Isolation and characteristics of galactosyltransferase from Golgi membranes of lactating sheep mammary glands.

The molecular and enzymic properties of galactosyltransferase from Golgi membranes isolated from homogenates of sheep mammary Golgi membranes have been investigated. The enzyme appears to be an intrinsic membrane component, not being solubilized by extraction with 0.1 or 1 M NaCl or with EDTA. Many solubilization procedures produce inhibition of the enzyme, whereas low concentrations (1%)) of Triton X-100 produce a stimulation of activity together with complete solubilization of the enzyme. The Triton-solubilized enzyme was purified by a combination of gel filtration and affinity chromatography, to give a product with a specific activity similar to those of previously characterized soluble galactosyltransferases. On polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate the purified enzyme showed two protein components, a major glycoprotein component of apparent molecular weight 69,UOO and a minor diffuse component of apparent molecular weight 53,000. Gel filtration of the enzyme directly solubilized from Golgi membranes on calibrated columns of Bio-Gel P-150 gave a major activity peak at M, = 65,000 and a shoulder at M,. = 55,999. It is concluded that the galactosyltransferase of Golgi mc,mbranes is larger by 10 to 13 x 19’ daltons than the most intact (M, = 50,000) of previously studied galactosyltransferases. In contrast, the enzymic properties of the Gnlgi membrane enzyme, assayed in the presence of Triton X-100, closely resemble those of soluble galactosyltransferases. Finally, we suggest that soluble galactosyltransferases may be produced by proteolytic cleavage of the membrane enzyme during membrane turnover in secreting tissues.

The molecular and enzymic properties of galactosyltransferase from Golgi membranes isolated from homogenates of sheep mammary Golgi membranes have been investigated.
The enzyme appears to be an intrinsic membrane component, not being solubilized by extraction with 0.1 or 1 M NaCl or with EDTA. Many solubilization procedures produce inhibition of the enzyme, whereas low concentrations (1%)) of Triton X-100 produce a stimulation of activity together with complete solubilization of the enzyme. The Triton-solubilized enzyme was purified by a combination of gel filtration and affinity chromatography, to give a product with a specific activity similar to those of previously characterized soluble galactosyltransferases.
On polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate the purified enzyme showed two protein components, a major glycoprotein component of apparent molecular weight 69,UOO and a minor diffuse component of apparent molecular weight 53,000. Gel filtration of the enzyme directly solubilized from Golgi membranes on calibrated columns of Bio-Gel P-150 gave a major activity peak at M, = 65,000 and a shoulder at M,. = 55,999. It is concluded that the galactosyltransferase of Golgi mc,mbranes is larger by 10 to 13 x 19' daltons than the most intact (M, = 50,000) of previously studied galactosyltransferases.
In contrast, the enzymic properties of the Gnlgi membrane enzyme, assayed in the presence of Triton X-100, closely resemble those of soluble galactosyltransferases.
Finally, we suggest that soluble galactosyltransferases may be produced by proteolytic cleavage of the membrane enzyme during membrane turnover in secreting tissues.
Galactosyltransferase (UDP-galactose, N-acetylglucosamine P-Cgalactosyltransferase) is commonly used as an enzymic marker for Golgi membranes from a number of cell types (l-3). In most tissues, the galactosyltransferase func-* This is a report of work initiated at the University of Leeds (U. K.) where it was supported by a studentship to C.A.S. from the Science Research Council, and continued at the University of Miami, supported by Grant GM 21363 from the National Institutes of Health.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked "'uduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. tions in catalyzing the synthesis of a galactosyl P-4-GlcNAc' linkage that is present in the oligosaccharide moieties of many secreted glycoproteins (4, 5); in the lactating mammary gland galactosyltransferase acts as the catalytic component of lactose synthase (EC 2.4.1.22), its monosaccharide binding properties being modified through interaction with the regulatory protein u-lactalbumin so that glucose, instead of being a poor acceptor substrate with a K,,, of 1 to 2 M, becomes a good substrate with a K,,, of about 1 mM (see Fiefs. 6 and 7).
The molecular and enzymic characteristics of galactosyltransferase have been extensively studied with soluble forms of the enzyme (M, = 40,000 to 50,000) that are found in secretions, principally milk and colostrum, although the enzyme form that functions in uiuo is membrane-bound (see Refs. 6 and 7). We have therefore undertaken a study of the galactosyltransferase present in Golgi membranes to determine the conditions under which it is solubilized, to ascertain its molecular size, and to compare its catalytic properties with those previously determined for naturally soluble forms of the enzyme. The results reported here indicate that the enzyme is an intrinsic membrane component, being solubilized partially by organic solvents and quantitatively by detergent (1% Triton X-1001, but not by high or low eoncentrations of sodium chloride nor by EDTA. The detergent-solubilized enzyme has been purified by gel filtration and affinity chromatography. Investigation of its molecular size by gel filtration and SDS-polyacrylamide gel electrophoresis indicates that it is larger by 13 to 15 x l@' daltons than the most intact naturally soluble galactosyltransferase (the colostrum enzyme), but, in the presence of low concentrations of detergent, has kinetic and regulatory properties closely similar to those of the soluble galactosyltransferases.

