Feline whey proteins: Identification, isolation and initial characterization of α-lactalbumin, β-lactoglobulin and lysozyme

Cornp. Biochem. Physiol. Vol.95B, No. 4, pp. 773-779, 1990 Printed in Great Britain

0305-0491/90$3.00+ 0.00 © 1990PergamonPress plc

FELINE WHEY PROTEINS: IDENTIFICATION, ISOLATION A N D INITIAL CHARACTERIZATION OF ~-LACTALBUMIN, fl-LACTOGLOBULIN A N D LYSOZYME JUDY A. HALLIDAY,* KEVIN BELL,* HUGH A. MCKENZIEt~ and DENISC. SHAW* *Department of Physiologyand Pharmacology, University of Queensland, St Lucia, Brisbane, QLD 4067, Australia (Tel: 07 377 3132); and tProtein BiochemistryGroup, John Curtin School of Medical Research, Australian National University, Canberra, A.C.T. 2601, Australia (Received 25 August 1989) Abstract--l. Both ~-lactalbumin and fl-lactoglobulin-likeproteins were detected in the whey fraction of

feline milk by immunoblotting with rabbit antisera to ct-lactalbumin and fl-lactoglobulin, respectively. 2. ~-Lactalbumin was found to occur in both glycosylatedand unglycosylatedforms in approximately equal concentrations. No polymorphism of feline ct-lactalbumin was found. 3. Feline fl-lactoglobulin-like proteins produced complex electrophoretic patterns that appear to be determined by three distinct loci. Between two and five genetic variants are expressed by each locus. 4. Lysozyme was detected at levels of approximately 1 mg/ml in skim milk. 5. The identifications of the proteins as ~t-lactalbumin,fl-lactoglobulin and lysozyme were confirmed by determination of N-terminal amino acid sequences.

INTRODUCTION Considerable information is available on the whey proteins of many eutherian mammals, in particular ruminants (McKenzie, 1971). In recent years the milk of metatherian and prototherian mammals has also been studied (McKenzie, 1971; Hopper and McKenzie, 1974; Bell et al., 1980; McKenzie et al., 1983). However, very little work has been done on the milk proteins of carnivorous mammals (Nagasawa et al., 1972; Swanson and Sanders, 1974; Lfnnerdal et al., 1981; Keen et aL, 1982). Most studies on carnivores have been concerned with changes in protein, fat and carbohydrate composition of the milk during the course of lactation. A/3-1actoglobulin-likeprotein has been described in the whey fraction of canine milk (Nagasawa et al., 1972; Pervaiz and Brew, 1986) but feline milk proteins have not been characterized prior to the present study. Our aim is to describe the occurrence of the whey proteins of feline milk, their isolation and initial characterization and in particular to determine whether or not there are counterparts of ~-lactalbumin and fl-lactoglobulin in the milk of this species. ct-Lactalbumin has been found in the milk of most mammals (McKenzie, 1971). Ebner et aL (1966) showed that it is an essential regulatory protein that modifies the function of galactosyltransferase (E.C. 2.4.1.38) to that of lactose synthase. Lysozyme is invariably present in milk but the concentration varies markedly between species (White et aL, 1988). It was first suggested by Brew and Campbell (1967) that ct-lactalbumin and lysozyme arose from a common ancestor. The sequence homology of the two proteins :~Present address: Chemistry Department, University College, University of New South Wales, Australian Defence Forces Academy, Canberra A.C.T. 2600, Australia.

