates are s-amylases (Vonk, 1960; Blandamer & Beechey, 1964). The lobster doubtlessly needs this enzyme for the digestion of glycogen. Our failure to detect appreciable quantities of amylase in a previous general survey of lobster enzymes (Brockerhoff et al., 1970) was largely due to choice of the substrate, the insoluble Remazolbrilliant Blue starch, which is readily attacked by hog pancreatic amylase but not by the enzyme of the lobster. REFERENCES ANDREWS P. (1964) Estimation of the molecular weights of proteins by Sephadex gelfiltration. Biochon. J. 91, 222-233. ARVYL. (1969) Les enzymes chez les crustac~s. Ann. Biol. 8, 9-10. BEmC~ELDP. (1955) Amylases. In Methods in Enzymology (Edited by COLOWICKS. P. & KAPLANN. O.), Vol. 1, pp. 149-150. Academic Press, New York. BLANDAMERA. H. & BImc~myR. B. (1963) Dextran-gel filtration in the purification of a crustacean o~-amylase. Nature, Lond. 197, 591-592. BLANDA~R A. & BImCH~ R. B. (1964) The identification of an ~-amylase in aqueous extracts of the hepatopancreas of Carcinus maenas, the common shore crab. Comp. Biochem. Physiol. 13, 97-105. BLA~A~R A. & BF_~CHEYR. B. (1966) The purification and properties of an a-amylase from the hepatopancreas of Carcinus maenas, the common shore crab. Biochim. biophys. Acta 118, 204-206. BROCKERHOFFH., HO~E R. J. & HWANGP. C. (1970) Digestive enzymes of the American lobster (Homarus americanus). J. Fish. Res. Bd Can. 27, 1357-1370. FINGERMA~ M., DOMINICZAKT., MIYAWAKIM., OGURO C. & YAMAMOTOY. (1967) Neuroendocrine control of the hepatopancreas in the crayfish Procambarus clarki. Physiol. Zo~L 40, 23-30. Biol. Abstr. 48, No. 62434. FISCHERE. H. & STEIN E. A. (1960) Alpha-amylases. In The Enzymes (Edited by Bo~a P. D., LARDYH. & M~ACK K.), 2nd edn. Vol. 4, pp. 313-343. Academic Press, New York. F~ATX L. & CAPUTOA. (1970) Purification of a-amylase from CsH mouse submaxillary gland by gel filtration. ]. Chromatog. 48, 547-550. GELGTTEB. (1964). Separation of pancreatic enzymes by gel filtration. Actu chem. seand. 18, 1283-1291. HOPPE-SEYL~.R F. (1876) l~ber Unterschiede im ehemischem Bau und der Verdauung h/Jherer und niederer Thiere. Pflugers. Arch. ges. Physiol. 14, 395--400. KOOIMAN P. (1964) The occurrence of carbohydrases in digestive juice and in hepatopancreas of Astacus fluviatilis Fabr. and of Homarus vulgaris M-E. ~t. cell. comp. Physiol. 63, 197-201. 11
302
M. B. WOJTOWlCZ AND H. BROCKERHOFF
KRISHNAMOORTHYR. V. & REDDYV. V. (1968) Hepatopancreatic amylase activity as a function of warm adaptation in a freshwater field crab. Experientia 24, 1019-1020. LowRY O. S., ROSEBROUGHN. J., FARRA. L. & RANDALLR. J. (1951) Protein measurement with the Folin phenol reagent, ft. biol. Chem. 193, 265-275. MILLER G. L. (1959) Protein determination for large numbers of samples. Analyt. Chem. 31, 964. NAGABHUSHANAMR. & SAROJINI R. (1968) Digestive enzymes in Diogenes bicristimanus (Crustacea: Decapoda). Brotdria 37, 155-172. PODOLERH. & APPLEBAUMS. W. (1971) The a-amylase of the beetle CaUosobruchus chinensis. Purification and action pattern. Biochem. J. 121, 317-320. RINDERKNECHTH., WILDING P. & HAVERBACKB. J. (1967) A new method for the determination of a-amylase. Experientia 23, 805. SATHER B. T. (1969) A comparative study of amylases and proteinases in some decapod crustacea. Comp. Biochem. Physiol. 28, 371-379. TAKAHASHI T., MORISHITA T. & TACHINO S. (1964) Studies on the digestive enzymes of spiny lobster, Panulirus japonicus (V. Siebold). Rep. Faculty Fish. Univ. of Mie, 5, 127-135. MONK H. J. (1960) Digestion and metabolism. In The Physiology of the Crustacea (Edited by WATERMANT. B.), Vol. 1, p. 296. Academic Press, New York. WIERSMA C. A. G. & VAN DER VEEN R. (1928) Die Kohlehydratverdauung bei Astacus fluviatilis. Z. vergl. Physiol. 7, 269-278. WOLVEKAMP M. S . P. (194-7) Sur la prdsence de deux amylases dans le suc gastrique des d6capodes..4ctualitds Biochim. 10, 19-21.
