Some monotreme milk “whey” and blood proteins

Comp. Biochem. Physiol. Vol. 9911,No. 1, pp. 99-118, 1991

0305-0491/91 $3.00+0.00 Pergamon Press plc

Printed in Great Britain

SOME MONOTREME MILK "WHEY" A N D BLOOD PROTEINS CARMEL G. TEAHAN,* HUGH A. MCKENzIE~ and MERVYN GRIFFITH$~ Protein Chemistry Group, John Curtin School of Medical Research, Australian National University, Canberra, ACT 2601, Australia (Received 17 September 1990)

Abstract--1. Eleetrophoretic studies are made of mature phase milk "whey" proteins and blood serum proteins of echidna (Tachyglossus aculeatus) and platypus (Ornithorhynchus anatinus). The echidna milk bands are designated A-M, those of platypus A--G. Some of the proteins are isolated and characterized. 2. Echidna band A protein has some similarity to high cystine "whey" proteins. Band E protein (apparent M,21,000) may be a fl-lactoglobulin-like protein. Band M is lysozyme. Band C is serum albumin. Bands G-K are transferrins. 3. Platypus milk bands A, C, D, F and G are isolated. Bands F and G are transferrins. 4. Lactose synthase and lytic activities are examined.

INTRODUCTION

Jordan and Morgan (1969) made some preliminary studies on the proteins of echidna milk. Grifliths et al. (1984) have made some observations on the constituents of platypus and echidna milk (especially the lipid complement) and Messer and Kerry (1973) and Messer et al. (1983) have considered the carbohydrate composition. Preliminary studies of echidna lysozyme have been described by Hopper and McKenzie (1974). Thus, our knowledge of the proteins of echidna and platypus milk is still not well developed. It is the objective of the present report to present a general study of the proteins of echidna and platypus mature milk (as well as some blood serum proteins) and to indicate some future directions, if the structure of these proteins and their temporal variation in lactation are to be more completely understood.

The work reported here is one of a series of comparative studies of the proteins of milk and blood of a variety of mammals, with the aim of eventually throwing light on the evolution of their structures and functions. During the past 30 years there has been a considerable accumulation of knowledge of both the qualitative and quantitative composition of bovine milk. In particular, the primary and secondary structures of the main caseins and "whey" proteins of bovine milk have been studied extensively. This knowledge of the bovine proteins may be used as a point of reference, not only for studies of the proteins of other eutherian mammals, but also for those of marsupials and monotremes. The bovine proteins have been reviewed by several authors, e.g. McKenzie (1970, 1971), Lyster (1972), Jenness (1979, 1982), Dalgleish (1982), Fox and Mulvihill (1982), Mepham et al. (1982), and Swaisgood (1982). There have been few studies of the milk proteins of the marsupials and monotremes (for general discussions o f these mammals, see Tyndale-Biscoe, 1973; Griffiths, 1978; Walton and Richardson, 1989). Green et al. (1980) have discussed changes in total solids and protein during lactation for the tammar wallaby. Studies of the carbohydrates of kangaroo and tammar wallaby milk have been reported by Messer and Mossop (1977), Messer and Green (1979), and Messer et al. (1984); of kangaroo "whey" proteins by Lemon and Bailey (1966), Hopper et al. (1970), McKenzie et al. (1983), and of tammar wallaby milk proteins by Green and Renfree (1982).

MATERIALS AND METHODS Materials General. Special attention was given to the purity of

water, which was double distilled, the final stage being in a still with a quartz condenser. Acetic acid and hydrochloric acid were redistilled analytical grade reagents. All reagents used in buffers, etc were analytical grade, except imidazole which was general purpose reagent, purified by sublimation in vacuo (by M. Maxted in this laboratory). Ammonium sulphate was special enzyme grade from Mann Research Laboratories, New York, in earlier work, and from BRL, Bethesda, MD, in later work. Urea was Aristar grade of BDH Chemicals Ltd. Electrophoresis reagents were as follows: polyacrylamide gel reagents from Eastman Kodak, Rochester, NY; hydrolysed starch from Connaught Laboratories, Toronto, Ontario; and staining dyes from Matheson, Coleman and Bell, East Rutherford, NJ. Dialysis casing was Spectrapor membrane from Spectrum Medical Industries Inc., Los Angeles, CA, that had been treated with 3% (v/v) redistilled acetic acid at 60°C and washed thoroughly with double distilled water. Nitrocellulose membrane (0.2#m) was from Schleicher and Schull, Dassel, FRG. Sephadex gels used in protein isolation were from Pharmacia, Uppsala, Sweden. Dowex-I resin (Dow) was from Sigma Chemical Co., St Louis, MO.

Present addresses: *Rayne Institute, Faculty of Clinical Sciences, University College London, University Street, London WC1E 6JJ, UK. tChemistry Department, University College, University of New South Wales, ADFA, Canberra, ACT 2600, Australia. :~CSIRO Division of Wildlife and Ecology, P.O. Box 84, Lyneham, ACT 2602, Australia. 99

100

CARMEL G. TEAHAN et al.

Reagents for amino acid analysis and protein and peptide sequencing were from Pierce Chemical Co., Rockford, IL and Beckman Instruments, Palo Alto, CA, and for HPLC from J. T. Baker Co., Phillipsburg, NJ. BNPS-skatole (2-(2'-nitrophenyl-sulphenyl)-3-methyl-3-bromoindolenine) and creosol were gifts from Dr H. Campbell. Enzymes. Trypsin-TPCK treated was from Worthington Corp., Freehold, N J; Staphylococcus aureus V8 protease from Miles Laboratories Inc., Elkhart, IA; Arg-C endoproteinase from Boehringer-Mannheim, Mannheim, FRG; chymosin (calf stomach), domestic hen egg-white lysozyme and bovine galactosyl transferase (EC 2.4.1.22) from Sigma Chemical Co., St Louis, MO. Anti-sera. Rabbit anti-equine fl-lactoglobulin was a gift from Dr T. K. Bell; anti-sera to platypus and echidna whey were raised in rabbits in the animal facility of this laboratory; rabbit anti-human lysozyme, anti-human fl2-microglobulin, and anti-bovine casein were from Calbiochem-Behring; rabbit anti-human lactoferrin, antihuman ~t-lactalbumin and anti-human serum albumin were from Nordic Immunology, Tilburg, The Netherlands; and rabbit anti-human transferrin was from Dako Immunoglobulins, Copenhagen, Denmark. Radiochemicals. UDP 14[C] galactose, ca 0.034mg/ml, 12.47 GBq (337 mCi)/mmol in ethanol-water (7 v: 3 v) was from New England Nuclear Research Products, Boston, MA; 59Fe(III) citrate, ca 10/tg 59Fe/ml, 3.7 MBq (100 # Ci)/ml in sodium citrate (10 g/I) was from Amersham International, Amersham, UK. Methods p H determination. A Philips PW9414 digital ion-activity meter and Philips or ElL electrodes were used in the determination of pH. The reference buffers and calibration procedure were in accordance with the recommendations of Bates (1973). Protein determination. The protein concentration of milk samples was estimated from micro-Kjeldahl nitrogen determination according to the procedure of McKenzie and Wallace as described by McKenzie and Murphy (1970). Estimates of protein concentration of protein fractions were made from u.v. absorption determinations at 280 nm using appropriate absorbance indices and correction for scattered light. An absorbance index of 12.0 (10 g/l) was assumed for total monotreme "whey" protein. Protein concentration. Protein solutions were concentrated using Amicon "Diaflo" ultrafiltration cells, initially using UM-2 membranes, but YM-2 membranes in later work. Gel electrophoresis. Starch gel electrophoresis was performed as described by McKenzie (1971). Caseins were examined in starch urea gels using the continuous buffer system (7M urea-0.1M Tris-0.19M glycine-0.001M DTI'-15% (w/v) starch, pH 8.9) of H. A. McKenzie and G. B. Treacy (personal communication). Whey proteins were examined in one or more of the following semidiscontinuous buffer systems of Ferguson and Wallace (1963) gel: 12-13% (w/v) starch, 0.014 M Tris-0.0029 M citric acid4).002 M LiOH4).0076 M H3BO3, pH 7.7 (20°C), electrode buffer4).lM LiOH4).38M H3BO 3, pH 8.5 (20°C); polyacrylamide gel electrophoresis by a modification of the method of Gahne et aL (1977) using the Tris-sulphate buffer system (0.047 M Tris-0.014M H2SO4, 12% (w/v) acrylamide--0.3% (w/v) Bis acrylamide, pH 8.0 (20°C)) of K. Bell and H. A. McKenzie (personal communication) and by the step gradient acrylamide Tris-glycinate system of Banyard and McKenzie (1982); and SDS-discontinuous polyacrylamide gel electrophoresis according to the method of Laemmli (1970). Starch gels were stained with nigrosine, polyaerylamide gels by the method of Diezel et al. (1972), and glycoproteins in SDS-polyacrylamide gels were stained with periodic acid-Sehiff's reagent (Fairbanks et al., 1971).