was confirmed by Brew et al. (1967) and subsequently it was established that the ct-lactalbumin gene probably arose as a result of a duplication of the lysozyme gene (Hall et al., 1982; Qasba and Safaya, 1984). Although the primary structures of fl-lactoglobulin from several species have been studied and the threedimensional structures of two forms of the bovine protein have been determined at medium resolution, its function has not been conclusively established. Its ability to bind hydrophobic molecules has long been established (Wishnia and Pinder, 1966). The binding of retinol by bovine fl-lactoglobulin (Fugate and Song, 1980), the similarity of the polypeptide chain folding of fl-lactoglobulin and retinol binding protein (Sawyer et al., 1985; Papiz et al., 1986), and their amino acid sequence homology, (GodovacZimmermann et al., 1985a; Pervaiz and Brew, 1985) are suggestive of a possible biological function for fl-lactoglobulin. In addition, it has been shown that I ~25 fl-lactoglobulin-retinol complexes bind specifically to cultured intestinal microvilli from week-old calves, but the binding is dependent on pH and ionic strength. As already indicated fl-lactoglobulin binds a variety of hydrophobic substances in vitro and significant amounts of free fatty acids and triglycetides under physiological conditions (Diaz de Villegas et al., 1987). MATERIALSAND METHODS Individual milk samples were obtained from 266 cats by hand milking followingthe intravenous injection of oxytocin (0.5 ml) to facilitate "letdown". In addition five cats were milked at intervals during the course of lactation. The milk was centrifuged (3000 rpm, 10°C, 10 min), the fat removed by suction and the skim milk stored at - 20°C until required for electrophoresis or isolation of individual proteins. The skim milk was diluted 1:3 v:v with distilled water prior to electrophoresis. Samples were analysed using four

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etectrophoretic systems at a constant running temperature of 10~'C:(i) isoelectric focusing (IEF) in the pH range 4.(~6.0; (ii) one-dimensional polyacrylamide gel electrophoresis (I DPAGE), pH 8.3, 13.5%T; (iii) linear gradient PAGE, pH 8.3, 10-20%T; and (iv) two-dimensional electrophoresis (2D-PAGE) incorporating IEF pH 4.0-6.0 in the first dimension and linear gradient PAGE in the second dimension. Acrylamide and bisacrylamide were BDH "Electran" grade. IEF was performed in ultrathin horizontal slab gels according to Patterson and Bell (1986). Diluted skim milk samples were applied on Whatman 3MM inserts 1.5cm from the anode for 20 rain. The conditions of electrophoresis were 20rain prefocus, 20min insert time and 90rain focusing time, at 12W constant power. The isoelectric points (pI) of the whey proteins were determined using the Pharmacia low pI calibration kit as described by Patterson and Bell (1986). The 13.5%T ID-PAGE and 10-20%T gradient 2D-PAGE gels were prepared as described by Pollitt and Bell (1983) except that the gel buffer was 9.38 mM Tris-17.8mM H2SO4, pH 8.3. For the 13.5%T 1D-PAGE, the diluted skimmed milk samples were applied on Whatman 3MM for 5 min at a constant current of 42 mA and the gels were run at a constant power of 60W for 210 min. 2D-PAGE was performed under the same conditions with the IEF first dimension strips being applied until the borate boundary passed through the strip. Gels were stained with Coomassie Brilliant Blue G250 in perchloric acid for 30 rain and destained in 5% acetic acid overnight (Holbrook and Leaver, 1976). Whey was prepared by acid precipitation of the casein by adjusting the pH of the milk to 4.2 with IM HCI, the precipitate formed was removed by centrifugation (10,000 g, 10 min, 10'~C) and the pH of the whey supernatant was adjusted to pH 7.0 with 1 M ammonia solution. The whey was dialysed against several changes of distilled water. Purified whey proteins were obtained by reverse phase high-performance liquid chromatography (RP-HPLC). A Brownlee Aquapre RP300 Cs (4.6 x 30 mm) column and a gradient of 0.1% trifluoroacetic acid (TFA)/acetonitrile (solvent B) in 0.1% TFA/water (solvent A) was used to separate the whey proteins. The gradient was 2 0 ~ 0 % B over 40 rain at a flow rate of 1 ml/min. Protein peaks were detected at 214 nm using a Waters detector (Model 441). Solvents were pumped by Waters pumps (Models M45 and 6000A) and the gradient was controlled by a Waters programmable gradient controller (Model 640). The relative molecular mass (Mr) of the individual whey proteins was determined by sodium dodecyl sulphate (SDS) electrophoresis in a Bio-Rad mini gel apparatus using the Laemmli (1970) buffer system. Purified whey proteins isolated by RP-HPLC were separated by electrophoresis along side Pharmacia low molecular weight standards and the M r of each whey protein was calculated from a standard curve. Proteins from 2D-PAGE (both stained and unstained) were electroblotted onto nitrocellulose in a Bio-Rad TransBlot apparatus (Model 250/2.5) at 70V for 10-15 rain in 10mM NaHCO3-3mM Na2CO 3 buffer pH 9.9 (Dunn, 1986). Immunoprobing of the membranes was performed according to the standard method outlined in the Bio-Rad protocol, using the following antisera raised in rabbits: anti-human ct-lactalbumin (Sigma) 1 : 200; anti-bovine fl-lactoglobulin (Nordic) l : 400; anti-porcine ct-lactalbumin 1 : 50; and anti-equine ~-lactoglobulin I 1:50. The latter two antisera were raised in rabbits by courses of four weekly intramuscular injections of 10 mg of the appropriate protein and incorporation of 0.5 ml of Freund's incomplete adjuvant in the first injection. One week after the final injections the rabbits were bled from the central ear artery and the sera used in blotting experiments without further purification. All blots were incubated with a second antibody (alkaline phosphatase conjugated goat anti-rabbit IgG) and positive cross reactions were identified as purple spots after development of an alkaline phosphatase reaction.

