Intestinal glycosidases of three species of seals

Comp. Bioehem. Physiol., 1968, Vol. 25, pp. 437 to 446. Pergamon Press. Printed in Great Britain

I N T E S T I N A L GLYCOSIDASES OF T H R E E SPECIES OF SEALS K. R. K E R R Y and M. M E S S E R * Gastroenterological Research Unit, Royal Children's Hospital Research Foundation, Parkville, Victoria, Australia

(Received 2 November 1967) A l m t e a c t m l . Intestinal glycosidase activities of six individual seals of three different species (Tasrnanian and New Zealand fur seals, Southern Elephant seal) were investigated. 2. Pancreatic amylase and intestinal lactase and o-nitro-phenyl-fl-galactosidase activities were present at very low levels in all the seals, including a suckling pup. Maltase and/somaltase activities were lower than those of other mammals. Sucrase and trehalase were absent. 3. Seal erythrocyte hexose-l-phosphate uridyltransferase and UDPglucose pyrophosphorylase activities were similar to those of normal human erythrocytes. 4. A sample of Tasmanian fur seal milk was analysed. It was found to contain a trace only of lactose. INTRODUCTION

TIeR MUCOSA of the small intestine contains enzymes which catalyse the hydrolysis of disaccharides such as maltose, /$omaltose, sucrose, lactose, cellobiose and trehalosc to their constituent monosaccharides. These disaccharidases arc found in a wide variety of mammals (Dahlqvist, 1964a), but recent observations by Sunshine & Kretchmer (1964) and Krctchmcr & Sunshine (1967) have shown that pups of three species of seals, viz. the California sea lion (Zal~hus californianus), the Stcller sea lion (Eumatopiasjubatus)and the Harbor seal (Phoca vitulina)arc exceptional in that they have no intestinal enzymes for the hydrolysis of sucrose, lactose, ccllobiosc and trchalosc. These findings raise the question whether absence of these enzymes is a feature c o m m o n to all species of seals (order Pinnipedia) and is found in adult as well as baby animals. The present study provides information on the intestinal disaccharidases and some other enzymes of three species of seals found in Australian waters; the T a s m a n i a n fur seal

(Arctocephalus tasmanicus),

the N e w Zealand fur seal and the Southern Elephant seal (Mirounga leonina). O f the six animals studied, four were adult. D a t a on the composition of a milk sample f r o m the T a s m a n i a n fur seal are included in this study.

(Arctocephalusforsteri)

* Present address: Department of Pediatrics, Stanford University School of Medicine, Palo Alto, California, U.S.A. 437

438

K.R. KERRYANDM. MEssza

METHODS AND MATERIALS A suckling Tasmanian fur seal pup and its mother were obtained from Seal Rocks, near Philip Island, Victoria, in November 1965. Another adult female was obtained from the same site in January 1966. An adult male New Zealand fur seal, and a weaned pup and adult male Elephant seal were obtained from Maequarie Island (Australian Antarctic Territory) in March 1966. The animals were killed by a shot through the head. Segments from different parts of the small intestine, and samples of pancreas, were removed as soon as possible after death and preserved in 0.9% NaCI in dry ice. Blood was collected in heparinized tubes and kept refrigerated. In the laboratory the tissue was thawed and the small intestinal mucosa scraped off. Intestinal mucosa and pancreas were homogenized in water to give 10% homogenates (wet wt./vol.). Disaccharidase activities of intestinal mucosa homogenates were assayed by a micro-modifieation (Burke et al., 1965) of the glucose oxidase method of Dahlqvist (1964b). A substrate concentration of 28 mM and pH of 5"8 was used for all assays; these conditions were found to be near optimal. Intestinal o-nitrophenylfl-galactosidase activity was assayed at pH 4"7 by a modification (Koldovsk9 et al., 1965) of the method of Lederberg (1950). Pancreatic amylase activity was measured by a saceharogenic method (Dahlqvist, 1962a). Erythrocyte hexose-l-phosphate uridyltransferase and UDPglucose pyrophosphorylase were assayed as described in Sigma Technical Bulletin No. 600 (Sigma Chemical Co., St. Louis). A sample of milk was obtained from a lactating Tasmanian fur seal by incision and drainage of the mammary gland. The milk was assayed for fat (including fatty acids) by the method of van de Kamer et al. (1949), for protein by the method of Lowry et al. (1951), using bovine serum albumin as standard, and for lactose by paper chromatography following extraction of the fat and removal of the protein (Pilson & Kelly, 1962). The milk was also assayed for total carbohydrate, using oreinol and sulphuric acid (Winzler, 1955), and for free glucose using a glucose oxidase reagent. RESULTS