MATERIALS AND METHODS
The sources of most material were reported in previous publications from this laboratory (8-10). UDP/Sepharose and cr-lactalbumin/Sepharose conjugates were prepared as described previously (8  Galactosyttransferase from Golgi Membranes otherwise quantitatively sedimented. The procedures were also tested for effect on enzyme activity. Subsequently, the only satisfactory procedure by this criterion was subjected to a more rigorous test of solubilization: the investigation of the enzyme size by gel filtration. Suspension of Golgi membranes in solutions of NaCl (0.1 or 1.0 M) or EDTA (1 mM) as well as treatment with ultrasonic vibrations at 0" for 15 to 60 min produced negligible solubilization of galactosyltransferase (<0.2%). Other extraction procedures, urea (2 M), SDS at several concentrations (>O.l%b), as well as pyridine and acetone at a number of concentrations, strongly inhibited enzyme activity. Treatment with trypsin (0.5% w/w protein for 30 min at 20") or steapsin (5% w/w for 30 min at 20%) resulted in a loss of activity. 1-Butanol at a concentration of 2% v/v (butanol/protein ratio of 4:l) solubilized 70% of galactosyltransferase, but at higher concentrations produced inhibition.
Deoxycholate also produced partial solubilization with enzyme inhibition. In contrast to all these procedures, treatment of Golgi membranes with a 1% (w/v) concentration of Triton X-100 released ga1actosyltransferase quantitatively into the supernatant. Triton X-100 had a stimulatory (2.5-fold at a concentration of 0.5 to 2.0% (w/v)) rather than inhibitory effect on galactosyltransfcrase activity in Golgi membranes. The quantitative solubilization of galactosyltransferase hy Triton X-100 (1% v/v solution) was not affected by varying in the Golgi protein to detergent ratio (w/w) from 0.1 to 1.0, by ionic strength (0 to 0.125 M NaCl), or by the presence of GlcNAc (20 mM) or 2 mercaptoethanol (3 mM). However, 5 mM GlcNAc and 3 mM mercaptoethanol were included in subsequent solubilization studies to help maintain enzyme stability. A number of procedures were investigated for purifying detergent-solubilized galactosyltransferase from Golgi membranes, and the following procedure was chosen as the most satisfactory on the criteria of effectiveness, yield, and simplicity. Because of a limited supply of Golgi membrane material, the procedure was used only on small batches of material. All steps in the purification procedure were performed at 4". Solubilization-Three to five milligrams or less of Golgi membrane protein was suspended in 1 ml of 25 mM sodium cadodylate buffer containing 1% (w/v) Triton X-100, 0.1 M NaCl, 5 mM GlcNAc, and 3 mu 2-mercaptoethanol, pH 7.4. After brief agitation with a Vortex mixer, the partially clarified suspension was centrifuged for 60 min at 10,000 x g at 4". Ninety-nine per cent of the galactosyltransferase activity was recovered in the supernatant.
Gel Filtration -The supernatant was loaded on a column of Bio-Gel P-150 (87 x 1.5 cm) equilibrated with 25 mM sodium cacodylate buffer containing 0.1 M NaCl, 5 mM GlcNAc, and 3 mM 2-mercaptoethanol, pH 7.4. The column was developed with the same buffer at a flow rate of 3.2 ml/h, and the emuent was collected in 2.0-ml fractions. The fractions were monitored by absorbance at 280 nm and assayed for galactosyltransferase activity. As shown in Fig. 1, a small proportion of the enzyme emerged in the void volume of the column but most (70% of the applied activity) was retarded, emerging as a peak and shoulder on the trailing edge of the main protein peak.