Western blotting and detection o f blotted proteins. Western blotting was performed according to Burnette (1981) and stained according to the methods of Burnette (1981) and Hancock and Tsang (1983). Antisera. Antisera were raised against platypus "total whey" and echidna "total whey". Antigen was emulsified with an equal volume of Freund's complete adjuvant and injected intramuscularly into the thighs of New Zealand White rabbits. Fourteen days later this procedure was repeated, but using Freund's incomplete adjuvant. Blood was collected from the marginal ear vein of each rabbit prior to the first injection, and 2 and 6 weeks after the second injection serum was prepared by centrifugation of clotted blood. The presence of antibodies was initially detected by double diffusion in agar gels (Ouchterlony, 1958). 59Fe autoradiograph. Iron (III)-binding proteins were identified by a method based on that of Bell et al. (1981). Protein solution (25 #1, containing 125-250/tg protein fraction, or "whey" fraction diluted one to five) was mixed with 56+59Fe solution (25/tl, solution containing 100/tCi 59Fe and 100 #g 56+59Fe per ml) and allowed to react overnight at 4°C. A duplicate set of samples without added 56+59Fe was also prepared. Both were subjected to polyacrylamide gel electrophoresis (Tris-glycinate system). The radioactive portion of the gel was dried and an X-ray film was exposed to the gel, the autoradiogram subsequently developed, and the remainder of the gel was stained by the method of Diezel et al. (1972). Lysozyme activity. Lysozyme activity of milk samples and protein fractions was determined by a modification (S. Kaminogawa and H. A. McKenzie) of the method of Parry et al. (1965). M. luteus cells were suspended in 0.012 M K2HPO4~).0546M KH2PO4, pH 6.2, 1=0.091, to give a final concentration of 15 mg/dl. The cell suspension (2.5 ml) was equilibrated at 25°C in a 10 mm quartz cell in a Cary Model 14 M-50 recording spectrophotometer. Sample solution (0.2 ml) was added and the contents mixed. The time rate of decrease in absorbance at 450 nm was recorded. The lytic activity was determined by use of calibration curve prepared for domestic hen egg-white lysozyme. Galactosyl transferase and lactose synthase activities. Galactosyl transferase and lactose synthase activities were determined by a modification of the method of Fitzgerald et al. (1970) which involves the incorporation of 14C galactose into N-acetyllactosamine and lactose, respectively. For the determination of galactosyl transferase activity, N-acetylglucosamine (100#1 0.03M, in 0.05M glycine0.0009 M NaOH4).02 M MnC12, pH 8.0) was mixed with pH 8.0 buffer (50#1), sample solution (50#1) (or bovine galactosyl transferase l pg/50pl) and UDP-galactose (10/tl, containing 5.92 nmol UDP-galactose = 5.88 nmol "cold" UDP-galactose + 0.04 nmol 14C-UDP-galactose, 26,000 d.p.m.). The reaction mixture was incubated at 37°C for 10min, and the reaction then stopped by applying the mixture to a small column of Dowex 1 (in C1- form). The N-acetyllactosamine and glucose were eluted from the column (UDP-galactose being retarded) into scintillation fluid (5 ml) and counted for 10 min in a Packard Tri-Carb liquid scintillation spectrometer. Controls included: water only, N-acetylglucosamine omitted, and UDP-14C galactose only. Lactose synthase activity was determined by mixing solutions of glucose (in place of N-acetylglucosamine), protein sample, galactosyl transferase and UDP-galactose ("hot" + "cold"), and proceeding as in the determination of galactosyl transferase activity. Appropriate controls were included. Amino acid analysis. The sample (ca 20 nmol) was hydrolysed by heating at 110°C for 22hr in 6 M HC1 (0.3-2.0 ml) in evacuated sealed tubes. A drop of aqueous phenol was added to each tube to help minimize loss of tyrosine. The hydrolysate was dried, dissolved in 0.66 M citric acid4).2 M NaC1 (pH 2.2) (0.5 ml) and an aliquot

Some monotreme milk "whey" and blood proteins applied to a Beckman amino acid analyser, modified extensively for single column methodology and nmol sensitivity. The eluates were reacted with ninhydrin and absorbanee determined at 440 and 570 rim. Cystine and cysteine contents were determined as cysteic acid following performic acid oxidation of the sample or as S-carboxymethylcysteine following reduction of the sample and carboxymethylation. The results of analyses were not corrected for incomplete hydrolysis or hydrolytic losses. Sequence determination. In earlier protein and peptide sequencing a Beckman Model 890C spinning cup sequencer was used as well as a Beckman Model 890M sequencer. In later work a Beckman Model 890M-2 sequencer capable of handling sample amounts as low as 60 pmol was employed. Polybrene was used as an inert carrier (Klapper et al., 1978; Tarr et al., 1978) in the spinning cup of all these sequencers. In the M series sequencers conversion of anilinothiazalinone products to phenylthiohydantoin-amino acids (PTH-amino acids) was automatic. The latter were identified by high performance liquid chromatography (HPLC) with the aid of a Hewlett-Packard 1084B HPLC system fitted with a reverse phase Beckman ultrasphere ODS column (4.6 m m x 200 ram) to resolve the PTH-amino acids. Their absorbance values were determined at 269 and 323 nm with a Beckman Altex Model 165 variable wavelength detector. N-Terminal amino acid residues were determined by direct sequencing on the protein. In some cases, sequencing of cleaved peptides was also performed: cyanogen bromide cleavage after Met by the method of Gross (1967), cleavage after Trp with BNPS-skatole (Fontana et al., 1973), cleavage after Lys and Arg with trypsin (Shaw, 1965), cleavage after Arg with endoproteinase Arg-C (Levy et al., 1972), cleavage after Glu with S. aureus V8 protease (Drapeau, 1977). Separation and detection o f peptides. Peptides were separated by two-dimensional peptide mapping on Whatman 3MM paper, the first dimension being

electrophoresis at pH4.7 (pyridine-acetic acid-water, lv: Iv:38v); the second dimension being ascending chromatography in butanol-acetic acid-pyridine-water (15v: 3v: 10v: 12v). Tryptophan containing peptides were detected by natural fluorescence under u.v. light, others by several procedures, including ninhydrin staining, and spraying with fluorescamine (FLURAM). The last was particularly useful for recovery of peptides for sequence determination. Collection and preliminary fractionation o f milk and blood. Samples of blood and milk were collected from Tachyglossus aculeatus multiaculeatus located on Kangaroo Island (off the coast of South Australia) and from Tachyglossus aculeatus aculeatus in southeastern and northern New South Wales (NSW) and the Australian Capital Territory (ACT). Details of samples, on which features of electrophoretic patterns, etc are based, are given in Table 1. Milk let-down was facilitated by intramuscular injection of oxytocin (Syntocinon, Sandvyg, 3-8 IU) ca 5 min prior to collection. Each sample was transferred by a specially made borosilicate glass pasteur pipette to a borosilicate glass vial and was held at ca 2°C after collection and centrifuged within 48 hr to remove fat (Sorvall RC5 centrifuge, SS34 rotor, 3000rpm, 3°C, 20 min). Some lactating females were transferred to the laboratory, and were fed during captivity with a daily diet of a custard comprising two hen eggs with 30 g powdered milk of low lactose content (Digestelact) and 200 ml water (Grifliths et al., 1984). Whole milk samples ranged in volume from 4 to 33 ml of which ca 40% (by volume) was skim milk (pH 7.1-7.2). Blood was obtained usually from a mammary gland vein. Serum was obtained by centrifugation after allowing blood samples to clot. All samples of blood and milk of Ornithorhynchus anatinus were taken from animals caught in the wild in the Shoalhaven River region of NSW (Table 2). The platypus was usually anaesthetized (ether) prior to milk collection,

Table 1. Summary of echidna milk samples studied in this laboratory Capture Milking Approx. lactation location Status date stage (days)* KI 80/13 Kangaroo Island Laboratory 19/09/80 27 23/09/80 31 29/09/80 37 03/10/80 41 07/10/80 45 10/10/80 48 13/10/80 51 16/10/80 54 20/10/80 58 23/10/80 61 27/10/80 65 07/11/80 76 KI 84/2 Kangaroo Island Laboratory 19/09/84 40 24/09/84 45 27/09/84 48 30/09/84 51 02/10/84 53 04/10/84 55 KI 6 9 / M 4 K a n g a r o oIsland Wild --/10/69 30 KI 7 0 / M 2 K a n g a r o Island o Wild --/09/70 40 KI 70/M3 Kangaroo Island Wild --/09/70 60 KI 73/1 Kangaroo Island Wild 27/10/73 50-80 KI 79/1 Kangaroo Island Wild 13/12/79 140 22/02/80 2OO KI 83/1 Kangaroo Island Wild 17/11/83 100 NS 73/1 S. New South Wales Wild 22/10/73 50 NS 69/1 N. New South Wales Wild --/10/69 70-80 NS 76/1 S. New South Wales Wild 01/04/76 Late AC 86/1 Australian Capital Territory Wild 08/01/86 154 AC 86/2 Australian Capital Territory Wild 09/01/86 155 28/01/86 174 *Estimate based on weight of pouch young, using a growth curve for echidna pouch young, devisedby M. E. Griffiths (pers. commn), for facilitatingthe correlation of weight and age. Late lactation is definedhere as ca 140-200 days post partum. Lactation lasts for ca 200 days (Abensperg-Traun, 1989; Gritl~ths et al., 1988). All samples are mature milk. Protein compositionin very early lactation is different(R. Joseph and M. Griffiths,pers. commn).Note that September-Octoberis spring and January is summer in the Southern Hemisphere. Animal designation

101

102

CARMEL G. TEAHAN e t

al.

