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Lactose synthetase activity of α-lactalbumins from several species
ARCHIVES
Lactose
OF
BIOCHEMISTRY
AND
Synthetase
BIOPHYSICS
Activity JANICE
Department
464-469
(1970)
of (r-Lactalbumins
M. LEY AND ROBERT
of Biochemistry, Received
138,
University February
of Minnesota,
5, 1970;
accepted
from
Several
Species’
JENNESS St. Paul, March
Minnesota
55101
3, 1970
The kinetics of the interaction of the A and B proteins of lactose synthetase was studied and found to conform to simple Langmuir adsorption theory. The affinity of the B proteins of six species for bovine A protein w&s measured, and the range of affinities was found to differ among species. Taking these factors into consideration, a procedure for utilizing the lactose synthetase reaction as the basis of a functional assay for cY-lactalbumin is outlined. Finally, preliminary evidence for a correlation between lactose levels and cu-lactalbumin levels in the milks of different species is presented. Lactose synthetase (UDP-galactose: Dglucose I-galactosyltransferase, EC 2.4.1.22) was the first enzyme of mammalian origin to be shown to be composed of two separate protein components (1). The A component of lactose synthetase is a galactosyltransferase, by itself capable of attaching galactose residues to N-acetylglucosamine acceptors (2). However, the presence of the B component alters the acceptor specificity so that galactose can be transferred to a glucose acceptor, resulting in lactose formation. The B component has been shown to be identical with the familiar milk protein, a-la&albumin (3). If increasing concentrations of B protein are added to a constant amount, of A protein in an assay mixture, the increase in reaction velocity describes a hyperbola (2)) suggesting a type of kinetics analogous to the MichaelisMenton description of the enzyme-substrate complex formation. For example, if the A and B proteins interact to form a rapidly associating and dissociating complex :
A+B*AB the reaction may be characterized dissociation constant &:
(1) by a
1 Scientific Journal Series No. 7160, Agricultural Experiment Station, University of Minnesota, St. Paul, Minnesota 55101.
(2) Furthermore, the total amount of A must be equal to that existing free in solution plus that bound in AB complex:
[AtI = [Al + WI
(3)
Equations (2) and (3) may be combined to eliminate [A] and rearranged to give the following result :
@I
[ABI
-@tl = & + [Bl
(4)
Since [AB] determines the velocity of lactose synthesis in an assay and [At] determines the maximal velocity that would be possible if all molecules of A were in the AB complex, Eq. (4) may be rewritten:
PI “‘,
= &
+ [B]
(5)
Therefore, the concentration of a-lactalbumin in the reaction mixture may be characterized by the experimentally measurable ratio, V/V, . The aims of this work were (1) to ascertain if saturation of A protein with B did proceed as predicted by simple adsorption theory, (2) to determine if the lactose synthetase
(u-LACTALBUMINS
AND
assay, using bovine A protein, could be used to investigate the homology of the cr-lactalbumins from various species and (3) to develop a procedure for determination of a-lactalbumin in the whey proteins of various milks. MATERIALS
AND
METHODS
Materials. The A and B proteins were prepared from a pooled bovine herd sample of milk. All centrifugations were done at 4” at 10,OOOg for 15 min. Five hundred milliliters of whole milk were centrifuged and the solid cream layer removed with a spatula. The casein was precipitated by the dropwise addition of 2 N HCl until the suspension reached pH 4.6. The suspension was centrifuged, and the precipitate was discarded. The supernatant fluid was made 40 mM in MnClz and 1 IILM. in mercaptoethanol. The suspension was centrifuged, and the precipitate was again discarded. The whey was fractionated with ammonium sulfate; the fraction precipitating between 35 and 75% saturation was saved and solubilized by brief dialysis against 0.02 N Tris-HCl buffer, pH 7.5, 5 nnvr in MgClt and 1 mu in mercaptoethanol. Ten milliliters of this crude preparation were applied to a Sephadex G-150 column, 3 X 160 cm, that had been previously equilibrated with the Tris buffer. Elution was carried out with the Tris buffer at the rate of 10 ml/hr. Three main peaks were obtained; the first consisted primarily of serum albumin