Seal intestinal glycosidase activities Table 1 summarizes the results of assays of the pancreatic amylase activities and of the maltase, isomaltase, sucrase, trehalase, lactase and o-nitrophenylfl-galactosidase activities of the proximal part of the small intestine of all the seals studied. The results are compared with published values for the mean intestinal disaccharidase activities of human jejunal mucosa. Pancreatic amylase activities of the seals were very low, especially in the Elephant seals. All the seals showed significant intestinal maltase and isomaltase activities. Both these activities were lower in the suckling pup than in the older animals. Levels of these enzymes in the adult animals were 5-20 per cent of those found in man.

439

INTESTINAL GLYCOSIDASRS OF SEALS

Intestinal sucrase and trehalase were entirely absent; these activities could not be detected in intestinal tissue from any of the seals even after prolonged incubation periods designed to detect upwards of 0.001 unit/g. TABLE I ~ I N T ~ T I N A L

OLYCOSIDASE ACTIVITY OF SEALS

Pancreatic

amylase Maltase Isomaltase Suerase Trehalase Lactase ONPGase* (Units/g wet wt. tissue) Tasmanian fur seal Suckling pup 26 Yearling 74 Adult 40 New Zealand fur seal Adult 16 Southern Elephant seal Weaned pup 0 Adult 1 Man (mean values) 4800 t

0"15 2"3 3"2

0"083 -1"6

0 0 0

0 0 0

0.021 0.055 0"051

-0.048

1"1

0"64

0

0

0"018

0"10

4"1 4"5

2-2 2.4

0 0

0 0

0"060 0.049

0"35 --

6"7~

6"8~

1.7§

2.7~

0"48 II

22~

One unit of amylase activity is that which liberates reducing groups equivalent to 1 pmole~f rnaltose/min. One unit of disaccharidase activity is that which hydrolyses 1 prnole bf substrate/min. * o-Nitrophenyl-/~-galactosidase. t Pig pancreas (Dahlqvist, 1962a). + Human adult jejunal rnucosa (Auricchio et aL, 1963). § Duodenal rnucosa of children (Kerry & Townley, 1965). I[Jejunal rnucosa from a 5-week-old dornestic cat (Hore & Messer, 1968). Lactase and o-nitrophenyl-~-galactosidase activities, though readily detectable, were very low in all animals, including the suckling pup. The highest lactase activity of intestinal mucosa of any of the six animals was 0.06 units/g. This was only 2.2 per cent of the mean value for man. No marked differences in the disaccharidase activities of various parts of the small intestine of the seals were observed. Seal intestinal maltase and isomaltase activities In other mammals the maltase activity of the small intestine has been shown to be due to more than one enzymatic component. Of the available methods for studying this multiplicity of intestinal maltases, heat inactivation has been found to be the most effective single method (see Discussion). Figure 1 presents heat inactivation curves for the maltase and /somaltase activities of intestinal tissue of Tasmanian fur seal. It is seen that no separation of the two activities was obtained. Attempts to achieve separation by changing the conditions of either temperature or pH were unsuccessful. No discontinuities in the curves, such as to suggest the presence of more than one enzymatic component (Dahlqvist, 1962b) were detected. Similar results were obtained with intestinal tissue from the New Zealand fur seal and the Elephant seal.