Affinity
Chrom.atography with a-Lactalbun~iniS~pharose -Solid NaCl and GlcNAc were added to the solution of enzyme, with stirring, to final concentrations of 1.0 M and 20 KIM, respectively. The material was loaded on to a second affinity column composed of 2.5 ml of Sepharose 4B/a-lactalbumin (2 mg of protein/ml of gel), equilibrated with 25 mM sodium cacodylate buffer containing 1.0 M NaCl, 20 mM GlcNAc, and 3 mM 2-mercaptoethanol, pH 7.4. After the loaded column had been washed with equilibrating buffer (30 to 40 column volumes), the enzyme was eluted with the same buffer devoid of GlcNAc. The presence of 1 M NaCl in the elution buffer was found to be essential for the recovery of membrane galactosyltransferase from cu-lactalbumin/Sepharose. It was necessary to repeat the final purification step to obtain optimal purification of the enzyme. A typical purification of the galactosyltransferase is outlined in Table II.

Purity and Molecular
Size of Membrane Galactosyltransferase

Affinity
Chromatography with UDPISepharose -The pooled material was stirred and 0.5 M MnCl, was added to a final concentration of 25 InM. The pH was readjusted to 7.4 with dilute NaOH. The material was loaded on a I-ml column The progressive purification of galactosyltransferase solubilized from Golgi membranes is illustrated by the SDSpolyacrylamide gel electrophoresis patterns shown in Fig. 2. The total Golgi membrane proteins and the fractions of material soluble and insoluble in 1% Triton X-100 have complex patterns, but distinct differences can be observed between the Triton-soluble and -insoluble fractions. Enzyme from the final purification stage, shown in Gel 7, is resolved into two components. Comparison of the mobilities of these components with those of standard proteins of known size gives apparent molecular weight values of 69,000 for the larger, well defined component and a molecular weight range of 53 to 55 x l@' for the lesser, diffuse component.
As the main component is a glycoprotein (indicated by positive staining with periodate-SchifI' reagent), the molecular weight value is likely to be an overestimate.
The molecular size of the enzyme was also investigated by analytical gel filtration.
While the galactosyltransferase activity of intact Golgi membranes was relatively stable, the solubilized and partially purified enzyme was very unstable. Enzyme directly solubilizcd from Golgi membranes with Triton X-100 was therefore used for gel filtration.
The column of Bio-Gel P-150 was calibrated with globular proteins of known molecular size. The elution position of the main peak of galactosyltransferase corresponded to a molecular weight of 65,000 while a shoulder on the trailing edge corresponded to the elution position of a 55,000 molecular weight species, in reasonable agreement with the mobilities on SDS gels of the components present in the purified enzyme.
The sedimentation coefficient of the solubilized membrane enzyme was also measured, as an additional indication of size, using the method of Yphantis and Waugh (14) as previously described (lo), where the depletion of enzyme activity from the upper compartment of a separation cell during centrifugation is determined. The sedimentation coefficient, derived from three separate runs of 30,45, and 60 min, gave a value for s~,,~(, of 3.63.

Enzymic Properties
Because of the inherent instability of the purified membrane galactosyltransferase, the enzymic properties were investigated using Golgi membranes in the presence of Triton X-100. A separate investigation with soluble galactosyltransferase from sheep colostrum showed that, while a small increase in activity is obtained in the presence of 1% Triton X-100, the apparent K,,, values for Mr?+, UDP-galactose, and N-acetylglucosamine, each determined at fixed concentrations of the other substrates, were unchanged in the presence of T&on. It would appear that a reasonably valid comparison can be made between the enzymic characteristics of the membrane enzyme in the presence of Triton and those published for the naturally soluble colostrum enzyme, based on experiments performed in the absence of detergent. Under the assay conditions used, enzyme activity was found to increase linearly with time and with the amount of added Golgi protein.