In preparation for amino acid sequencing native proteins were electroblotted from the 2D-PAGE patterns on to either polybrene-treated glass fibre (GF/C) (Vandekerckhove et al., 1985) or polyvinylidene difluoride (PVDF) (Matsudaira, 1987; Moos et al., 1988). Individual proteins were located by fluorescamine staining, excised from the sheets and placed directly into the reaction chamber of an Applied Biosystems pulsed liquid phase sequencer, Model 477A. A standard TFA-treated GF/C disc was positioned under the excised blotted protein and wet with polybrene prior to sequencing (Board et al., 1988). Sequencing was performed using Edman chemistry and the phenylthiohydantoin derivatives were detected by an on-line Applied Biosystems HPLC Model 120A. Feline lysozyme was obtained by preparative IEF in a Bio-Rad Rotophor apparatus using "buffalytes" pH 4--8 (Pierce) to generate the pH gradient. As lysozyme has a basic pI, those fractions at high pH were pooled. The lower molecular weight lysozyme was separated from contaminants such as transferrin and lactoferrin by ultrafiltration through a 30 kDa cut-off membrane (Amieon). Lysozyme was electrophoretically pure when analysed by SDS electrophoresis. Lysozymal activity of the pure sample and skim milk was determined by measuring the decrease in turbidity of a Microcoecus lysodeikticus cell suspension with time at 450 nm (McKenzie and White, 1986). Lactose synthase activity was determined using t4C UDP galactose (Amersham) and bovine galactosyl transferase (Sigma) in a reaction with feline milk as the source of ~-lactalbumin (Fitzgerald et al., 1970). RESULTS

Separation of feline skim milk by p H 8.3, 13.5%T, horizontal P A G E resolved the non-casein (whey) proteins into four m a j o r zones. These proteins were easily detected by Coomassie Brilliant Blue G250 staining a n d were designated A - D , with zone A proteins h a v i n g the fastest mobilities to the a n o d e and zone D the slowest (Fig. 1). Zone D was identified as serum a l b u m i n by electrophoretic a n d immunological criteria. Zones A, B a n d C migrated anodally well clear o f serum a l b u m i n a n d this was consistent with their being milk-specific whey proteins. A m i n i m u m acrylamide c o n c e n t r a t i o n of 13.5%T was necessary to ensure t h a t the most anodally migrating group of proteins (zone A) was separated from the borate b o u n d a r y . E x a m i n a t i o n o f milk samples from different cats was u n d e r t a k e n to unravel the complexity of the patterns. Careful e x a m i n a t i o n of the zone A proteins with respect to migration, occurrence a n d concentration indicated t h a t zone A was comprised of three groups of proteins, designated 1, 2 a n d 3 in order of decreasing mobility towards the a n o d e (Fig. 1). The patterns were quite complex with group 1 having a m i n i m u m of five variants with one of the variants migrating in the same region as the less anodal group 2 variants. Similarly, group 2 proteins, which appeared to consist of a m i n i m u m of five variants, also h a d a variant which migrated within the group 1 region. In addition, several of the variants within these two groups tended to comigrate u n d e r these conditions, m a k i n g identification difficult. The migrations o f group 3 proteins overlapped with those o f group 2 b u t not to the p o i n t of confusing the typing of these two groups. F o r example, group 3 was c o m p o s e d of two variants with group 2 variants migrating between t h e m (Fig. 1).