440

K . R . KERRYAND M. M.I~SER

These results suggest that seal intestine contains only one maltase, and that the same enzyme catalyses the hydrolysis of both maltose and/somahose. Further evidence pertaining to the latter point was obtained from experiments in which maltose and/somaltose were incubated with seal intestinal homogenate either separately or in mixture. Less glucose was produced when the two substrates were present in mixture than when they were incubated separately (Table 2). I n contrast, the a m o u n t of glucose found after incubation of seal intestine with a mixture of lactose and maltose was equal to the sum of that formed from lactose I00 ! 8O 7O 6O

A 30.

+°'

I 61 °

10.

•

10 20 HEATING T I M E (Idln]

3G

FIG. 1. Heat inactivation of the maltase (A) and/somaltase (@) activities of homogenates of intestinal mucosa of a Tasmanian fur seal. The experiments were done using 10 mM sodium phosphate buffer, pH 7"0. TABLE

2--EFFECT OF SEAL mTESTZNAL

HOMOGENATE

(TASMANIAN

FU'~ S~AL) ON THE

HYDROLYSISOF MALTOSEAND DISACCHARIDES SEPARATELYAND IN MIXTURE Glucose (pg/tube) Substrate (14 m M )

Sucrose Lactose Isomaltose Maltose Maltose -b sucrose Maltose % lactose Maltose -/- isomaltose

Found

Expected*

Found x 100 Expected

0 0"2 3"9 6.6 6"3 6"4 5.2

----6"6 6"8 10-5

----95 94 S0

* Sum of glucose produced from disaccharide and maltose when incubated separately.

INTESTINAL GLYCOSIDASI~ OF SEALS

441

and maltose incubated separately. These results suggest that maltose and/somaltose compete for the same enzyme, whereas maltose and lactose are hydrolysed by different enzymes. Since sucrose had no effect on the hydrolysis of maltose (Table 2), the active site of seal intestinal maltase (/somaltase) appears to have no affafity for sucrose.

Ultracentrifugation of seal intestinal homogenate It was of interest to obtain information on the distribution of the seal intestinal glycosidases between the pellet and the soluble fraction following ultracentrifugation. To this end, intestinal homogenate from an Elephant seal was centrifuged at 105,000g for 90 min, the supernatant fraction was removed, and the pellet was rehomogenized in 0"9% NaC1. It was found that 90 per cent of the lactase, 40 per cent of the o-nitrophenyl-~-galactosidase and 85 per cent of the maltase and /wmaltase activities of the original homogenate were recovered in the pellet, the remainder being in the soluble fraction.

Composition of milk of the Tasmanianfur seal There is much evidence that the milk of aquatic mammals has an unusually high fat content and that in several species of seal the milk contains no lactose (King, 1964; see also Discussion). Since we were fortunate in being able to collect a small sample of milk from a lactating Tasmanian fur seal, analyses of the most important constituents of this milk were carried out. The milk, which was yellowish-grey in colour, had the consistency of thick cream and a strong, fish-like smell. Its fat content was 49 per cent, protein 12 per cent, total solids 64 per cent and total (including bound) carbohydrate 0.2 per cent. The milk contained 0.04 per cent free glucose. Only a trace of lactose could be detected chromatographically. By comparing the intensity of the spot with that produced with a known amount of lactose it was estimated that the concentration of lactose was 60-80 mg/ 100 ml milk.

Seal erythrocyte hexose-l-phosphate uridyltransferase and UPDglucose pyrophosphorylase activities In view of the absence of lactose from seal milk, it was of interest to determine the activities of enzymes involved in galactose metabolism. The assay methods TABLE 3--SEAL ERYTHROCYTE HEXOSE-1-PHOSPHATE URIDYLTRANSFERASE AND U D P G L U C O S E PYROPHOSPHORYLASE ACTIVITIES

Uridyltransferase Pyrophosphorylase (Units/g haemoglobin) Elephant seal (weaned pup) Elephant seal (adult) New Zealand fur seal (adult) Man (normal range)