Table 2. Summary of platypus milk samples studied in this laboratory Approx. Animal Capture Milking lactation designation location Status date stage 82/010 82/921 82/918 83/014 83/171 83/948 83/151 83/414 84/450 84/445 84/974 84/014 84/442 84/160 84/459 84/945 84/460 84/457

New South Wales

Wild

let-down being induced by injection of oxytocin (2 IU) 10 rain prior to milking. Sample volumes of whole milk ranged from 0.2 to 15.5 ml. Each sample was transferred to a glass vial (as for echidna) and held at 4°C for 48 hr prior to skimming, except December 1984 samples which were frozen and held at - 20°C for ~ 76 hr prior to thawing and skimming. Adult animals were anaesthetized prior to collection of blood samples (1.0-1.5 mi) from the venous sinus located at the tip of the bill.

18/12/82 18/12/82 19/12/82 19/02/83 16/12/83 17/12/83 17/12/83 17/12/83 16/12/84 16/12/84 17/12/84 16/12/84 16/12/84 18/12/84 18/12/84 18/12/84 18/12/84 18/12/84

mid

The echidna and platypus skim milk samples were fractionated further into "whey protein" and "casein" fractions by centrifugingat 48,200 g (max.) for 30 min at 3°C in an SS-34 rotor in Sorvall R C 5 centrifuge.The relativelylarge casein micellcspresent in monotreme milk (H. A. McKenzic and M. C. Taylor, pers. commn) favour their separation from "whey proteins" at lower R C F than for bovine casein micelles (105,000g, 90 m in). This does not, of course, give a completcly "clearcut" separation, the "whey protein"

ca

Echidna skim-milk

Echidna total 'whey protein'

I

Heparin-Sepharose (pH 7.4) F III

FI

FII

Lysozyme fraction Band M(9)

I DEAE-Sephadex A-25 (pH 7.3) F II,FIII~ F IV

FI

I Sephadex G-100 superfine (pH 7.3)

FI

FII

FIII

Bands G-K (1) (Transferrin)

FV

I

I

CM-Sephadex C-50 (pH 5.0)

Sephadex G-100 superfine (pit 7.3)

FIII

F II

FI

Band A(7,8)

I

Sephadex G-100

Band B(4)

superfine (pH 7.3)

F III

Band A(7,8)

F II

FI Band B(4)

(a) Fractionation Procedure I Fig. l(a)

FIII

FIV

Band C(2) (Serum albumin)

103

Some monotreme milk "whey" and blood proteins Echidna skim-milk

Echidna total 'whey protein'

I

Iteparin-Sepharo~ (pH 7.4) FIII FI

FII

Lysozyme Band M(9)

I

DEAE-Sepimdex A-50 (pH 7.3)

'

FI~

I

II

F VI, F VIII

t F II

FIII

¢

I

DEAE-Sephacel

FI FII

FIII FIV FV

Band g(6)

FVII

FV

FIV

Bands G-K(1) (Transferrin) Sephadex G-100 (pH 7.3)

,

II

FII

FIX

I Sephadex G-100 (pH 7.3)

FI

t

F II

FIII

Band N 5 )

FI Bands G-K (i) Transferrin

+ others

Band A(7,8)

I DEAE-Sephacel (pH 7.3) Purified Band A(7,8)

(b) Fractionation Procedure II Fig. l(b) Fig. 1. Fractionation procedures I and II for echidna "whey" proteins. In both procedures the "whey protein" fraction was first passed through a column of Heparin-Sepharose. The eluted "lysozyme depleted" fraction was then subjected to further fractionation. The volumes of this fraction used varied from ca 2.5 to 6.5 ml, containing ca 200-480 mg total protein. The ion exchange chromatography columns were 16 mm x 280-400 mm. Elution was with a salt gradient, 0-0.1 M NaC1 in starting buffer, with a final application of 1 M NaC1. The columns for gel filtration were 28 m m × ca 700 ram. When the sole objective of the fractionation was the isolation of transferrin the Heparin-Sepharose step was omitted in both procedures I and II. However, in procedure II the final purification was on DEAE-Sephadex A-25.

fraction, so obtained, will contain some monomeric and lower polymeric forms of the caseins. In the present paper the term "whey protein" fraction refers to the fraction obtained as described above. The "whey protein" fraction was stored at - 2-4°C if used within 2 days, otherwise it was frozen and held at -20°C. Casein fractions were stored at - 20oc. Isolation of"whey" proteins. In the course of this work a variety of fractionation procedures were developed. Only those used in attempts to characterize individual proteins are considered here. Flow diagrams summarizing the procedures for the fmctionation of echidna "whey proteins" are given in Figs la and lb, and for platypus in Fig. 2. The buffer systems used were: 0.05 M Tris-0.047 M HC1, pH 7.1; 0.05 M Tris-0.045 M HCI, pH 7.3; 0.005 M sodium diethylbarbiturate-0.004 M HC1-0.05 M NaC1, pH 7.4; and 0.05 M CH3COONa-0.021 M CH3COOH, pH 5.03. The pH values are given for 20°C. The temperature coefficient

for the Tris buffer is appreciable: -0.028/°C. In general, the fractionation procedures were performed at 2°C. RESULTS AND DISCUSSION

Appearance and protein content o f milk samples Both echidna and platypus whole milk and skim milk samples differ appreciably in appearance from bovine milk. The echidna milk samples are pink in colour and very opaque; in contrast the platypus milk samples are creamy-yellow in appearance. The centrifuged echidna " w h e y " is very similar in appearance to solutions o f ovotransferrin, but the corresponding platypus " w h e y " is very pale in colour. T o t a l protein contents for milk and " w h e y " and "casein" fractions are given for echidna and platypus

CARMELG. TEAHANet al.

104

Platypus skim milk

Total 'whey protein'

1

I Sephadex

G-100

I

1

DEAE-SephadexA-25

Phenyl-Sepharose (pH 7.5)

(superfine, pH 7.3)

(pH 7.3)

, FI

FII FIII

FIV

FII HII

FI

I

I

A-25 (pH 7.3)

A-25 (pH 7.3)

DEAE-Sephadex DEAE-Sephadex

Band D(6)

FI

FI

Band C(5)

I

DEAE-Sephadex A-50(pH 7.1) FIII

FII Band A(7)

FII, ~

FI, II, IV-VII

I DEAE-SephadexA-50 (pH 7.1) Tf rich

I SephadexG-75 (superfine, pH 7.1) Band G(1)

Fig. 2. The procedures used in the fractionation of platypus total "whey" protein. Columns and buffers were generally similar to those used for echidna "whey" fractionation. However, the fractionation on the Phenyl-Sepharose column (16 mm x 100 mm) was performed at 20°C. The sample applied to the column was in Tris-HCl buffer (pH 7.5) containing 0.001 M Na 2 EDTA; elution being performed initially in this buffer, followed by buffer in which the Na 2 EDTA was replaced by 0.001 M CaC12. Three fractions were eluted with the first buffer, no additional protein being obtained in the buffer containing Ca(II). in Tables 3a and 3b, respectively. The total protein and casein contents of echidna milk appear to be high up to 48 days and then to fall to lower levels (but there are insufficient samples to be confident of this). The exact state of lactation for each platypus sample is not known and it is not possible to speculate on any temporal trend.

Enzymic activities Echidna milk exhibited pronounced lytic activity, as was observed earlier by Hopper and McKenzie (1974). In the present studies unsatisfactory results were obtained in lytic activity determinations on platypus milk using the modified method of Parry et al. (1965, see Methods). Following the subsequent development of a highly sensitive method by McKenzie and White (1986) for the determination of lytic activity, it was shown that platypus milk only exhibits weak lytic activity. In contrast, appreciable lactose synthase activity was detected in all the platypus milk samples examined. The levels of galactosyl transferase activity in the echnida milk samples were appreciable. However, the levels of endogenous lactose synthase activity were low. From experiments in which addition of bovine ct-lactalbumin was made, it was concluded that low levels of ctlactalbumin, or other modifier protein, in echidna

milk were the limiting factor in the lactose synthase activity.