Key Word Index,--Amylase; Homarus americanus; digestion; lobster amylase.
ISOLATION AND SOME PROPERTIES OF THE DIGESTIVE AMYLASE OF THE AMERICAN LOBSTER (HOM.4RUS
AMERICANUS) M. B. WOJTOWICZ and H. BROCKERHOFF Fisheries Research Board of Canada, Halifax Laboratory, Halifax, Nova Scotia
(Received 14 September 1971) Al~tract--1. Gastric juice of lobster contains an amylase (2 per cent of the total protein) which can be isolated by affinity chromatography on a dextran gel followed by chromatography on a polyacrylamide gel. 2. The specific activity of the enzyme towards starch is 90 (/zmolesmaltose/ min per mg protein), its molecular weight 41,000 and its optimum of activity is at pH 5"2. 3. The purified enzyme is activated by sodium chloride, but does not require added calcium.
INTRODUCTION THE PRESENCEof amylase (alpha-amylase: alpha-l,4-glucan 4-glucanohydrolase, E.C. 3.2.1.1) among the digestive enzymes of crustacea has been recognized for a long time (Hoppe-Seyler, 1876; Wiersma & van der Veen, 1928; Arvy, 1969). Generally, crab enzymes have been investigated (Blandamer & Beechey, 1963; Krishnamoorthy & Reddy, 1968; Nagabhushanam & Sarojini, 1968; Sather, 1969) though there have been qualitative investigations of amylase in the stomach juice of crayfish (Kooiman, 1964; Fingerman et al., 1967) and lobster (Wolvekamp, 1947; Kooiman, 1964) and the digestive tract of spiny lobster (Takahashi et al., 1964). There is a general agreement that the enzymes are alpha-amylases (Vonk, 1960; Blandamer & Beechey, 1966). The isolation of a crustacean amylase has not yet been reported although the enzyme of a crab has been concentrated twenty to forty times (Blandamer & Beechey, 1966) and the amylase of an insect has been isolated recently (Podoler & Applebaum, 1971). In a recent survey on the digestive enzymes of the American lobster we failed to detect a noteworthy amylolytic activity (Brockerhoff et al., 1970). We have since found that appreciable activity could be demonstrated by using soluble starch as a substrate in place of Remazolbrilliant Blue starch. We isolated the enzyme by affinity chromatography on a dextran gel, followed by filtration on a non-retarding gel, and determined some of its characteristics. 295
296
M. B. WOJTOWICZAND H. BROCKERHOFF MATERIALS AND METHODS
Gastric juice of lobster The animals were kept in running sea water at 9-12°C and ted with chopped herring and mackerel. Gastric juice was collected (7-10 days after feeding) by suction with a plastic tube, filtered through glass wool and kept at 2-3°C before analysis, or adjusted to pH 8, dialyzed against water and lyophilized.