Feline whey proteins zone

4-

J,

Fig. 1. 13.5%T, pH 8.3, PAGE patterns of nine individual feline skim milks stained for protein and demonstrating the separation into four major zones (A D) and the marked heterogeneity of proteins in zone A. Note the overlapping of systems 1, 2 and 3.

While the 13.5%T PAGE method resulted in excellent separation of the whey proteins of feline milk, the limitations described above made it unsuitable for reliable typing of all samples or for the comparative analysis of samples from different individuals. Isoelectric focusing was adopted as a more suitable alternative as variants with pls differing by as little as 0.01 pH units could be reliably separated and typed.

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IEF, pH 4.0-6.0, separated the proteins into five zones, I-V (Fig. 2a and b). Zone V proteins had high isoelectric points and focused against the cathode. The major protein in this zone was identified as lysozyme by N-terminal sequencing studies and lysozyme activity determinations. Zone IV proteins occurred as a single band with a pI of 4.66 in all samples (Fig. 2). However, this single band was resolved into three proteins with M r values of 20,000, 19,000 and 14,000 (Fig. 3 IV1, IV 2 and IV3, respectively) using 2D-PAGE. These are also equivalent to the bands in zones B and C in Fig. 1. No genetic variants of these proteins have been detected. Zones I, II and III formed complex patterns and exhibited marked variation between samples (Fig. 2). These are the same proteins as the zone A, 1-3 proteins depicted in Fig. 1. Five variants, designated IA--IE in order of increasing mobility to the cathode, were observed in zone I. These proteins had pls ranging from 4.48 (IA) to 4.67 (IE). Variants IA and IB differed by only 0.05 pH unit but could still be consistently separated (Fig. 2a, lane 6). Variants IA_D migrated anodally to zone IV while I E appeared as a rather diffuse band cathodal to zone IV. Zone II was found to be composed of five variants, designated IIA_E in order of increasing pls. The pls varied from 4.78 (IIA) to 5.07 (liE). In zone III only two variants, I l i A and III c, with pls of 4.78 and 5.01, were observed. Despite the fact that the proteins of zones II and III overlapped, individual protein bands in both systems were easily distinguishable. Under these IEF conditions serum albumin appeared as a very diffuse band underlying zone IV. The electrophoretic profiles of the whey from five cats were studied at intervals throughout lactation. The patterns for individual cats remained qualitatively identical throughout lactation (results not shown).

+

1

a)

2

3

4

5

6

7

8

b) Fig. 2. (a) IEF, pH 4q5, patterns of eight individual feline skim milk samples stained for protein illustrating the separation into five zones, designated I-V (zone V not shown). All observed variants in zones I, II and III are shown with the typings of samples 1-8 being IDD Ilco IIIcc; InD IIAElilac; Icc IIBEIIIAA;IBD IIEE IIIAA;IOO Ilcc IIIcc; lAB IIDE IIIAA;IBE IIDE IIIAA;IAC IIBc IIIAA.(b) A schematic representation of all observed variants from zones I (stippling), 1I (ruling), III (filled) and IV (open).

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+

IEF .......................................................