Casein electrophoretic patterns The purpose of the present investigation was to investigate the "whey" proteins. However, a few observations were made on casein patterns for echidna and platypus. As observed previously in this laboratory by H. A. McKenzie and co-workers, no evidence was obtained of cleavage of echidna caseins by chymosin (under conditions of cleavage for bovine). The pattern for the echidna proteins differed in detail from bovine patterns. However, there was some similarity in overall distribution of the bands. The profile of platypus casein bands was quite distinct from that of the echidna, and was unaffected by chymosin. Echidna "whey protein" electrophoretic patterns Examples of echidna and platypus "whey protein" and blood serum gel electrophoretic patterns are shown in Figs 3 and 4. The Ferguson-Wallace system patterns for echidna "whey proteins" (Fig. 3a) are more complex than the corresponding patterns for platypus (Fig. 4a, discussed below). The echidna bands studied in the present work have been designated A - M in order of decreasing mobility. In

105

Some monotreme milk "whey" and blood proteins

Animal KI 80/3

KI 83/1 KI 84/2

Table 3a. Proteincontentof echidnamilk Approx. Total protein "Whey" stage of g/dl g/dl protein Date of lactation Whole Skim g/dl Milking (days) milk milk skim milk 3/10/80 41 12.4 21.5 6.4 10/10/80 48 11.9 20.5 n.d. 13/10/80 51 8.4 15.0 6.0 16/10/80 54 9.4 13.9 6.1 17/11/83 100 14.4 24.0 n.d. 30/09/84 51 9.7 14.6 5.5 2/10/84 53 9.7 14.2 6.4 4/10/84 55 9.5 14.7 6.5

Table 3b. Proteincontentof platypusmilk Total protein "Whey" Approx. g/dl g/dl protein Date of stage of Whole Skim g/dl Animal milking lactation milk milk skim milk 84/160 18/12/84 mid 6.4 9.2 7.7 84/459 18/12/84 8.6 13.6 n.d. 84/945 18/12/84 7.5 12.5 n.d. 84/014 16/12/84 9.4 19.3 n.d. 84/450 16/12/84 8.2 20.1 n.d. 84/974 17/12/84 4.4 9.2 n.d. 84/460 17/12/84 7.8 13.5 n.d. n.d. = not determined. addition to these bands there is a group of fast moving bands, labelled ~t, shown in the pattern of Fig. 3a. The pattern shown was chosen in order to show these bands, because they are either not seen in the patterns of some samples, or are rather weak in others. Their nature, and the extent to which some of them may be caseins has not been investigated, nor do we know whether the variation in the ct region is temporal during lactation or not. It does not seem to be temporal in the limited number of samples studied. It will be noted that some of the bands (notably C and I) have mobilities similar to bands in the serum (Fig. 3b). Most of the other bands appear to have no counterpart in the serum. The zone labelled M is cathodic in migration and is very diffuse in this (Ferguson-Wallace) buffer system. It is related to the presence of lysozyme (see

below). The main temporal variation observed is in the five bands G H I J K and is both qualitativeand quantitative. Only four of them (G-J) are evident in the example shown in Fig. 3a. These bands were shown to be due to iron-binding proteins (see below). Some nine bands (designated I-9) were clearly resolved, and some five diffuse bands were seen, in SDS-gradient polyacrylamide gels (10-20 g/dl). The proteins range in apparent molecular weight from 15,500 to 89,000 (Fig. 3b). In the course of the present study it was possible to relate some of these bands to those in the conventional Ferguson-Wallace system. The designations are shown in parentheses in Fig. 3a.

Variations in iron (IIl)-binding bands of echidna In the period 1969-1986 milk samples were taken from eight lactating echidna (Tachyglossus aculeatus multiaculeatus) from Kangaroo Island, and from five echidnas (Tachyglossus aculeatus aculeatus) in southern New South Wales and ACT. However, it was only possible to make observations on the

Casein protein g/dl skim milk 13.9 n.d. 7.8 7.3 n.d. 7.0 7.4 7.0

Casein protein g/dl skim milk 2.5 n.d. n.d. n.d. n.d. n.d. n.d.

temporal variation in proteins during a given lactation cycle for four of them. The most evident variation in milk protein electrophoretic pattern in the Ferguson-Wallace buffer system was in the region (bands G - K ) where transferrins would be expected. By comparison with the echidna serum protein and bovine transferrin patterns and from 59Fe-binding experiments, it was concluded that these bands are due to transferrins and not lactoferrins. A maximum of five bands was seen in this region of the echidna milk and "whey protein" patterns, while a single major band together with two very minor transferrin bands were detected in the serum patterns (Figs 3a-b). A schematic representation of the banding observed in this region of the "whey protein" electrophoretic patterns in Ferguson and Wallace starch gels is given in Fig. 5. Since many of the samples had been run on separate gels the patterns were normalized using the serum albumin band as a reference point. Details of the origin of the samples are given in Table 1. The skim milk and "whey" fraction transferrin bands have been labelled 0-4 (equivalent respectively to K - G ) in order of increasing mobility in the starch and polyacrylamide systems used. A series of samples was taken from a Kangaroo Island echidna (KI 80/13) held in the laboratory between 27 and 76 days of lactation. These were run on Ferguson and Wallace starch gels subsequent to sampling (Fig. 5). Later, they were run also on a 12% discontinuous polyacrylamide gel, pH 8.0. The order of mobility of the band in this system is the same as in the other system, but the ratios of the mobilities are slightly different. Four transferrin bands (labelled 1--4 in order of increasing mobility) were observed on Ferguson and Wallace gel electrophoresis of a sample at ca 27 days lactation. Bands 1 and 4 were very faint, band 2 appearing to be the dominant band. Band 2 had the sample electrophoretic mobility as the major

106

CARMELG. TEAHANet

al.

ECHIDNA origin

10%

4-

94

A (7,8)

1 2 3

B (4) C (2) D E (6)

67

4 5 43

F (5) G Hi (1) J

30

L

6

20.1

origin M(9)

14.4

+ 20% WHEY

SERUM

a)

WHEY SERUM

MW

b)

Fig. 3. Gel electrophoretic patterns of echidna total "whey" and serum proteins. (a) Ferguson-Wallace semi-discontinuous buffer system-starch gel patterns (details in experimental section). (b) Laemmli system SDS-gradient polyacrylamide gel (10-20%) patterns. echidna serum transferrin band. By 31--41 days lactation, a fifth band (band 0) is observed running behind band 1. Also, band 1 had increased in prominence over this time period. By c a 45 days lactation band 4 had disappeared. In samples of c a 48 and 76 days lactation band 0 had increased in prominence, band 2 remaining the dominant band. In summary, the band of greatest mobility diminished and was no longer detectable on the gel and a new band, with a lower mobility than any of the bands previously seen, appeared and increased in prominence during the lactation period studied. A second series of samples was taken from another Kangaroo Island echidna (KI 84/2) over the period

c a 40-55 days lactation. The pattern at 40 days is shown in Fig. 5; no significant changes were observed in the transferrin pattern over this period. The four bands in the transferrin region are bands 1--4, bands 2 and 3 appearing to be codominant. This pattern may be indicative of early, as opposed to late, lactation. A sample taken from a Kangaroo Island echidna (KI 83/1) at c a 100 days lactation, and from another, KI 73/1 at c a 50-80 days, exhibited a 0, 1, 2, 3 pattern, band 2 appearing to be the dominant band. Bands 0, 1 and 2 were observed in samples taken from another Kangaroo Island echidna (KI 79/1) at 140 and 200 days.

Some monotrem¢ milk "whey" and blood proteins

107

PLATYPUS origin

10%

! A (7) B

94 1 2

67

3 4 E

5

43

F (1) G

30

origin

7 8

20.1

14.4

WHEY

20%

SERUM

a)

b)

WHEYSERUM MW

Fig. 4. Gel elcctrophoretic patterns of platypus total "whey" and serum proteins. (a) Ferguson-Wallace semi-discontinuous buffer system-starch gel patterns. (b) Laemmli system SDS-gradient polyacrylamide gel (10-20%) patterns. A single sample taken from a New South Wales echidna (NS 76/1) in late lactation (probably just prior to weaning) also exhibited the 0, 1, 2 pattern. The three bands were codominant (usually the band intensity increases going from 0 ~ 2 ) . Since the sample was obtained very late in the lactation season, this probably accounts for the prominence of band 0. Two samples from an ACT echidna (ACT 86/2) taken at ca 154 and 174 days exhibited the 0, 1, 2, 3 pattern. These findings would appear to support the hypothesis that a shift occurs in the banding pattern during the course of lactation, the fast moving bands decreasing in intensity with the concomitant appearance and increase in intensity of band 0. However, caution must be exercised in this interpretation, given

the limited number of samples available and the lack of precise knowledge of the stage of lactation at which the samples were taken. Many more samples, taken over an extended period, will be required before a final correlation can be made between protein patterns and stages of lactation. The limited number of individual samples available, representing various stages of lactation, does not permit variation between individuals to be identified. Furthermore differences based solely on origin could not be distinguished between the mainland echidna (aculeatus) samples and the Kangaroo Island samples (multiaculeatus) studied. As already stated, the major serum transferrin band corresponds in mobility to band 2 of the milk transferrin bands. The transferrin system observed in serum samples appears

108

CARMEL G . TEAI-IAN

et al.