Amylase standard Alpha amylase AA (Mann Research Laboratories, New York) from hog pancreas, nominal activity 690 units/rag, measured activity (see below) 460 units/mg.
Assay of activity The procedure of Bernfeld (1955) was used: Incubation of a 1% starch solution at 25°C, reaction of the liberated reducing groups with 3,5-dinitrosalycilic acid; reading of the optical density at 540 #m against a blank prepared at zero time ; calibration with maltose (0"3-3"0/zmoles). All activities are given in international units:/~moles maltose released/rain. Specific activities are units/rag protein. In a few experiments the activity was determined on an insoluble preparation of starch labeled with Remazolbrilliant Blue R (Calbiochem, Los Angeles) according to Rinderknecht et al. (1967), with shaking at 37°C, filtration at the end of the incubation and reading the absorbance at 595 t,m. Different incubation times, up to 2 hr, yielded a flattening curve from which the initial velocity was estimated as the tangent at time 0.
Protein determination Protein was determined by the method of Lowry et al. (1951) using the modifications described by Miller (1959).
Disc electrophoresis The purification of the amylase was followed by disc electrophoresis. The Canalco Model 12 apparatus (Canalco, Rockville, Maryland) and the RSD disc standard reagent kit were used, and the basic procedure described for the apparatus was followed. The separating gel of 7% acrylamide, polymerized with ammonium persulfate, was equilibrated and run at pH 9"0-9"5, with 10-150/zg sample protein, for 30-45 min at 5 mA per tube. The gel was fixed with 12% triehloroacetic acid for 30 min and stained with Coomassie Blue,
0.05%. RESULTS
Stability of the enzyme A previous failure to detect significant amylase activity in lyophilized gastric juice (Brockerhoff et al., 1960) led us to suspect the stability of the enzyme. However, the amylase is quite stable. On dialysis of fresh juice (pH 5) against cold water no activity was lost. Adjustment to p H 8 and storage at 4°C overnight, dialysis at p H 8 and lyophilization led to almost quantitative recovery.
Assay of amylase in lobster gastric juice T h e activities of dialyzed and lyophilized juice were measured at p H 5.2 as a function of the time of incubation and of the amount of protein added for assay
DIGESTIVEAMYLASEOF THE AMERICANLOBSTER
297
(Fig. 1). From the figures it can be read that one unit or less of the enzyme will give linear response in the assay for incubation times up to 12 min. & /-~
//
°
LI.I O9 0 I-" o ,-I
0 . 6 mg PROTEIN
.~°
/ I 3
I I 6 9 MINUTES
I 12
12Min
LI.I _JlO 0
,('/-
9Mi.
a..
,
o.z
o,s
Lo
mg
PROTEIN
L~
FIo. 1. Digestion of soluble starch by lobster gastric juice as a function of time (A) and protein concentration (B). One ml 1% starch at pH 5.2, 25°C (Bernfeld, 1955).
Specific activity and quantity of amylase in gastric juice Assays of gastric juice of two to four individual lobsters during the course of 4 months gave specific activities from 1.2 to 1.8 units/mg, an average of 1.5 units/mg. Considering that the specific activity of the pure enzyme is 90, it follows that 1.3-2.0 per cent of the gastric proteins consist of amylase. The activity of the isolated enzyme against soluble starch is 20 per cent of that of the commercial hog pancreatic amylase (sp. act. 460). However, if the lobster enzyme was incubated with Remazolbrilliant Blue starch its activity was only 0.07 per cent of that of the hog enzyme (against this substrate).
Isolation of the amylase; molecular weight; purity Lyophilized gastric juice, 0.25 g in 3 ml 0.1 M ammonium acetate, pH 7, was applied to a 2.5 x 90 em eolunm of Sephadex G-100 and eluted with the same buffer at a temperature of 4°C and a flow rate of 10 ml/hr, and 5-ml fractions were collected (Fig. 2). The amylase is eluted in the region of proteins of an estimated molecular weight around 13,000; this is due to the retarding effect of the carbohydrate matrix of the dextran gel on the enzyme (Gelotte, 1964). The total recovery
298
M.B.
