The region of bovine a-lactalbumin which interacts with bovine colostrum galactosyltransferase in the lac- tose synthase complex has been examined by differen ...
Lactose Synthase AN INVESTIGATION OF THE INTERACTION SITE OF a-LACTALBUMIN FOR GALACTOSYLTRANSFERASE BY DIFFERENTIAL KINETIC LABELING* (Received for publication, April 24, 1979, and in revised form, December 4, 1979)
Richard H. Richardson and Keith Brew$ From the Department of Biochemistry, University of Miami, School of Medicine, Miami, Florida33101
The region of bovine a-lactalbumin which interacts review) consists of two proteins, galactosylt,ransferase and awith bovine colostrum galactosyltransferase in the lac- lactalbumin, which together catalyze the synthesis of lactose tose synthasecomplex has been examined by differen- in the lactating mammarygland (Reaction 1). tial labeling of the amino groups withacetic anhydride. UDP-D-galactose + D-glucose + lactose + UDP (1) a-Lactalbumin was acetylated in the free state and in a 1:l complex with galactosyltransferase in the pres- Galactosyltransferase isfound in a number of tissues besides ence of Mn2+,UDP-glucose, and N-acetylglucosamine the mammary gland and is an intrinsic component of the with trace amounts of high specific activity [3H]acetic membranes of the Golgi apparatus. In these other tissues, anhydride and was separated from each solution by gel galactosyltransferase catalyzes the transfer of galactose from filtration. The sample modified in the presence of ga- UDP-galactose to terminal GlcNAc’ residues in the oligosaclactosyltransferase contained 74% as much 3H label as charide moieties of glycoproteins. The enzyme can also catathat modified in the free state; both samples contained lyze the transfer of galactose to the monosaccharide GlcNAc less than 0.2 acetyl groups/protein molecule. A portion and tooligomers of GlcNAc, but glucose is an extremely poor of each sample was mixed with an aliquot of a-lactalsubstrate with a K,,, of about 2 M. @-Lactalbumin,which is bumin that had been acetylated with excess [‘4C]acetic anhydride under denaturing conditions to facilitate the produced in significant amounts only by the lactating mamdetermination of the relative reactivitiesof individual mary gland, effectively reduces theK , of galactosyltransferase amino groups through 3H/’4Cratios. The protein sam- for glucose by 3 orders of magnitude, so that lactose synthesis ples were completely acetylated with unlabeled acetic is catalyzed at physiological concentration of glucose. Kinetic anhydride, reduced, carboxymethylated, cleaved with and binding studies indicate that a-lactalbumin affects the cyanogen bromide to give the two cyanogen bromide binding of glucose to galactosyltransferase through its ability fragments (CN1 and CN2), which were further sub- to bind synergistically withthe monosaccharide to theenzyme cleaved enzymically. Radioactive peptides each con- to form1:l complexes. Thus, glucose (and other monosacchataining each of the 12 lysines and the a-amino group rides) tighten the binding of a-lactalbumin and vice versa. were purified by column chromatography and located The elucidation of the primary structures of bovine and a in the sequence by amino acid analysis. Examination of number of other a-lactalbumins has clearly established that the 3H/14Cratios from the a-lactalbumin modified in they arehomologous with lysozymes of the chicken egg white free solution revealed a range of reactivities of the type (3).However, little resemblance can be seen in functional amino groups, which are related to theirenvironments properties of a-lactalbumins and lysozymes, a-lactalbumins in previously proposed models for the structureof the having no catalytic activity, and lysozymes having no ability protein. By comparison with this control sample, the to affect monosaccharide binding with galactosyltransferase. protein modified in thecomplex showed major perturDespite a considerable number of previous chemical modibations in reactivity in only two groups, lysine 5, which fication studies (see Ref. 2 ) , little specific information about was reduced in reactivity by a factor of 3, and lysine 114, which was increased in reactivity by a factorof 2. the nature of functional areas in the a-lactalbumin molecule The amino groups of these residues are proximally has been obtained. Such information is needed to develop an situated in an areaof the a-lactalbuminmolecule neigh- understanding of its regulatory effects on galactosyltransferboring the region corresponding to one end of the active ase and of the structural basis of its functional differences site cleft region in the homologous lysozymes. The ori- from lysozyme. We report here the results of an alternative chemical apgin of the differential reactivities and their relevance to the structure,function, and evolution of a-lactalbu- proach to theidentification of functional areas in a-lactalbumin based on a procedure developed by Kaplan et al. (4) and min are discussed. Bosshard et al. ( 5 ) .In this, a-lactalbuminin free solution and in a complex with galactosyltransferase was modified with trace amounts of [“Hlacetic anhydride. By determining the The lactose synthaseenzyme system (see Refs. 1 and 2 for extent of labeling of the individual amino groups of a-lactal* This work was supported by Grant GM 21363 from the National bumin (a-NH2 and 12 lysyl €-amino groups) under these two Institute of General Medical Sciences, National Institutes of Health. conditions, two NH2 groups whose reactivities are perturbed The costs of publication of this article were defrayed in part by the on complex formation have been identified. These appear to be hereby payment of pagecharges. Thisarticlemusttherefore marked “aduertisement” in accordance with 18 U.S.C. Section 1734 be in close proximity in the three-dimensional structureof ulactalbumin. The results suggest that a region in a-lactalbumin solely to indicate this fact. corresponding to partof the active site cleft region in lysozyme $Recipient of Research Career Development Award No. KO4 GM00147 from the National Institute of General Medical Sciences, National Institutes of Health.