Animal KISO/13

KI80/13

KI80/13 K180/13 KI80/13 KI80/13

K I 8 0 /1 3

K184/2

KI8~1

KI73/1

K179/1

N S 7 6 / 1 AC86/2

I~te of 19/9/80 milking

23/9/80

29/09/80

7/11/80

19/9/84

17111/83

27/10/73

22/2/80

114176

76

40

Stage of lactation

27

31

37

3110/80

41

7110/80

#*5

10/10/80

48

100

50-80

late

9/1/86

late

155

(days)

+

34 2 10

m m

m m

m

34

m m m

m m /

m m

12 0

ORIGIN m

Fig. 5. Schematic of normalized electrophoretic patterns of echidna milk transferrins (see text). KI 80/13, KI 84/2, KI 83/I, KI 73/1, KI 79/1 signify Kangaroo Island echidnas. NS 76/1 and AC 86/2 signify southern New South Wales echidna and Australian Capital Territory echidna, respectively. Details with respect to dates of milking are given in Table 1. simpler than the milk transferrin system. A single major transferrin band is visible for serum samples on Ferguson and Wallace starch gels, with two very faint bands on either side. Immunology of echidna "whey" and serum proteins A brief study was made of the immunology of echidna "whey" and serum proteins. The proteins were separated by SDS-polyacrylamide gel electrophoresis of "whey" fraction and serum, respectively. They were transferred ("blotted") onto nitrocellulose paper and probed with antisera as described in the Methods section, bound antibody being detected by the binding of 12~I-labelled S. aureus protein A. None of the separated proteins reacted with rabbit anti-human lactoferrin, rabbit anti-human fl2-microglobulin and anti-equine fl-lactoglobulin. These results are consistent with the apparent absence of the proteins in echidna, but, of course, do not constitute proof of their absence. Band 1 (M r ca 78,000) of echidna "whey" and the corresponding band of echidna serum in the SDS-polyacrylamide gel system gave a reaction with rabbit anti-human transferrin. This is consistent with the conclusion that this zone reflects the presence of transferrin in echidna milk. Band 2 (Mr ca 70,000) in the SDS-polyacrylamide system, and what appears to be its equivalent in serum, cross-reacted with rabbit anti-human serum albumin. Protein from a low molecular weight zone (M, ca 15,000) cross-reacted with both rabbit anti-human ~t-lactalbumin and rabbit anti-human lysozyme. It was not possible to conclude whether there were one or two proteins in this zone, i.e. it was not possible to determine whether the reaction was due to the presence of an echidna lysozyme or ~-lactalbumin, or both. There was also some evidence of the presence of monomeric caseins in the "whey" fraction on the basis of some cross reaction with anti-human caseins.

Isolation and properties of echidna proteins Band A protein. Two preparations of echidna band A protein were made using fractionation procedure I (Fig. la), one being isolated from DEAE-Sephadex A-25 Fraction IV, the second from Fraction V. A third preparation was made using procedure II (Fig. lb). It is not possible to show all the elution profiles and electrophoretic patterns. A typical elution profile for lysozyme depleted total "whey" protein obtained in procedure II is slaown in Fig. 6. Fractions III and IV, which principally contained band A protein and transferrins, were subjected to gel filtration on Sephadex G-100, the elution profile and electrophoretic patterns of fractions being shown in Figs 7a-c. Fraction II which was rich in band A protein was further purified on DEAE-Sephacel, band A protein being primarily in the first fraction (Fig. 7d-f). Band A protein isolated from Fraction IV of procedure I and from combined Fractions III and IV of procedure II showed only a single band in SDS-polyacrylamide gel, in polyacrylamide gel (pH 8.0) and starch gel (pH 7.7) electrophoresis. A puzzling feature of the preparation from Fraction V in procedure I, was that, while it gave a single band in SDS-polyacrylamide gel electrophoresis, it showed a major and minor band in FergusonWallace starch gel electrophoresis. Nevertheless, this preparation showed only a single N-terminal amino acid sequence, indicating that the effect was probably one of apparent, rather than real heterogeneity. Bands 7 and 8 of total whey are rather poorly resolved on SDS-polyacrylamide gels and the band A protein is rather diffuse in this system. Hence it has not been possible to determine unequivocally whether bands 7 or 8 correspond to band A. An apparent molecular weight of ca 17,800 has been assigned tentatively to band A protein. It does not react with periodic acid Schiff's reagent and there is no evidence of hexosamines in amino acid analyses. Hence, it is unlikely to be a glycoprotein. Band A protein is not active in the lactose synthase system.

Some monotreme milk "whey" and blood proteins

109

ss"

NaO ( M ) (--)

4,, J ~ t,o S

280

, ,,-"

(--)

O. 1 2

//

0.2

."

A

I J

0.08

, 600

1200

1800

2400

3000

VOLUME (too

Fig. 6. Elution profile for the ion exchange chromatography of lysozyme-depletedechidna total "whey" protein on a DEAE-Sephadex A-50 column (procedure II). The composition of some individual fractions is indicated in Fig. lb. The amino acid analysis, assuming ca 160 residues, is shown in Table 4. The sequence of the first 25 N-terminal residues was determined using 30 nmol native protein. The following sequence was obtained: 10 Val-Asn-Pro-Lys-Pro-Glu-Lys-Ala-Gly-Ser

first 25 residues of native protein had identified a methionine at position 19. The two sequences distinguished are as follows: I

12

1. Val-Asn-Pro-Lys-Pro-Glu-Lys-Ala-Glu-Ser-(Cys)-Pro 20

25

26

2. Asp-Pro-Asn-Asp-(Cys)-Gln-Val-

20

-(Cys)-Pro-Leu-Ser-Val-Leu-Glu-Asn-Met-Asp 25

Pro-Asn-Asp-(Cys)-Gln Residues 11 and 24 could not be identified unequivocally by HPLC, but it is likely that 1/2 cystine residues occur at these positions. Native protein (ca 70 nmol) was cleaved after tryptophan residues with BNPS-skatole. The cleaved protein (ca 10 nmol) was sequenced through 15 cycles to ascertain the extent of the cleavage and the number of peptides generated. Two sequences were detected: the N-terminal sequence and a second sequence which ran as follows: 10 Asp-Ala-Glu-Asp-Asn-Ile-?-?-?-? 15 Asn-Val-Gln-?-Lys Amino acid analysis of the protein indicated the presence of ca four methionine residues. Cleavage after methionine residues should, therefore, yield five peptides which might be sequenced for all or part of their length. The protein (ca 280 nmol) was reduced and carboxymethylated prior to the cleavage reaction. However, this resulted in almost total loss of protein at the dialysis step with only ca 10 nmol being recovered. This was cleaved with cyanogen bromide and the mixture sequenced through 12 cycles. Five sequences appeared to be present, but only two of them could be distinguished. It was possible to align these two peptides since N-terminal sequencing of the

Since substantial loss of protein was incurred at the reduction and carboxymethylation step, performic acid oxidation was chosen as the method of cleaving disulphide bonds subsequent to a second attempt at cyanogen bromide cleavage of the protein. However, poor yields of all but the first two sequences were obtained using this method. Comparison of the partial sequence and amino acid composition of band A protein with those of other proteins did not enable unequivocal identification of the protein, which is further considered in the Discussion. Band B protein. Band B protein was isolated from Fraction II from CM-Sephadex C-50 in fractionation procedure I (Fig. la). This fraction did not exhibit heterogeneity on Ferguson-WaUace gel electrophoresis or polyacrylamide gel electrophoresis. It was shown that band B corresponds to band 4 on SDS-polyacrylamide patterns, and the latter enabled a crude M, value of 55,000 to be estimated. The amino acid composition was determined and is given in Table 4. Band B could be stained with periodic acid-Schiff's reagent and hexosamines were detected in amino acid analyses. Hence, it is concluded that it is a glycoprotein. The sequence of the first 15 residues of the Nterminus was determined on the native protein and found to be: 10 Gly-Val-Ala-Gln-Pro-Thr-Leu-Gly-?-Gly15

Asp-Gly-Phe-Pro-Phe

110

CARMELG. TEAFIANet al.