-10%

ii ¸ Ill

20%

-

"l" Fig. 3. Two-dimensional (IEF, pH 4.0~.0, 10 20%T PAGE, pH 8.3) protein-stained pattern of a single milk sample of type Ico llce lilac. Note the heterogeneity of the zone IV proteins. The three proteins, IVy, IV2 and IV3, exhibit apparent M r differences but no charge shift. Immunoblotting techniques were used in an attempt to identify the zone I, II, III and IV proteins. Following 2D-PAGE of skim milk samples possessing all recognized variants of zones I - I I I (Fig. 3), the patterns were electroblotted onto nitrocellulose. The blots were probed with rabbit antisera to bovine and equine I fl-lactoglobulins and porcine and human

-lactalbumins. Rabbit antiserum to human ~-lactalbumin showed a strong positive cross reaction with all three proteins (IV~ 3), both native and denatured, from zone IV. This indicated that all three proteins which had the same pI (4.66) are ~-lactalbumin-like proteins. A less strong but nevertheless positive cross reaction was obtained with the antiserum to porcine -lactalbumin. Similarly all variants from zones I, II and III, both native and denatured, reacted positively with rabbit antisera to bovine and equine I fl-lactoglobulins, indicating that all three groups are fl-lactoglobulinlike proteins. The relative molecular masses of zone I, II and III proteins were determined to be in the range 18,000-20,000, providing further evidence for the identification of these proteins as fl-lactoglobulinlike proteins. At this stage it has not been possible to obtain family data to determine the inheritance of zone I, II and III proteins. In each of the postulated zones, the variants occurred singly or in pairs with both bands in approximately equal concentrations. This suggests that the genes controlling the fl-lactoglobulin-like proteins within each zone are codominant alleles. The phenotypic and genotypic frequencies of all three fl-lactoglobulin systems were calculated assuming a codominant allelic system of inheritance for each system. These are presented in Table 1 with the most frequent alleles for fl-lactoglobulin I, II and III being D (0.444), E (0.492) and A (0.761), respectively. The N-terminal sequences of proteins from zones I (Io), II (Ilo), III (IliA), IV and V are detailed in Table 2 with those of bovine A and equine I fl-lactoglobulins, human and equine A c~-lactalbumins and human, equine and bovine milk lysozymes. Zones I D, II o and III A showed appreciable homology with the bovine and equine fl-lactoglobulin proteins, confirming that the three groups (I, II and III) of proteins are fl-lactoglobulin-like. Feline [3-1actoglobulins I and III had 23 of the first 24 residues in common; the only variation was at position 18 with Met in I and Thr in III. However fl-lactoglobulin II proteins were quite different, having only 16 or 17 residues in common with the other feline fl-lactoglobulin. All three fllactoglobulin types had a Met at position 7 and Val-Ala-Gly at positions 15-17, which is characteristic of fi-lactoglobulins except for kangaroo fllactoglobulin-like protein (Godovac-Zimmermann

Table 1. Phenotypic and genotypic frequencies of the feline fl-lactoglobulin proteins fl-Lactoglobulin 1 frequencies Phenotypic Genotypic

fl-Lactoglobulin II frequencies Phenotypic Genotypic

A AB AC AD AE B BC BD BE C CD CE D DE E

A AB AC AD AE B BC BD BE C CD CE D DE E

0.023 0.015 0.026 0.053 0.008 0.083 0.086 0.158 0.004 0.116 0.162 0.004 0.252 0.011 0.000

A 0.073 B 0.214 C 0.256 D 0.444 E 0.013

Number of milk samples: 266.