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The abbreviation used is: GlcNAc, N-acesylglucosamine.
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Interaction a-Lactalbumin ofSite
may function in interacting with galactosyltransferase and monosaccharides. EXPERIMENTAL PROCEDURES'
Materials-Bovine a-lactalbumin and bovine colostrum galactosyltransferase were prepared as described previously (9). Galactosyltransferase was estimated by measurement of activity using a specific activity of 18 units/mg (6). ['HIAcetic anhydride (5.3 Ci/mmol) was purchased from Amersham/Searle and [l-"C]acetic anhydride (8.6 mCi/mmol) from New England Nuclear. Cyanogen bromide and pyridine were the products of J. T. Baker, iodoacetic acid was the product of Aldrich, and N acetyl-D-glucosamine and 2,5-diphenyloxazole (PPO) wereSigma products. Triton X-100, 1,4-bis[2-(5-phenyloxazolyl)]benzene(POPOP), and toluene were obtained from Research Products International. Reagents for manual Edman degradation (phenylisothiocyanate and anhydrous heptafluorobutyric acid, both of Sequanal grade) were purchased from Pierce. Thermolysin ( A grade) was purchased from Calbiochem and trypsin-TPCK (L-1-tosylamido-2-phenylethyl chloromethyl ketone) (228 units/mg) and a-chymotrypsin (65 units/mg) from Worthington. Aminex A-5 of particle size 13 f 2 p was purchased from Bio-Rad Laboratories and Partisil-10 ODS-2 from Reeve Angel. " H Acetylation of a-Lactalbumin in Free Solutionand in a Complex with Galactosyltransferase-Two identical reaction mixtures were prepared except for the presence of galactosyltransferase (2.80 mg; 56 nmol; 2 p ~ in) one. Both solutions contained 0.795mg (56 nniol; 2 p ~ of) bovine a-lactalbumin, 0.3 mM UDP-Glc, 1 mM MnClr, and 0.2 M NaCl in 0.02 M Tris-HCl buffer, pH 8.0. The total volume of each solution was 28 ml. T o each was added ["Hlacetic anhydride (1.23 pmol, 8.3mCi) in 110 pl of dry, freshly distilled acetonitrile. The modification was performed at 0°C and the reaction was left for 4 h to complete. "H-acetylated a-lactalbumin was isolated from the mixture by gel filtration. Each reaction mixture was applied, in two separate lots (14.0 ml), to columns (2.5 X 90 cm) of Bio-Gel P-60 equilibrated and eluted with 0.1 M NH.,HCO,Ia t 4°C. Fractions of 7.5 ml were collected and aliquots of 10 pl were taken for scintillation counting (see Fig. 1). The combined fractions were freeze-dried and stored at -20'C. Determination of Radioactiuity-"H/'JC ratios for individual lysine residues were determined by counting an aliquotof the purified peptide in 7.5 ml of scintillation fluid and a total of 0.75 ml of water. Samples containing pyridine werefirst dried in a desiccator with PzOs and redissolved in H,O. The overlap of counts in the 'H channel was 33R, whereas there was no overlap of "H into I4C. Counts were corrected for this overlapbeforecalculation of ratios. Ratios for residues from a-lactalbumin modified in free solution are denoted by r,, whereas ratios for a-lactalbumin modified in complex with galactosyltransferase are denoted by r,. A protection factor, R, is defined by R = r,/r