This sequence does not appear to be homologous to any known major whey protein. Band C protein. Band C protein was isolated from Fraction IV from the CM-Sephadex C-50 column in procedure I (Fig. la). It exhibited absence of heterogeneity in Ferguson-Wallace gel electrophoresis and in SDS-polyacrylamide gradient gels. It was shown that band C corresponds to band 2 on SDSpolyacrylamide gels, has an apparent M r ca 70,000, and is not a glycoprotein. The mobility of band C protein is the same as that of echidna serum albumin, and appears to have a similar molecular size to bovine serum albumin on SDS-polyacrylamide gels. Its amino acid composition was similar to those of bovine serum albumin and human serum albumin. The sequence of the first 30 residues of the N-terminus was determined, and it is compared with those of the bovine and human proteins in Fig. 8. The homology between the sequences of bovine and human serum albumin in the first 30 residues is 83% and between either of these albumins and echidna band C protein is 63%. Thus, it is concluded that band C protein is echidna serum albumin. Band D protein. Echidna band D protein was not isolated, Band E protein. The mixture of proteins obtained in Fraction II from DEAE-Sephadex A-50 in fractionation procedure II included band E protein. This fraction was subjected to chromatography on a column of DEAE-Sephacel, and band E protein was isolated from Fraction IV. The protein gave a single band on electrophoresis in the Ferguson-Wallace system. It corresponded to band 6 on SDS-polyacrylamide gradient gels and appears to have a M, of ca 21,000. Also it does not seem to be a glycoprotein and its mobility is unaffected on treatment with 2-mercaptoethanol, indicating absence of interchain SS linkages, The amino acid composition (assuming 190 residues total) is shown in Table 4. The sequence of the first 45 residues of the N-terminus was determined on the native protein (ca 60nmol) and is shown in Fig. 9. Peptides were prepared by cleavage of the protein after tryptophan residues with BNPSskatole and by cleavage of the reduced and carboxymethylated protein after methionine residue with cyanogen bromide. Sequence studies of these peptides enabled the sequence of a further 23 residues to be determined, the results being summarized in Fig. 9. Comparison of the partial sequence of the first 68 residues, out of a total of ca 190 residues, with sequences in a data bank did not enable unequivocal identification of the protein. However, D. C. Shaw (pers. commn) has drawn our attention to the fact that the sequence of residues 9-22 meets one of the criteria of North (1989) for the family of proteins of which fl-lactoglobulin is a member. Band F protein. Fractions VII, IX from D E A E Sephadex A-50 in procedure II (Fig. lb) contained band F protein together with a mixture of other proteins. While gel filtration on Sephadex G-100 resulted in some further fractionation, band F protein (Fraction III) still exhibited some heterogeneity. It appeared to be equivalent to band 5

on SDS-polyacrylamide gels (hi, ca 52,000). No further studies were made of this protein. Bands G - K protein. As already indicated these bands are due to transferrin and they exhibit temporal variation during lactation. Their properties, together with the isolation from milk and blood serum, are discussed elsewhere (Teahan and McKenzie, 1990). Band L protein. This protein was not isolated. Band M protein. This diffuse cathodic band in the Ferguson-Wallace system is due to lysozyme and its isolation and properties will be discussed elsewhere by Teahan et al. (to be published). The possible presence o f o~-lactalbumin in echidna milk

It has been established elsewhere that echidna milk contains oligosaccharides of lactose (Messer and A 280 1 cm (_)

o. a

0.4

~l) I

/

100

II

Ill

$

200

I 300

VOLUME (ml)

a)

i~!¸ !iV!

EW

II b)

c) Fig. 7(a-c)

I

II

Some monotreme milk "whey" and blood proteins

111

280

A

lcm

NaCI (M)

(--)

(--)

0.2

0.08

0.1

0.04 NaCl gradient

s

~

si ~ " 1.N.c,

~200

400

600

l

~1

800

1000

VOLUME (ml)

d)

origin

origin

e)

1

2

3

f)

1

2

a

Fig. 7(d-f) Fig. 7. (a) Elution profile of combined Fractions III and IV ex DEAE-Sephadex A-50, procedure II (Fig. 6) on a Sephadex (3-100 column. (b) Electrophoretic patterns on polyacrylamide gel (12%), pH 8.0, of Fractions I and II. Protein stained with Xylene Brilliant Cyanin G. (c) SDS-gradient polyacrylamide gel (10-20%) of echidna whey protein (EW) and Fractions I and II. (d) Elution profile of Fraction II ex Sephadex G-100 on a DEAE-Sephacel column. (e) Acrylamide (12%) gel electrophoretic pattern (pH 8.0) of 1. Total "whey" protein, 2. Major fraction, 3. Minor fraction. (f) Ferguson-Wallace system (pH 7.7) starch gel electrophoretic patterns of fractions equivalent to those in (e).

CARMELG. TEAHAN et al.

112

Table 4. Amino acid composition of some echidna proteins (number of residues per

molecule) Residue

Band A

Asp + A s n Glu + G l n His Lys Arg Pro Cys/2 Met Ser Thr Gly Ala Leu Val lie

18 28 2* 14 3 18" 24% 4T 9 2 6 7 8 8 4

Tyr Trp Phe Total

2* n.d. 2 160 (c)

Band B 70 31 16" 21 14 22* 10t 20~ 40 26 44 45 52 23 16

16" n.d. 28 492 (c)

Band C 61 72 14" 56 16 23* 27t 5t 38 26 25 66 63 38 4

19" n.d. 28 582 (c)

BSA (a) 54 79 17 59 23 28 35 4 28 34 16 46 61 36 14

19 2 27 582 (c)

HSA (b) 53 82 16 59 24 24 35 6 24 28 12 62 61 41 8

18 1 31 585 (c)

Band E 29 28 2* 16 7 11" 4~ 8 7 16 4 5 18 14 9

5* n.d. 8 190 (c)

*Analysis of native protein.

tAnalysis of oxidized protein; all other analyses are means of analysis of native and oxidized protein. (a), (b): composition from amino acid sequence of Brown and Shockley (1982) and Dugaiczyk et aL (1982), respectively;(c): composition on basis of total number of residues assumed; (d): total no. residues from sequence; n.d.: not determined. Kerry, 1973; Hopper and McKenzie, 1974). We have shown that it exhibits weak lactose synthase activity in confirmation of the observations of Hopper and McKenzie (1974). The latter workers isolated two lysozymes from echidna milk: lysozyme I from Tachyglossus aculeatus multiaculeatus and iysozyme II from Tachyglossus aculeatus aculeatus. Both lysozymes exhibited lytic activity, but lysozyme I appeared to act, albeit weakly, as modifer protein in the lactose-synthase system, with either bovine or echidna galactosyl transferase. Using the limited samples available, no success was achieved in isolating a conventional ~-lactalbumin from the echidna fractions. In the present work we noted that the

isolated echidna lysozyme reacted weakly with antiserum to human ~-lactalbumin. This was in contrast to the markedly stronger reaction of the total " w h e y " protein with these antibodies. Hence, we made a further search for an ~-Iactalbumin in the total " w h e y " protein. Because of the low levels of endogenous lactose synthase activity, the concentration of ~-lactalbumin (if any) is likely to be at a low level. Gel filtration of total " w h e y " protein (ca 5 ml) on Sephadex G-100 superfine, at p H 7.3, resulted in four fractions (I-IV). Lysozyme lytic activity was present in all fractions. This may have been due to similar effects to those observed by Whitaker (1963) in the gel

1

i0

Band C

Asp-Ala-Gln-Lys-Ser-Glu-Leu-Gly-His-Arg

BSA

Asp-Thr-His-Lys-Ser-Glu-Ile-Ala-His-Arg

HSA

Asp-Ala-His-Lys-Ser-Glu-Val-Ala-His-A~g

Band C

Tyr-Lys-Glu-Leu-Gly-Glu-Asp-His-Phe-Lys

BSA

Phe-Lys-Asp-Leu-Gly-Glu-Glu-His-Phe-Lys

HSA

Phe-Lys-Asp-Leu-Gly-Glu-Glu-Asn-Phe-Lys

2O

3O Band C

Ala-Leu-Ala-Leu-Val-Thr-Phe-Ser-Gln-Tyr

BSA

Gly-Leu-Val-Leu-Ile-Ala-Phe-Ser-Gln-Tyr

HSA Ala-Leu-Val-Leu-Ile-Ala-Phe-Ala-Gln-Tyr Fig.8.~mpa~s~n~fN-~nnina~sequences~fechidnami~k~and~pr~tein(band~)withth~fb~vine serum albumin(BSA) and humanserum albumin(HSA).