0.000 0.000 0.008 0.011 0.053 0.008 0.019 0.022 0.034 0.049 0.083 0.241 0.049 0.192 0.233

A 0.036 B 0.045 C 0.224 D 0.203 E 0.492

fl-Lactoglobulin III frequencies Phenotypic Genotypic A AC C

0.594 0.334 0.071

A C

0.761 0.239

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Table 2. N-terminal sequencesof feline wheyproteins from zones I-IV 1

5

10

fl-Lactoglobulins Zone I D ZoneIID Zone IIIA *BovineA tEquineI

A A A L T

T T T I N

V L V V I

P P P T P

L P L Q Q

T T T T T

-Lactalbumins Zone IV :~Bo~ineA §Human IJEqt/ineA

K E K K

Q Q Q Q

F L F F

T T T T

K K K K

Lysozymes Feline milk ¶ H u m a n milk **Equine milk ttBovine milk

K K K K

I V V K

F F F F

T E S Q

K R K R

15

20

M D G M E D M D G M K G M Q D

L L L L L

D D D D D

L I L I L

Q R Q Q Q

K Q K K E

V V V V V

A G M W A G T W A G T W A G T W A G K W

H H H Y H

S M A M S M A M S M A M S L A M S V A M

(C) C C C

E E E E

L V L L

S F S S

Q Q Q Q

V E L V

L L L L

K K K K

D D D S

M L 1 M

D K D D

G G G G

I A V S I A V T

(C) C C C

E E E E

L L L L

A A A A

R R H R

K T K T

L L L L

R A K R K A K K

E L Q L

G M D G M D E M D G L D

G G G G

Y G Y G Y G Y K G G G G

L L L L

P P P P

F Y F Y

( ) Cys is assumed as no PTH derivatives were observed for this residue. *Braunitzer et al. (1973). tGodovac-Zimmermann et al. (1985b). :~Brew et al. (1970). §Findlay and Brew (1972). IIKaminogawa et al. (1984). ']Joll6s and Joll~s (1972). **McKenzie and Shaw (1985). ttWhite et al. (1988).

and Shaw, 1987). fl-Lactoglobulin I showed 63% positional identity with both bovine fl-lactoglobulin A and equine fl-lactoglobulin I in the first 24 residues while fl-lactoglobulin II showed 54% positional identity with bovine A and 58% with equine I fl-lactoglobulins. In all the feline fl-lactoglobulins residue 20 was found to be His which is also found in the equine and porcine fl-lactoglobulins (GodovacZimmermann et al., 1987). Feline c~-lactalbumin of M r 14,000 (zone IVa) showed 92% positional homology with human ~-lactalbumin in the first 24 residues (Table 2). There was also 63 and 83% positional homology between the feline protein and bovine A and equine A ct-lactalbumins, respectively. Preliminary lactose synthase activity determinations of a mixture of all three zone IV proteins showed that the protein, identified as ~-lactalbumin, could act as a specifier in the lactose synthase system. Lysozyme which was isolated from zone V showed appreciable homology with human, equine and bovine milk lysozymes, with 13 (human), 14 (equine) or II (bovine) residues out of the first 20 being conserved. Feline lysozyme was determined to occur in a concentration of approximately 1 mg/ml in feline skim milk, assuming that the specific activities of feline lysozyme and hen egg white lysozyme are equivalent. Optimum feline milk lysozyme activity occurred between pH 7.0 and 7.4 in imidazole-HCl buffer (ionic strength 0.05). DISCUSSION

The major whey proteins in feline milk have been identified as fl-lactoglobulins by immunoblotting with specific antisera and N-terminal sequence studies. This group of proteins has been found to be genetically heterogenous in several species including cattle, sheep, pig and dolphin (Bell, 1962; Bell and McKenzie, 1967; Kessler and Brew, 1970; Pervaiz and Brew, 1985). However the high degree of heterogeneity found in feline fl-lactoglobulins is unique. Despite initial sequence studies being limited to Nterminal analyses of only the most common variants of feline fl-lactoglobulin, several distinctive features