2"2 7"4 2'5 1-7-9"0

12 22 26 1"7-45

442

K. R. KERRYAND M. M ~ s ~

used were the same as those applied in suspected cases of human galactosemia (congenital hexose-l-phosphate uridyltransferase deficiency). T h e results (Table 3) indicate that hexose-l-phosphate uridyltransferase and UDPglucose pyrophosphorylase were present in seal erythrocytes at levels similar to those found in normal human red cells. DISCUSSION

Intestinal glycoddase activity in pinnipeds In the present investigation, no intestinal sucrase or trehalase activity was detected in pup or adult specimens of any of the three species of seals; this observation is identical with the previous findings of Sunshine & Kretchmer (1964) and Kretchmer & Sunshine (1967) with pups of the California and SteUer sea lions and the Harbor seal. In a recent investigation, Herber & Peterson (1967), however, detected traces of sucrase activity in the intestinal mucosa of baby and adult walruses (Odobenus r. divergens); these authors did not assay trehalase. In the three species of seals studied by us, low levels of intestinal lactase and o-nitrophenyl-fl-galactosidase activities were found in all animals. Kretchmer & Sunshine (1967) found a small amount of lactase activity in the intestine of a pup Harbor seal, and Herber & Peterson (1967) found traces of intestinal lactase in baby but not adult walruses. Sunshine & Kretchmer (1964), however, were unable to detect either lactase or o-nitrophenyl-fl-galactosidase activity in the California and Steller sea lion. The animals studied by these three groups of investigators include representatives of all three families of the order Pinnipedia: Otariidae (California sea lion, Steller sea lion, Tasmanian fur seal, New Zealand fur seal); Odobenidae (walrus); and Phocidae (Harbor seal, Southern Elephant seal). T h e available evidence therefore strongly suggests that complete or virtual absence of intestinal sucrase, trehalase and lactase is a feature common to all species of this order. In this regard pinnipeds differ from other mammals which have been investigated, the only known exception being two species of the cat family (domestic cat and lion), in which absence of intestinal trehalase has been observed (Hore & Messer, 1968). All the pinnipeds which have been studied have been shown to have significant intestinal maltase activity (Sunshine & Kretchmer, 1964; Kretchmer & Sunshine, 1967; Herber & Peterson, 1967; present investigation). Intestinal isomaltase activity was found in walruses (Herber & Peterson, 1967) and in the seals studied by us. However, both maltase and/mmaltase were present at levels considerably below those found in man or the rat, and pancreatic amylase activities were very low in the seals studied in the present investigation. The intestinal maltase and isomaltase activities of other mammals have been shown to be due to more than one enzymatic component. In man, whose intestinal disaccharidases have recently been intensively investigated, the presence of at least four maltases, designated by Dahlqvist (1962b) as maltases Ia, Ib, II and III, has been established by means of heat inactivation (Dahlqvist, 1962b; Messer & Kerry,

INTESTINAL GLYCOSIDASHS OF SBALS

443

1967; see also Auricehio et aL, 1965). T h e major part of the/somaltase activity was found to be due to maltase Ia, and almost all the suerase activity to maltase Ib. In the present investigation, no dear evidence for the existence of more than one seal intestinal maltase or/somaltase could be obtained by heat inactivation, but the possibility remains that seal intestine contains two or more ~-glueosidases with similar heat stabilities. Since the seal intestine had no sucrase activity it contains no enzyme corresponding to human maltase Ib. In so far as no separation between the maltase and /somaltase activities could be achieved, seal intestinal maltase resembles human maltase Ia.