113

Some monot~memilk"whey"and blood proteins 10

1

Ala-Ala-Asp-Val-Ser-Gly-Lys-Pro-Ile-Gln-Thr-Glu-Lys-Leu

20 Lys-Gly-Gln-Trp-His-Thr-Ile-Ser-Met-Ala-Thr-Asn-Glu-Met

30

40

Lys-Leu-Ile-Glu-Lys-Asp-Gly-Thr-Met-Arg-Phe-Phe-Phe-Lys

5O ? -Val-Val-Pro-Arg-Asn-Ile-Asp-Glu-Leu-Ile-Val-

60 Leu-Lys-

Sequence

sequence

? -Lys- ? - ? -Glu-Thr

analysis

of whole protein

of reduced and carboxymethylated

fragments Sequence

68

? -Glu-Asn-Asn-

N-terminal

? -Met

cyanogen bromide

........

of peptides

following

cleavage

of the native protein

by BNPS-skatole Unidentified

residues

displaced

dashed

in a given peptide lines,

and,

are shown by

if not identified

in any

peptide, by ?. Fig. 9. Partial amino acid sequence of echidna milk band E protein. The strategy of the sequence determination is summarized in the figure.

filtration of domestic hen egg-white lysozyme, i.e. interaction of lysozyme with Sephadex and/or other proteins present. Galactosyl transferase activity was detected in Fraction II only, both on the basis of synthesis of N-aeetyllactosamine from added N-acetylglucosamine and UDP-galactose and of synthesis of lactose from added bovine ct-lactalbumin, glucose and UDPgalactose. CBPB gg/I--H

Bovine galactosyltransferase was added to column fractions in an attempt to detect the presence of an echidna ~t-lactalbumin. Low levels of activity were detected in Fractions I and II (200-700 cpm above background) and even lower in Fraction III (200--400cpm, compared with 4000cpm for 1 #g bovine ~t-lactalbumin). Activity in Fraction III (where ~t-lactalbumin might have been expected on the basis of molecular size) was also determined when

114

CARMELG. TEAHANet al.

echidna galactosyltransferase (from Fraction II) was substituted for the bovine transferase. However, the count (per min) was still only 200-400 above background. (The latter was appreciable, ca 1800 cpm, i.e. when galactosyl transferase, 14[C]-UDP-galactose and glucose were incubated together in the absence of exogenous ~-lactalbumin.) The possibility that Fraction II contained both echidna galactosyl transferase and an ct-lactalbumin was also investigated. Lysozyme was first removed from this fraction by passage through a column of Heparin-Sepharose. The effluent was devoid of lysozyme by lytic activity tests, but exhibited considerably increased lactose synthase activity. The activity was further enhanced when bovine galactosyl transferase was added. This was consistent with the presence of a putative ct-lactalbumin. Hence, a further fractionation of Fraction II (containing ca 120 mg total protein) was attempted by ion-exchange chromatography on a column of DEAE-Sephadex A-25. Three Fractions (designated II.l, II.2, II.3) were resolved. Gel electrophoretic analysis of each of these fractions indicated that Fraction II.1 contained transferrin and that Fractions II.2 and II.3 were a mixture of proteins. Lactose synthase activity (using bovine galactosyl transferase) was confined to two tubes in Fraction II.2. The relevant tube contents were pooled (ca 17mg protein), dialysed against buffer (0.05 M Tris4).043 M HC1-0.035 M Na 2 EDTA, pH 7.5) concentrated and fractionated on a column of Phenyl-Sepharaose. Most of the protein passed through the column unretarded in the elution buffer (Tris-HC1, pH 7.5, 0.001 M Na2 EDTA). A second fraction contained _< 10% of the total protein. No further protein was eluted on the application of a buffer containing calcium (II). The minor fraction was the only fraction exhibiting lactose synthase activity. It was heterogeneous on SDS-polyacrylamide gel electrophoresis. No band of M, ca 15,000 was detected by Coomassie Blue staining. On Western immunoblotting using rabbit anti-human human ct-lactalbumin antibody, no such band was detected on India ink staining. However, reaction with the antibody was detected by autoradiography. The fraction did not exhibit lytic activity. These results are considered further in the General Discussion. Platypus "whey" protein electrophoretic patterns As already indicated, the Ferguson-Wallace gel electrophoretic patterns (Fig. 4a) of platypus milk are less complex than those of echidna. Seven bands A ~ 3 , in order of decreasing mobility, were resolved. These bands are all anodic, no cathodic band being observed in the milk samples. Bands D, E, F and G all have bands of equivalent mobility in platypus serum (Fig. 4a). A diffuse streak was apparent in the cathodic region of the serum patterns. Eight bands of appreciable staining intensity were found in SDS-polyacrylamide gradient electrophoretic patterns, ranging in apparent Mr from ca 19,000 to 79,000. The bands are labelled 1-8 in order of decreasing molecular size in Fig. 4b. The pattern was not altered by pre-treatment of the "whey" proteins with 2-mercaptoethanol. Each of four of

these bands had a counterpart in the serum pattern (Fig. 4b). Iron (III)-binding bands of platypus On the basis of mobility and 59Fe autoradiography it was concluded that bands G and F in the platypus total "whey" protein electrophoretic patterns are transferrins. Both bands have counterparts in the serum patterns. These proteins are discussed elsewhere by Teahan and McKenzie (1990). Immunology of platypus "whey" proteins

Platypus total "whey" and serum cross-reacted with rabbit anti-human serum albumin and rabbit anti-human serum transferrin. There was no crossreaction evident between any platypus "whey" protein and the following: rabbit anti-human lactoferrin, rabbit anti-human fl:microglobulin, rabbit antihuman lysozyme, rabbit anti-human ct-lactalbumin and rabbit anti-equine fl-lactoglobulin. Isolation and properties of platypus proteins

Several of the major "whey" proteins of platypus were isolated and attempts made to characterize them. The main procedures for fractionation are summarized in the schematic diagram of Fig. 3. Band A. Fraction IV from gel filtration of total "whey" protein on Sephadex G-100 was rich in band A protein, but also contained some band D protein. Further fractionation on DEAE-Sephadex A-25 resulted in a fraction (II) showing a single band, equivalent to A, on Ferguson-Wallace gel electrophoresis. However, two bands of very similar molecular size were resolved on SDS-polyacrylamide gradient gels. Band A corresponded to band 7 on the gradient gel, indicating a Mr ca 20,000. Band C. This band is rather diffuse in FergusonWallace gel electrophoretic patterns. Protein corresponding to it was isolated by hydrophobic chromatography as outlined in Fig. 3. It was hoped in this fractionation to be able to isolate a platypus ct-lactalbumin, but only band C protein was obtained. Band C protein was shown to be equivalent to band 5 of the SDS-polyacrylamide gradient gel pattern and to have a Mr ca 43,000. Band D. The first stage of the isolation of band D protein was similar to the first stage of isolation of band A protein. The former was contained in Fraction III, which was further purified by ion exchange chromatography (Fig. 3). Band D protein was found to correspond to band 6 on SDS-polyacrylamide gradient gels. It was shown to have a M r ca 24,000. Bands F and G. Bands F and G are due to iron (III)-binding proteins, transferrins and their characterization is described elsewhere, as already indicated. The presence platypus milk

of ct-lactalbumin and lysozyme in

An attempt was made to isolate platypus lysozyme by adsorption on Heparin-Sepharose. All of the "whey" protein passed through the column unretarded. Anti-human lysozyme did not exhibit any reaction with any of the proteins on Western immunobiotting. It appears that platypus milk only contains very low levels of lysozyme, as is also

Some monotreme milk "whey" and blood proteins indicated by its weak lytic activity. The levels of lactose synthase activity in platypus milk were higher than those for echidna milk, but still much weaker than for cow milk. Attempts to isolate platypus a-lactalbumin were not completely successful. GENERAL DISCUSSION

As indicated in the Introduction a considerable amount of information has now been obtained on the composition of bovine milk, especially with respect to its proteins and the high level of genetic variation that they exhibit. Thus, the composition of bovine milk provides a suitable benchmark for comparison of the components of the milk of other mammals. Both mature echidna and platypus milk are rich in solids, echidna milk containing approximately 48.9% (w/w) solids and platypus milk 39.1% (w/w) solids (Griffiths et al., 1984). In the case of eutherian milks it has been found that the solids content of the milk is related to the suckling regimen (Ben Shaul, 1962; Jenness and Sloan, 1970). Griffiths (1968, 1978) has noted that echidna young are suckled at intervals of several days, large volumes of milk (as much as 20% of the young bodyweight) being consumed at each suckling. Given the correlation between infrequent suckling and milk high in solids, it may be inferred, in the absence of direct evidence, that platypus young also suckle at intervals of several days given the high solids content of the milk. Thus, the suckling regimen of these monotremes is distinctly different from that of the bovine. Comparing the protein content of milk of various species, both echidna and platypus milk are among the richest, being similar to rabbit, guinea-pig, rat and marsupial milk (Table 5). Furthermore, the growth rate of echidna young (0.4 g/ml milk consumed) compares favourably with the growth rate of fast-growing species such as guinea-pig (0.4 g/ml) and rabbit (0.3-0.6 g/ml) and is considerably higher than the rate of growth of slow-growing species such as cattle (0.1 g/ml) and sheep (0.13 g/ml) (Green et al., 1985; Mepham and Beck, 1973; Cowie, 1969; Yates et al., 1971; MacFarlane et al., 1969). The milk of both echidna and platypus contain caseins. In contrast to the bovine, where the casein Table 5. Averageprotein concentrationsin the milk of severalspecies* Protein g/100ml Species whole milk Cow 3.6 Goat 2.9 Sheep 5.5 Pig 4.8 Horse 2.5 Man 1.0 Rat 8.4 Guinea-pig 8.1 Rabbit 13.6 Common opossum 8.4 Brush possum 9.2 Red kangaroo ca 7 Echidna 10.7 Platypus 7.5 *Based on results of Jenness and Sloan (1970), Bergman and Housley (1968), Gross and Bolliger(1959), Lemon and Barker (1967), McLean et al. (1984). and present work.