WoJTowlez
AND
H. BROCKERHOFF
of activity was 70 per cent; the middle of the peak (70 per cent of the recovered activity) was collected; a twelvefold purification had been achieved.
A
A /"~\
0
I-(,9 Z w ._1 <
B
I13.. 12)
te
Ve / Vo FIa. 2. Gel filtration of lobster gastric juice proteins on Sephadex G-100 (A) followed by Bio-Gel P-100 (B). Protein (280m/z): , amylase activity: The amylase from the Sephadex column was concentrated to 0.1 vol by ultrafiltration, dialyzed against water, lyophilized and dissolved in buffer, 0.1 M NaC10.05 M Tris, pH 8. It was then applied to a column 2.5 x 90 cm, at 4°C, of Bio-Gel P-100 (Calbiochem, Los Angeles), and eluted with the same buffer (Fig. 2). The effluent was monitored at 280/zm. The column had previously been calibrated (Andrews, 1964) with molecular weight markers (Fig. 3). The amylase was eluted at a V J V o ratio of 1.42 corresponding to a molecular weight of 41,000. A similar determination on Bio-Gel ¢, 0
)4 I-r ¢.9
-e~rum
Albumin Ov0]bumin e~e ~ e
Ld el." ._1 (.)
td -J 0
I 1.2
Chymotrypsinogen~ e ~ I I I L4 1.6 I8 Ve / Vo
FIG. 3. Determination of the molecular weight of lobster amylase on a Bio-Gel P-100 column, calibrated with standard M.W. markers.
FIG. 4. Disc electrophoresis on polyacrylamide gel, pH 9, of crude amylase from a Sephadex G-100 separation (left) and of pure amylase after Bio-Gel P-100 filtration (right) (stain: Coomassie Blue).
DIGESTIVE AMYLASE OF T H E AMERICAN LOBSTER
299
P-200 (a gel with larger pores) gave an identical value. Since the Bio-Gels are polyacrylamides and free of carbohydrates they will not interact with amylase, and the determined molecular weight will be near to the true one. The compounds of low molecular weight that contaminated the amylase after the Sephadex filtration were now widely separated from the enzyme (Fig. 2A and B). The absorbent material that appeared in the void volume may consist of denaturated (unfolded) protein. The yield of activity in the collected middle of the amylase peak was 50 per cent, so that the overall yield, from gastric juice, was around 25 per cent. By taking wider fractions the yield of pure enzyme could undoubtedly be increased. The overall purification from the gastric proteins (about 1 unit/mg) was approximately 100-fold. Figure 4 shows disc gel electrophoretic patterns (pH 9) of a crude amylase (Fig. 4, left) from a Sephadex G-100 separation and of the final preparation (Fig. 4, right).
Optimal pH of activity The enzyme was assayed in the following buffers: Na-acetate, 0.02 M, pH 4.05-5; Na-Tris-maleate, 0.02 M, pH 5.2-8.0; Na-phosphate, 0.02 M, pH 6.0-7.0. Activities measured at the same pH but with different buffers were similar. The optimal pH was found to be around 5.2 (Fig. 5).
/\
->
."
I..-
o
x,.
J
W
>
/
F-
/
\
\
_J hi
fll~
o
I 4
I 5
I 6
I 7
8
pH FIG. 5. pH activity profile of lobster amylase. The pH profile against the insoluble Remazolbrilliant Blue starch showed a peak around pH 5, rather inprecise because of the very low activity of the enzyme against this substrate.