and the proteins eluting in the void volume, the second was composed mainly of p-lactoglobulin, and the third was almost exclusively a-lactalbumin. The fractions eluting between the first and second peaks were assayed for A protein activity; those fractions showing high activity were subdivided and frozen under nitrogen at -20”. This preparation was stable .Eor several months. Once thawed, it could be stored in a refrigerator for about a week although it (continued to lose activity. For the preparation of a standard supply of bovine a-lactalbumin, the third peak from the rechromatochromatography was Sephadex graphed on a Sephadex G-150 column, 3 X 160 cm. The peak fractions from this second column were desalted with Sephadex G-25, using the centrifuge-basket technique, and freeze-dried. The whey proteins used in the study were prepared from the skim milks by acid precipitation of the casein followed by exhaustive dialysis of the whey and freeze-drying. ol-Lactalbumin was isolated from the white-tailed deer (Odocoileus virginianus), pig (Sus serofa), and rat (Rattus norvegicus) whey proteins by two successive Sephadex G-150 chromatography steps. Human (Homo
LACTOSE
SYNTHESIS
465
sapiens) cu-lactalbumin was prepared by Miss Nancy Phillips of this laboratory and the goat (Capra hircus) or-la&albumin was a gift from Dr. A. Sen, Bose Institute, Calcutta. Methods. The lactose synthetase assay reaction was carried out in 6 X 5C-mm culture tubes. The assay components, made up in 50 mM glycine buffer, pH 8.5, were added with a 56~1 syringe (Hamilton Instruments, Whittier, California). The 40-~1 of assay mixture contained 0.03 pmole uniformly labeled in the UDP-[%I -galactose, galactose moiety (New England Nuclear), containing approximately 6000 cpm, 3 rmoles n-glucose, 1.6 pmoles MnClz, 7 ~1 of the A protein preparation, and various amounts of B protein. The tubes were covered with Paralilm and incubated in a 37” water bath for 30 min. The reaction was stopped by the addition of 20 11 of 150 mM EDTA containing 1% lactose to act as a cold carrier in the subsequent steps. Twenty microliters of t,he reaction mixture were then spotted on an Eastman Chromagram Sheet No. 6965 (Distillation Products Industries, Rochester, New York) and developed by ascending technique with an ammonium acetate: ethanol solvent (1 vol 1 M ammonium acetate, pH 3.8, containing 0.5yo EDTA plus 2 vol 95yo ethanol). After development, the Chromagrams were oven-dried at lOO”, and the sections of the Chromagram corresponding to a lactose standard were cut out, and the cellulose powder from them was scraped into conical centrifuge tubes. Four milliliters of water were added and the tubes were allowed to stand for several hours with occasional mixing. The cellulose was then centrifuged down and 3.0 ml of the supernatant fluid were washed through an AG-1-formate column, 0.33 X 5 cm, that had been previously equilibrated with lactose. Eluate and wash were transferred to a planchet, evaporated on a hot and counted with a gas-flow planchet plate, counter (Nuclear Chicago Planchet Counting System No. 4338). A blank, containing all components except ol-lactalbumin, was used to correct for endogenous A protein activity. Under these conditions, the assay remained linear for at least 60 min. RESULTS
The effect of increasing concentrations of B protein on reaction velocity is shown in Fig. 1. To test if the data describes a true hyperbola, two methods of linear transformation were employed. Figure 2 shows the data plotted according to the method of Lineweaver-Burke, and Fig. 3 shows the same data plotted on a Hofstee plot. The four points on the linear portion of the
466
LEY
AND
JENNESS
Assays were also performed using the whey proteins of the six species as the source of B protein. The solutions of whey proteins were first heat-treated by holding in a 65” water bath for 3 min to inactivate any A protein activity they might have otherwise contributed to the assay. Final whey protein concentrations in the assay mixture ranged from 200-2000 pg/ml. The data from each experiment was plotted on either Lineweaver-Burke or Hofstee plots and the Kd values graphically obtained are tabulated in Table I. DISCUSSION 1. The effect of adding increasing amounts of B protein (~lactalbumin) to a constant amount of A protein in the lactose synthetase assay procedure. FIG.