Nature of the seal intestinal lactase Recent evidence suggests that mammalian small intestine contains at least two enzymes acting on /8-galactosides. One has a pH optimum between 5 and 6, generally has greater activity towards lactose than other/S-galactosides, and is found predominantly in the pellet following ultracentrifugation; the second is optimally active below pH 5, usually has greater activity towards heterogalactosides such as o-nitrophenyl-~-galactoside than lactose, and is found mainly in the supernatant fraction after ultracentrifugation (Doell & Kretchmer, 1962; Dahlqvist & Asp, 1967; Hore & Messer, 1968). T h e first enzyme appears to be localized in the brush border of the intestine (Koldovsky et al., 1965), i.e. the probable site of hydrolysis of disaccharides in vivo (Crane, 1966); the functional significance of the second, "soluble" ~-galactosidase is unknown. Since the intestines of the seals studied in the present investigation had significant o-nitro-phenyl-/3-galactosidase as well as lactase activities (Table 1) it was considered that both activities might be due to a "soluble" ~-galactosidase, and that an enzyme similar to the insoluble, brush border lactose of other mammals might be absent. However, it was found that 90 per cent of the lactase activity of intesfinal tissue from an Elephant seal was recovered in the pellet following ultracentrifugation; this observation, as well as the fact that the intestinal lactase of all the seals was optimally active at pH 5.8, suggests that this enzyme is similar to the brush border lactase of other mammals. Furthermore, since the lactase and o-nitrophenyl-/3-galactosidase activities did not distribute themselves between the two ultracentrifugal fractions in identical fashion, it would seem that the seal intestine, like that of other mammals, contains at least two/~-galactosidases.

Absence of lactosefrom the milk of pinnipeds; implications T h e lactose content of the milk of terrestrial mammals ranges between 2 per cent (rabbit) and 7 per cent (man) (Altman& Dittmer, 1961). In the present investigation a sample of milk from a Tasmanian fur seal was found to contain less than 0.1 per cent lactose. Sivertsen (1941 ; quoted by Pilson & Kelly, 1962) found no lactose in the milk of the Harp seal (Pagophilus groenlandica) and the Hooded seal (Cystophora cristata). Prison & Kelly (1962), using improved methods of analysis, demonstrated complete absence of lactose in California sea lion milk. Pilson (1965) has briefly noted that the milk of the Steller sea lion, the Northern

AAA B I

I

K. R. KERaYAND.M. M~sm~

fur seal (Callortn'nus ursinus) and the walrus contains no lactose; however, chromatographically detectable amounts of lactose were found in the milk of species of three genera of the family Phocidae, viz. Phoca, Phagopilm and Mirounga. Absence of lactose from the milk of the walrus has been confirmed by Herber & Peterson (1967). On the other hand, Jenness et al. (1964) found "moderate" amounts of lactose in the milk of the Weddell seal (Leptonychote ~oeddelli), while Amoroso et al. (1951), using an unspecified method of analysis, found as much as 2'6 per cent lactose in the milk of the grey seal (Halichoerus grypus). Thus, with one or two possible exceptions, the milk of pinnipeds is devoid of nutritionally significant amounts of lactose. One consequence of this is that in the suckling pinniped, blood glucose levels must be maintained entirely by gluconeogenesis, presumably from protein; Sunshine & Kretchmer (1964) found the blood glucose content of the California sea lion pups to be within limits considered normal for man. Since baby pinnipeds have no exogenous source of galactose, this monosaccharide, which is found in mammalian nervous and other tissues in the form of galactolipids and mucopolysaccharides, must be supplied entirely from glucose (Kalckar, 1965). In the present investigation one of the enzymes involved in the conversion of glucose to UPDgalactose---UPDglucose pyrophosphorylase---was found to be active in seal erythrocytes at levels similar to those found in human red cells. In the absence of dietary lactose, enzymes for the metabolism of dietary lactose would seem superfluous. However, Mathai et al. (1966) have shown the enzymes galactokinas e and hexose-l-phosphate uridyltransferase to be present in the liver and erythrocytes of the Californian sea lion. The presence of the latter enzyme in seal erythrocytes was previously demonstrated by Sunshine & Kretchmer (1964) and confirmed in the present investigation. Mathai et al. (1966) have suggested that the persistence of enzymes of galactose metabolism in the sea lion may be related to the relatively high galactose content of mucopolysaccharides of various aquatic organisms.