Activation by sodium chloride The enzyme was dialyzed exhaustively against water until the molarity measured by conductivity (if assumed to be due to NaCI) was reduced to 10-4. This preparation was strongly activated by NaC1, to a maximum of almost fifteen times at the optimal concentration of 0.05-0.1 M (Fig. 6).
300
M . B . WOJTOWlCZ AND H. BROCKERHOFF
MAX
F>
-
/
I--
o
o.° I -4
I -;3
I -2
I -I
LOG (. NcICI) FIG. 6. Activation of lobster amylase by sodium chloride.
Influence of calcium ion Semi-purified enzyme from the Sephadex filtration was used for this experiment. It was dissolved in 0.01 M sodium ethylenediaminetetra-aeetate (EDTA) and dialyzed against the 500-fold volume of 0.01 M EDTA. This process was repeated three times. The influence of calcium was tested by the addition of calcium acetate in concentrations of 0.025-0.075 M; these have been reported by Blandamer & Beechey (1964) to activate the amylase of a crab. No increase of activity could be observed. The addition of 0.05 M NaC1 to the same preparation restored the activity of the enzyme, but the simultaneous addition of calcium had again no activating effect. In fact, at 0.05 M concentration, calcium acetate inhibited the enzyme by 50 per cent. DISCUSSION The isolation of an amylase by consecutive gel filtration on a retarding and non-retarding gel was recently described by Frati & Caputo (1970) for the proteins from mouse submaxillary gland; a pure enzyme was obtained by those authors. The facility with which amylase can be isolated from the very complex protein mixture of lobster gastric juice underlines the general applicability and simplicity of this method. The amylase of the lobster has many features in common with previously described amylases of crustacea (Vonk, 1960; Arvy, 1969) and amylases in general (Fischer & Stein, 1960). Its pH optimum is acidic, pH 5.2; similar optima have been reported for crayfish stomach juice (Wiersma & van der Veen, 1928; Wolvekamp, 1947) and spiny lobster stomach extract (Takahashi et al., 1964), although pH 7 for hepatopancreas (Blandamer & Beechey, 1964), and pH 8 for midgut gland of crabs (Sather, 1969) have also been given. The enzyme is activated
DIGESTIVE AMYLASE OF THE AMERICAN LOBSTER
301
by chloride ion like all amylases (Fischer & Stein, 1960) but activation by calcium cannot be demonstrated, presumably because the enzyme binds its obligatory calcium so tightly that it cannot be removed by dialysis against EDTA (Fischer & Stein, 1960). The molecular weight of the lobster amylase, 41,000, is in the expected region; that of the amylase of hog pancreas, e.g. is 45,000 (Fischer & Stein, 1960). The enzyme appears to be rather resistant towards attack by the proteinases of the gastric juice, lobster trypsin and the low molecular weight proteinases (Brockerhoff et al., 1970). There is a general consensus that the amylases of invertebrates are s-amylases (Vonk, 1960; Blandamer & Beechey, 1964). The lobster doubtlessly needs this enzyme for the digestion of glycogen. Our failure to detect appreciable quantities of amylase in a previous general survey of lobster enzymes (Brockerhoff et al., 1970) was largely due to choice of the substrate, the insoluble Remazolbrilliant Blue starch, which is readily attacked by hog pancreatic amylase but not by the enzyme of the lobster. REFERENCES ANDREWS P. (1964) Estimation of the molecular weights of proteins by Sephadex gelfiltration. Biochon. J. 