The fact that the data from the hyperbolic plot (Fig. 1) could be satisfactorily applied to a Lineweaver-Burke or a Hofstee plot
60 h
40 > 20
01 0
8
*
8
0.20
*
’
0.40
8
0.60
(
8
0.80
“‘bl 0
0.04
0.08
0.12
“LB1 FIG. 2. Replot of the data from the hyperbolic plot (Fig. 1) according to the method of Lineweaver-Burke (l/V = l/V, + (Kd/Vm) (l/[BJ)). The Kd value is obtained from the r-intercept, which is equal to - 1/Kd.
hyperbola at low velocities were omitted from the Hofstee plot since the velocity per is approximately conunit protein (V/B) stant at these a-lactalbumin levels. The Kd values graphically obtained from the two plots are 62 and 66 pg per ml, respectively. Assays were then done using various concentrations (20-500 pg/ml) of the a-lactalbumins from other species as the source of B protein. The data from each of the experiments were plotted on either LineweaverBurke or Hofstee plots. Table I lists the & values graphically obtained with the a-lactalbumins of six different species.
FIG. 3. Replot of the data from the hyperbolic plot (Fig. 1) according to the method of Hofstee (V = V,(V/[Bl) (Kd)). The & value is obtained from the slope of the line.
TABLE Kd VALUES
Animal
cow Goat Deer Pig Rat Human
OF SIX HOMOLOGOUS
Kd Of or-lactalbumin bg/mO
64 70 74 103 111 118
I
a-LACTALBUMINS AND WHEY PROTEINS” Kc, Of whey protein GdmU
328 222 440 800 1330 148
THEIR
a-Lactalbumin in whey protein (%)
20 32 17 13 8 80
a The & values were determined in the lactose synthetase assay with bovine A protein. The a-lactalbumin content was calculated from the respective Kd values as outlined in the Discussion section.
a-LACTALBUMINS
AND
(Figs. 2 and 3) is evidence that the saturation of A protein with B could be adequately described by a Langmuir-type adsorption isotherm. Thus a particular a-lactalbumin concentration may be characterized by a particular ratio of V/V, . V, is determined graphically from the Lineweaver-Burke or Hofstee plot of velocities obtained with various concentrations of or-lactalbumin. Equation (5) shows that when the concentration of I3 protein is equal to the constant & , then the ratio V/V, will be 34. Furthermore, since V/y, is a ratio, it does not depend on absolute reaction velocities and hence is not sensitive to day-to-day fluctuations in the potency of the A protein. Thus when a given amount of bovine A protein is present in an assay mixture, one-half maximal velocity will be observed when 64 fig/ml of bovine a-la&albumin is present. This concept of the interaction of the A and B proteins conflicts somewhat with the observations of other workers. Brodbeck and Ebner (1) reported being able to titrate either the A or B protein in the presence of a constant amount of the other. Although they did not publish any saturation curves, it would not be possible from our model to truly saturate B protein with A. However, it would be possible to assay for low concentrations of a-la&albumin in the presence of a constant amount of A protein or to assay units of A protein activity in the presence of saturating amounts of a-la&albumin. Likewise, Palmiter (4) reported a linear response in his assay system when increasing amounts of B protein were added, up to a point at which he felt the A subunit became limiting. At this point the increase in reaction velocity abruptly leveled off. Recently, the existence of a third component of lactose synthetase has been proposed (5). The author has suggested that in vivo and in crude preparations, a Z component acts to prevent the dissociation of the AB complex. However, when the preparations were further purified, the Z component was presumably lost, so that the system then obeyed a simple chemical equilibrium:
LW” I-L = [A]“[B]”
(6)
LACTOSE
SYNTHESIS
467
When increasing concentrations of a crude B protein preparation were added to several different concentrations of crude A protein preparation, a family of curves, roughly approximating hyperbolas, was obtained. The author ascribed this to the saturation of A protein with B protein in the presence of the Z component which prevented dissociation of the AB complex. The curves flattened when the concentration of B protein reached saturation levels. When the same experiment was repeated with purified A and B proteins, a family of straight lines was obtained. The author attributed this to behavior according to Eq. (6), where increasing the concentration of B protein in the presence of a constant amount of A results in a linear increase in [AB]. The fallacy in thii reasoning lies in the fact that the A term in Eq. (6) denotes the concentration of A existing free in solution. [At], not the concentration of free A, remains constant when