Relationship between intestinal disaccharidases and diet It seems reasonable to suppose that the complete absence or very low levels of intestinal lactase activity in pinnipeds is related to the absence of lactose from the milk of the mother animals. Similarly, the absence of intestinal sucrase and trehalase, and the relatively low levels of enzymes involved in the digestion of starch (intestinal maltase and/somaltase, pancreatic amylase) may be related to the highly carnivorous dietary habits of these animals (Hore & Messer, 1968). However, the nature of these relationships is unclear. Deren et al. (1967) have shown that the amount of carbohydrate in the diet can influence the levels of intestinal sucrase and maltase in the rat, but these effects can be regarded as shortterm adaptations. When pups of the California and Steller sea lion were fed lactose or sucrose (Sunshine & Kretchmer, 1964), they developed clinical signs similar to those described for infants with congenital or acquired disaccharidase

INTESTINAL GLYCOSIDAS~ OF SEALS

4~5

deficiency (see, for example, Anderson et aL, 1963; Burke et aL, 1965; Kerry & Townley, 1965). Hence, absence of intestinal lactase in animals whose milk contains normal amounts of lactose would be a severe selective disadvantage, and this situation is therefore unlikely to be found in mammals. However, the same reasoning cannot be applied for the hypothetical reverse situation, i.e. the presence of normal levels of intestinal laetase in animals whose milk contains no lactose. One may speculate that for as yet unknown reasons, pinnipeds "lost" lactose from their milk during the course of evolution; under these circumstances, loss of the ability to digest lactose would not be a handicap. One could go further and argue that loss or repression of synthesis of non-functional proteins, such as lactase, sucrase and trehalase, particularly from a constantly and rapidly regenerating tissue such as the intestinal mucosa, might be of some selective advantage. This advantage, though slight, might be sufficient to cause loss of these enzymes over long periods of evolutionary time. Acknowledgements---We would like to thank Drs. R. Herber and M. L. Peterson for permission to quote their unpublished manuscripts. We are grateful for the assistance given by the Australian National Antarctic Research Expeditions, and particularly for allowing one of us (K. R. Kerry) to accompany a relief expedition to Macquarie Island in March 1966. We are also grateful to the Department of Fisheries and Wildlife, Victoria, for supplying material captured at Seal Rocks, and in particular to Mr. R. M. Wameke of that Department for his help in capturing seals. We wish to thank Miss P. Hore for assaying o-nitrophenyl-~-galactosidase, Miss I. Oehlmann for determining erythroeyte hexose-l-phosphate and UDPgiueose pyrophosphorylase activities, and Miss D. Boden and Miss S. Miller for technical assistance. We are grateful to Dr. Charlotte M. Anderson for her advice and encouragement. This investigation was supported by a grant from the National Health and Medical Research Council of Australia. REFERENCES ALTMANP. L. & DITTMER D. S. (editors) (1961) Blood and Other Body Fhdds. Biological Handbooks, Federation of American Societies for Experimental Biology, Washington, D.C. AMOROSO E. C., GOFFIN A., H~LLRy G., MATTI-IEWSL. H. & M A ~ S D. J. (1951) Lactation in the grey seal..Z Physiol. 113, 4P (abstract). ANDERSONC. M., M ~ E R M., TOWNLm"R. R. W. & FREEMANM. (1963) Intestinal sucrase and isomaltase deficiency in two siblings. Pediatrics 31, 1003-1010. AURICCHIOS., RUBINOA., TOSI R., SEMENZAG., LANDOLTi . , KISTLERH. & PRADm~A. (1963) Disaccharidase activities in human intestinal mucosa. Er~rcnol. biol. din. 3, 193-208. AtmlCCmO S., S ~ Z A G. & RUSINOA. (1965) Multiplicity of human intestinal disaccharidases--II. Characterization of the individual maltases. Biochim. biophys. Acta 96, 498-507. BURKE V., KERRY K. R. & ANVmmONC. M. (1965) The relationship of dietary lactose to refractory diarrhoea in infancy. Aust. paedlat..~. 1, 147-160. CRANE R. K. (1966) Enzymes and malabsorption: a concept of brush border membrane disease. Gastroenterology 50, 254-262. DAHLQVISTA. (1962a) A method for the determination of amylase in intestinal content. Scand. y. din. Lab. Invest. 14, 145-151. x5

446

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M gss~

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