91, 222-233. ARVYL. (1969) Les enzymes chez les crustac~s. Ann. Biol. 8, 9-10. BEmC~ELDP. (1955) Amylases. In Methods in Enzymology (Edited by COLOWICKS. P. & KAPLANN. O.), Vol. 1, pp. 149-150. Academic Press, New York. BLANDAMERA. H. & BImc~myR. B. (1963) Dextran-gel filtration in the purification of a crustacean o~-amylase. Nature, Lond. 197, 591-592. BLANDA~R A. & BImCH~ R. B. (1964) The identification of an ~-amylase in aqueous extracts of the hepatopancreas of Carcinus maenas, the common shore crab. Comp. Biochem. Physiol. 13, 97-105. BLA~A~R A. & BF_~CHEYR. B. (1966) The purification and properties of an a-amylase from the hepatopancreas of Carcinus maenas, the common shore crab. Biochim. biophys. Acta 118, 204-206. BROCKERHOFFH., HO~E R. J. & HWANGP. C. (1970) Digestive enzymes of the American lobster (Homarus americanus). J. Fish. Res. Bd Can. 27, 1357-1370. FINGERMA~ M., DOMINICZAKT., MIYAWAKIM., OGURO C. & YAMAMOTOY. (1967) Neuroendocrine control of the hepatopancreas in the crayfish Procambarus clarki. Physiol. Zo~L 40, 23-30. Biol. Abstr. 48, No. 62434. FISCHERE. H. & STEIN E. A. (1960) Alpha-amylases. In The Enzymes (Edited by Bo~a P. D., LARDYH. & M~ACK K.), 2nd edn. Vol. 4, pp. 313-343. Academic Press, New York. F~ATX L. & CAPUTOA. (1970) Purification of a-amylase from CsH mouse submaxillary gland by gel filtration. ]. Chromatog. 48, 547-550. GELGTTEB. (1964). Separation of pancreatic enzymes by gel filtration. Actu chem. seand. 18, 1283-1291. HOPPE-SEYL~.R F. (1876) l~ber Unterschiede im ehemischem Bau und der Verdauung h/Jherer und niederer Thiere. Pflugers. Arch. ges. Physiol. 14, 395--400. KOOIMAN P. (1964) The occurrence of carbohydrases in digestive juice and in hepatopancreas of Astacus fluviatilis Fabr. and of Homarus vulgaris M-E. ~t. cell. comp. Physiol. 63, 197-201. 11
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KRISHNAMOORTHYR. V. & REDDYV. V. (1968) Hepatopancreatic amylase activity as a function of warm adaptation in a freshwater field crab. Experientia 24, 1019-1020. LowRY O. S., ROSEBROUGHN. J., FARRA. L. & RANDALLR. J. (1951) Protein measurement with the Folin phenol reagent, ft. biol. Chem. 193, 265-275. MILLER G. L. (1959) Protein determination for large numbers of samples. Analyt. Chem. 31, 964. NAGABHUSHANAMR. & SAROJINI R. (1968) Digestive enzymes in Diogenes bicristimanus (Crustacea: Decapoda). Brotdria 37, 155-172. PODOLERH. & APPLEBAUMS. W. (1971) The a-amylase of the beetle CaUosobruchus chinensis. Purification and action pattern. Biochem. J. 121, 317-320. RINDERKNECHTH., WILDING P. & HAVERBACKB. J. (1967) A new method for the determination of a-amylase. Experientia 23, 805. SATHER B. T. (1969) A comparative study of amylases and proteinases in some decapod crustacea. Comp. Biochem. Physiol. 28, 371-379. TAKAHASHI T., MORISHITA T. & TACHINO S. (1964) Studies on the digestive enzymes of spiny lobster, Panulirus japonicus (V. Siebold). Rep. Faculty Fish. Univ. of Mie, 5, 127-135. MONK H. J. (1960) Digestion and metabolism. In The Physiology of the Crustacea (Edited by WATERMANT. B.), Vol. 1, p. 296. Academic Press, New York. WIERSMA C. A. G. & VAN DER VEEN R. (1928) Die Kohlehydratverdauung bei Astacus fluviatilis. Z. vergl. Physiol. 7, 269-278. WOLVEKAMP M. S . P. (194-7) Sur la prdsence de deux amylases dans le suc gastrique des d6capodes..4ctualitds Biochim. 10, 19-21.
Key Word Index,--Amylase; Homarus americanus; digestion; lobster amylase.