increasing concentrations of B are added to a constant amount of A protein. Thus Eq. (6), developed as outlined in the opening paragraphs of the paper, would predict that if the experiment were carried to sufficiently high concentrations of B protein, a family of hyperbolas, not straight lines, would be obtained. Similarly, the results obtained with the crude preparations of A and B would not require invoking of the Z component to explain the flattening of the curves. If the assay procedure outlined above, utilizing bovine A protein, is used to assay for the a-lactalbumins of other species, it is first necessary to ascertain if the B proteins of various species all exhibit the same affinity for bovine galactosyltransferase. Brodbeck, Denton, Tanahashi, and Ebner (3) had concluded that the B proteins of the eight species that they investigated were at least qualitatively equivalent, based on the measurement of reaction velocity obtained with a single low concentration of B protein and a constant amount of bovine A protein. Apparently the authors did not take into consideration the hyperbolic nature of the saturation curve. Thus their measurements of reaction velocity at a single cy-lactalbumin concentration would be influenced both by the affinity of the various B proteins for bovine A protein and by the maximal
46s
LEY
AND
velocities possible with the various AB complexes. Table I shows that the six B proteins tested in our procedure did not all have the same affinity for bovine A protein. Rather, the range of affinities for the bovine galactosyltransferase roughly correspond to the phylogenetic divergence of the organisms from the bovine. Goat and deer, both ruminants, have the lowest dissociation constants, while pig, rat, and human have higher & values. It is of interest at this point to compare the enzymic activities of the various B proteins with their immunological cross-reaction. If the B proteins of different species are tested with rabbit antisera prepared in response to crystallized bovine cr-lactalbumin, cross-reaction is observed only with the ruminant cu-lactalbumins (3, 6). This contrasts with the continuum of enzymic reaction observed when both ruminant and nonruminant B proteins are tested with bovine A protein. The fact that ar-lactalbumin has a functional role as a “specifier protein” makes it unique for the study of evolutionary homology. The non-ruminant a-lactalbumins have undergone sufficient amino acid substitution so that their conformation is no longer recognized by antisera made in response to bovine or-lactalbumin. However, they are still capable of interacting with bovine A protein, although with considerably less affinity than the ruminant cr-lactalbumins. From the Kd values of the various a-lactalbumins and the Kd values of their homologous whey proteins, the percentage of a-lactalbumin in each of the whey proteins could be calculated. For example, the Kd value of bovine whey protein, or the concentration of bovine whey proteins that resulted in one-half maximal velocity, is 328 pg/ml. This concentration of whey protein must then contain 64 pg/ml of &a&albumin, since that is the concentration of or-lactalbumin that produced one-half max-’ imal velocity in the experiments with Lu-lactalbumin alone. Thus the concentration of a-lactalbumin in this sample of bovine whey is 64/328 or 20 %. The validity of this
JENNESS TABLE LACTOSE
Rat
CONTENT CONTENT
2.8 4.8 4.6 4.6 5.5 7.0
cow Goat Deer Pig Human a Our
II
AND a-LACT~LBUMIN OF SIX MILKS
1.3 0.6 0.7 1.0 2.0 0.6
8 9 10 (L a 9
8 20 32 17 13 80
0.10 0.12 0.22 0.17 0.26 0.48
analyses. 1 E
human
LACTOSE,
and
FIG. 4. Correlation the a-lactalbumin
g/l00
ml milk
between the lactose levels of six milks.
levels
approach was checked by the following experiment. Various concentrations of the purified bovine ol-lactalbumin (20-150 pg/ ml) were again assayed, but this time each assay mixture also contained 191 pg/ml of bovine whey protein. The whey protein was assumed to contribute 38 pg/ml of a-lactalbumin (20% of its weight) to the assay. If this percentage were correct, and if there were no activators or inhibitors of the AB complex in the whey protein, then the Kd value generated by this experiment should be the same as that obtained in the experiment with bovine cy-lactalbumin alone. When the data from the experiment were plotted on a Hofstee plot, the Kd value graphically obtained was 65 wg/ml, in good agreement with values obtained previously. Recently, a model has been proposed whereby the flux of cr-lactalbumin through the secretory cell of the mammary gland is responsible for the regulation of lactose synthesis at various stages of lactation (7).