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fraction is the major protein fraction, the "whey" protein fraction exceeds the casein fraction in the monotremes. The size of the casein micelles is considerably greater than that of the bovine. When chymosin was allowed to react with the monotrcrae casein fractions no evidence was obtained of the release of a para-K-casein fraction. These experiments were done at times in which there would be complete reaction for bovine x-casein. However, further experiments should be made in the future with much longer reaction times. Even though samples were collected at intervals over a period of 15 years, the number available was still limited. No evidence of genetic polymorphism was obtained, in contrast to other species. However, greater numbers of samples will need to be examined before any definitive conclusions can be reached. It is now known that /3-1actoglobulin, contrary to earlier views, occurs in the milk of a variety of mammals. A ~-lactoglobulin-like protein (band E protein) has been characterized in the milk of one marsupial, the grey kangaroo (GodovacZimmermann et al., 1987). In the present work the echidna "whey" protein, band E, has some characteristics of/~-lactoglobulin-like proteins. Its apparent Mr, indicating 190 residues, may be too high: it possibly has only ca 160 residues which would make it more typical (cf. the kangaroo protein). However, further work will need to be done before any final conclusions can be drawn. Bovine milk contains appreciable levels of ctlactalbumin (McLean et al., 1984), very low levels of lysozyme (White et al., 1988) and appreciable levels of transferrin and lactoferrin (Groves, 1965; Masson and Heremans, 1971). In contrast, the level of lysozyme is high in human exocrine secretions. The question of the occurrence of ~-lactalbumin in echidna milk is still not fully resolved. Hopper and McKenzie (1974) were unable to isolate an ~-Iactalbumin from the milk of the echidna. However, they found two lysozymes, I and II, from the milk of Tachyglossus aculeatus multiaculeatus and Tachyglossus aculeatus aculeatus, respectively. The occurrence of these two lysozymes has been confirmed by Teahan et al. (1991) in this laboratory. They have been isolated and their amino acid sequences determined. The ability of echidna lysozyme to bind calcium (II) has been demonstrated qualitatively (R. Tellan and D. C, Shaw, pers. commn). In view of its structure and ability to bind calcium (II) it is reasonable to assume that echidna lysozyme should be able to act as modifier protein in the lactose synthase system. However, as will be considered elsewhere, the recent preparations did not exhibit this property. Some evidence was obtained in the present work, albeit indirect, of the presence of an ~-lactalbumin or ~-lactalbumin-like protein in echidna milk. Further attempts to identify and isolate this putative protein are necessary. However, they would be facilitated considerably by the development of a more satisfactory and sensitive method for the determination of lactose synthase activity, comparable to the method recently developed in this laboratory for the determination of lytic activity. When the current studies on attempts to fractionate ~-lactalbumin were performed in the presence

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and absence of calcium (II) in elution buffers, they electrophoretic methods (see e.g. Gordon et al., 1988). were predicated on a simple interpretation of the behaviour of ~t-lactalbumin under these conditions. Recent studies by Kronman (1989) indicate a far SUMMARY more complex situation and further fractionation attempts should be planned in the light of Kronman's 1. Electrophoretic studies are made for mature work. milk "whey" proteins and blood serum proteins In contrast, the levels of lysozyme are low in the of the echidna (Tachyglossus aculeatus) and the platypus and red kangaroo (McKenzie et al., 1983). platypus (Ornithorhynchus anatinus). The milk proFurther work is needed on these lysozymes, exploittein bands are designated in terms of their order of ing the new method of determination of lytic activity. decreasing mobility in the Ferguson-Wallace semiAt the same time further studies are needed on discontinuous buffer-gel system (pH 7.7). The bands platypus 0t-lactalbumin, especially if the structural are related to those resolved in a SDS-polyacrylevoluition of lysozyme and ct-lactalbumin is to be amide gradient gel system. Some bands have counterfurther elucidated (McKenzie and White, 1991). parts in the blood serum patterns. The echidna milk Echidna milk contains only transferrin, which electrophoretic patterns are more complex than those exhibits greater heterogeneity than the transferrin of of platypus. Proteins corresponding to some of the echidna blood serum and platypus milk transferrin bands are isolated and characterized. (see also Jordon and Morgan, 1969). In contrast to 2. The echidna bands resolved in the Fergusonbovine milk, lactoferrin does not appear to be present Wallace system are designated A - M . Band A protein in milk of either monotreme. (Further studies of has an Mr ca 18,000 and 25 residues of its N-terminal monotreme transferrins are reported elsewhere by sequence have been determined. Band B protein has Teahan and McKenzie (1990).) an Mr ca 55,000 and 15 residues of its N-terminal Echidna milk band C protein is also in common sequence have been determined. Band C protein is with a corresponding protein in echidna blood, namely serum albumin. Band E protein has an Mr ca 21,000. serum albumin. In contrast, proteins of echidna bands The sequence of the first 68 N-terminal residues has A, B and E appear to be milk specific. They have been determined. It has not been possible to relate Mr values of 17,800, 55,000 and 21,400 respectively. unequivocally proteins of bands A, B and E to known Attempts to relate band B protein to other known milk milk proteins. However, band A protein has some proteins have not been successful so far. resemblance to the cystine rich proteins of mouse, rat Band A protein exhibits some degree of homology and camel milk; and band E protein to the group of with a whey acidic protein precursor from the milk of proteins that includes fl-lactoglobulins. Bands G - K the rat and mouse (Piletz et al., 1981; Hennighausen are transferrins and the patterns exhibit temporal and Sippel, 1982; Hennighausen et al., 1982). variation during lactation. A diffuse cathodic band Residues 1-24 of the echidna band A protein appear M is due to lysozyme. The possible occurrence of to have nearly 30% homology with residues 73-96 of a-lactalbumin in echidna milk is discussed. the rat protein, and 33% homology with the corre3. The electrophoretic bands of platypus milk are sponding residues of the mouse protein. A similar designated A to G. Their Mr values range from 19,000 protein to the mouse and rat proteins, which is also to 79,000. Proteins corresponding to bands A, C and rich in cystine and is a phosphoprotein, has been D have been isolated and preliminary characterizcharacterized in camel milk by Beg et al. (1986). In ation made. Bands F and G are transferrins. furlher work on band A protein attention should be 4. The occurrence of lactose synthase and lytic given to the possibility that it contains phosphoserine activities in echidna and platypus milk is discussed. residues. The occurrence of these proteins indicates that novel proteins, not evident in bovine milk, will Acknowledgements--Acknowledgements are due to the adbecome increasingly evident in other species. Although the overall electrophoretic pattern of the ministrators for the protection of fauna in the Australian platypus milk "whey" proteins is simpler than the Capital Territory, and in the States of New South Wales and South Australia for granting access for the collection of the echidna pattern, studies of their characterization are echidna samples, and to the State of New South Wales for at an earlier stage. However, at least four of them access for collection of platypus samples. Thanks are due appear to be milk specific. to the following for assistance during the course of this In future work on both echidna and platypus work: V. J. Muller, L. Mark, M. Huddy, S. Smit and proteins, considerable attention will need to be given K. McAndrew. We also thank D. C. Phillips and D. C. Shaw for their interest. One of us (Carmel Teahan) is to methods of fractionation. It has long been known, for example, that lysozyme, under a variety of con- grateful to the Australian National University for the award ditions of pH, etc. interacts with itself and other of a post-graduate research scholarship. proteins (for review see McKenzie and White, 1991). Whitaker (1963) first drew attention to its interaction REFERENCES with Sephadex column materials. Both ~t-lactalbumin and lysozyme are labile and, if the activities of the Abensperg-Traun M. (1989) Some observations on the monotreme proteins are to be sufficiently preserved to duration of lactation and movements of a Tachyglossus enable reliable quantitative determinations of these aculeatus acanthion (Monotremata: tachyglossidae) from activities, gentle methods of fractionation suitable Western Australia. Aust. Mammal. 12, 33-34. for small quantities must be used. In certain cir- Banyard M. R. C. and McKenzie H. A. (1982) The fractionation and characterization of bovine tear proteins, cumstances this will preclude the use of HPLC especially lactoferrin. Mol. cell. Biochem. 47, 115-124. methods, and recourse will be needed to the newer

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