have emerged. It appears that the feline fl-lactoglobulins are the products of three separate loci. Proteins from the feline fl-lactoglobulin II locus have eight residues out of the first 24 that are different from those of I. All genetic variants sequenced prior to equine fl-lactoglobulin II could be accounted for by assuming single-point mutations which changed up to 1.2% of the total chain residues. It has been shown that the two equine fl-lactoglobulins have 70% homology and that 23% of the exchanged residues can only be accounted for by two-point mutations (Godovac-Zimmermann et al., 1985b). Our N-terminal sequence data suggest that a similar situation occurs in feline milk with the existence of at least two fl-lactoglobulin loci and allelic variants at each locus occurring possibly as a result of several single-point mutation amino acid substitutions. The electrophoretic patterns indicated that in fact three loci exist with allelic variation at each locus. Histidine is present at position 20 in all the feline fl-lactoglobulins. Equine and porcine fl-lactoglobulins appear to have His at position 20, and electrophoretic mobilities of the native feline fl-lactoglobulins in gels indicate that they are monomeric. It has been suggested that primary sequence similarities between fl-lactoglobulin and plasma retinol binding proteins may be significant in defining a function for fl-lactoglobulins (Pervaiz and Brew, 1985; Godovac-Zimmermann et al., 1985a). Fugate and Song (1980) demonstrated that fl-lactoglobulin could bind retinol in vitro and suggested that the Trp residue at position 19 may be a binding site for the fl-ionone moiety of retinol. The Trp residue at position 19 is conserved in the feline fl-lactoglobulin and in all other fl-lactoglobulins sequenced to date (Godovac-Zimmermann et al., 1987; North, 1989). Studies of the primary structure of the feline fl-lactoglobulins will aid in the characterization of fl-lactoglobulins and may be useful in elucidating the biological function and genetic evolution of this group of proteins. There has been a recent report of a human placental protein that shows 55% positional identity with horse fl-lactoglobulin I in the primary amino acid sequence (Julkunen et al., 1988). The nucleotide sequence of the placental protein was

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found to be similar to that of sheep fl-lactoglobulin including a portion of D N A that codes for an 18-amino-acid signal peptide. These findings are particularly interesting as fl-lactoglobulin is not expressed in human milk. The occurrence of the three types of/3-1actoglobulins in feline milk is intriguing as it is possible that the three different feline /3lactoglobulins may bind different ligands. Further sequence studies of the feline fl-lactoglobulins are in progress. The presence of ~-lactalbumin was detected by immunoblotting with rabbit antiserum to human and porcine ~-lactalbumins. Only one genetic variant of ~-lactalbumin has been observed in feline milk although electrophoretic analysis of this variant indicated that two of the three protein bands were glycosylated (Figs 2 and 3). As no charge shift was observed in the electrophoretic separation of glycosylated feline ~-lactalbumin, it is likely that the carbohydrate side chain contains neutral sugars and not sialic acid. Glycosylation of ~-lactalbumin has been described in the rat (Prasad et al., 1979), rabbit (Hopp and Woods, 1979), cow (Brew et al., 1970) and goat (Brew, 1972). The required sequence for carbohydrate binding (Asn-X-Ser/Thr) is present at position 45 in all these ~-lactalbumins. The extent of glycosylation varies, with rat and rabbit ~-lactalbumin being fully glycosylated and bovine and caprid being partially glycosylated. The differing degrees of glycosylation are thought to be influenced by the residue at position 46 which is glycine in rat and rabbit ~-lactalbumin but is aspartic acid in bovine and caprid ~-lactalbumin (Hopp and Woods, 1979; Shewal et al., 1984). The N-terminal sequence of feline ~-lactalbumin showed 92 and 63% positional homology with human and bovine ~-lactalbumin, respectively. The structurally important Cys at position 6 is conserved within the first 24 residues. The modifier action of -lactalbumin is believed to be mediated by a hydrophobic interaction between the two molecules, and among the residues which have been suggested are Trp-60, Tyr-103, Ile-95 and Trp-104 (Mitranic et al., 1988). These residues have not yet been determined for the feline protein. Feline ~-lactalbumin shows 61% homology with feline milk lysozyme in the first 18 residues (allowing for the deletion of two residues in ~-lactalbumin relative to lysozyme; Table 2). Acknowledgements--This work was carried out while one of us (J.H.) was the recipient of a research scholarship provided by the Australian Stud Book, Alison Road, Randwick, New South Wales 2031, Australia.

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