Cation Activation
In the absence of added cations, Golgi membranes galactosyltransferase shows a low, but significant level of activity the enzyme preparation then only shows activity in the presence of added cations. As previously reported for colostrum galactosyltransferase, Zn'+ , Fe'+ Co"+ and Cd" all supported enzyme activity although at lo'wer rates than obtained with Mn". Mg" * or CaY+ did not support activity but Ca ?+ did stimulate galactosyltransferase activity in the presence of 20 PM Mn'+. These properties are similar to those previously observed with colostrum galactosyltransferase (9).

Kinetic Properties
For the evaluation of the kinetic parameters of the membrane galactosyltransferase, initial velocities were measured at varying concentrations of one substrate or activator and a series of fixed concentration of a second substrate while maintaining other assay components at fixed concentrations. As the purpose of the study is not to accomplish a complete steady state kinetic analysis of the enzyme, but to effect a comparison with previous data, not all possible combinations of substrates and activators were varied for the reactions catalyzed by the enzyme (N-acetyllactosamine synthesis, lactose synthesis in the presence of a-lactalbumin, and transfer of galactose to glycoprotein).
Selected pairs of substrates and activators were varied to elucidate specific parameters and kinetic features.
The following combinations were used. 1. for transfer to GlcNAc (N-acetyllactosamine synthesis): (a) Mn" and UDP-galactose, at a fixed concentration of GlcNAc (20 mM); (6)  The investigation of lactose synthesis and glycoprotein synthesis were insufficiently detailed to give more than apparent kinetic parameters and patterns of Lineweaver Burke plots. These parameters and patterns can be compared with those previously obtained with soluble galactosyltransferase under similar conditions. The data from the kinetic experi- merits, apart from combination l(a), which gives nonlinear replots (81, and combination 3, which is a single Lineweaver-Burk plot, were subjected to computer analysis by fitting to appropriate rate equations as described previously. Only the data for varying concentrations of the substrates UDP-galactose and GlcNAc are shown (Fig. 3). These give in double reciprocal plots a set of parallel lines and lit best to the rate equation u = ((VA BY(KJ3 + Ka + AB)) as found for the soluble galactosyltransferases under these conditions (9, 15). The other patterns are closely similar to those previously reported for human milk (15) and bovine colostrum galactosyltransferase (9).
For lactose synthesis at varying concentrations of o-lactalbumin and glucose, an asymmetric intersecting kinetic pattern was obtained, where Lineweaver-Burk plots with glucose as variable substrate at a series of fixed concentrations of (Ylactalbumin intersect precisely on the vertical aiis, but for OL-  lactalbumin as variable substrate at a series of fixed concentrations of glucose, the lines intersect to the left of the l/u axis. The values for kinetic parameters obtained from these data are summarized in Table III. For N-acetyllactosamine synthesis, the apparent parameters were corrected in "true" values utilizing the rate equation v = VABCIK,KJK, + K*K,A + K,,K,C + K,AB + K,AC + K.BC + ABC where: A, B, and C are concentrations of Mn'+, UDP-galactose, and GlcNAc respectively; K,, and Kib are dissociation constants for Mn2+ and UDP-gala&se from E 1 Mn'+ and E. Mn2+ * UDP-gala&se, respectively; and K, , K,, and Kc are the Michaelis constants for the appropriate substrates. The procedures for correction to the true values have been described previously (15). For lactose synthesis, the K,,, value for o-la&albumin must be zero (Kc), but apparent values for Kidac and K,(, (the K,,, for glucose) can be deduced. The data for Mn*+ and UDP-gala&se have been divided into two sections as Lineweaver-Burk plots for Mn*+ as variable substrates, or secondary plots against l/Mn'+ show a break in