LU-LACTALBUMINS
AND
Thus it wa,s of interest to investigate the possibility that a-lactalbumin might play a role in determining the lactose levels of the milks of various species. The lactose content and the w’hey protein content of several milks are listed in Table II. From the percentage of a-lactalbumin in the whey protein, obtained as outlined above, and the whey protein content of the milk, the (Ylactalbumin. content of the milk was calculated. The correlation between the lactose levels and the whey protein levels in the milks is diagrammed in Fig. 4. The linear correlation coefficient between the two sets of measurements is +0.95, indicating that perhaps cu-lactalbumin does have a role in determining the lactose levels of the milks of these six species. ACKNOWLEDGMENTS The authors thank H.kR. Warner for throughout the course
Dr. J. E. Gander and Dr. their helpful discussions of this investigation.
LACTOSE
SYNTHESIS
469
REFERENCES 1. BRODBECK, U., AND EBNER, K. E., J. Biol. Chem. 241, 762 (1966). 2. BREW, K., VANAMAN, T. C., AND HILL, R. L., Proc. Nat. Acad. Sci. U.S.A. 60, 491 (1968). 3. BRODBECK, U., DENTON, W. L., TANAHASHI, N., AND EBNER, K. E., J. Biol. Chem. 242, 1391 (1967). 4. PALMITER, R. D., Biochem. J. 113,409 (1969). 5. PALMITER, R. D., Biochim. Biophys. Acta 178, 35 (1969). 6. LYSTER, R. L. J., JENNESS, R., PHILLIPS, N. I., AND SLOAN, R. E., Comp. Biochem. Physiol. 17, 967 (1966). 7. BREW, K., Nature London 222, 671 (1969). 8. Cox, W. M., AND MUELLER, A. J., J. Nutr. 13, 249 (1937). 9. MACY, I. G., KELLY, H. J., AND SLOAN, R. E., “The Composition of Milks,” Nat. Res. Council Pub. No. 254 (1953). 19. GAMBLE, J. A., ELLIS, N. R., AND BESLAY, A. K., “The Composition and Properties of Goat’s Milk as Compared with Cow’s Milk.” U.S. Dept. Agr. Tech. Bull. 671 (1939).
Lactose
OF
BIOCHEMISTRY
AND
Synthetase
BIOPHYSICS
Activity JANICE
Department
464-469
(1970)
of (r-Lactalbumins
M. LEY AND ROBERT
of Biochemistry, Received
138,
University February
of Minnesota,
5, 1970;
accepted
from
Several
Species’
JENNESS St. Paul, March
Minnesota
55101
3, 1970
The kinetics of the interaction of the A and B proteins of lactose synthetase was studied and found to conform to simple Langmuir adsorption theory. The affinity of the B proteins of six species for bovine A protein w&s measured, and the range of affinities was found to differ among species. Taking these factors into consideration, a procedure for utilizing the lactose synthetase reaction as the basis of a functional assay for cY-lactalbumin is outlined. Finally, preliminary evidence for a correlation between lactose levels and cu-lactalbumin levels in the milks of different species is presented. Lactose synthetase (UDP-galactose: Dglucose I-galactosyltransferase, EC 2.4.1.22) was the first enzyme of mammalian origin to be shown to be composed of two separate protein components (1). The A component of lactose synthetase is a galactosyltransferase, by itself capable of attaching galactose residues to N-acetylglucosamine acceptors (2). However, the presence of the B component alters the acceptor specificity so that galactose can be transferred to a glucose acceptor, resulting in lactose formation. The B component has been shown to be identical with the familiar milk protein, a-la&albumin (3). If increasing concentrations of B protein are added to a constant amount, of A protein in an assay mixture, the increase in reaction velocity describes a hyperbola (2)) suggesting a type of kinetics analogous to the MichaelisMenton description of the enzyme-substrate complex formation. For example, if the A and B proteins interact to form a rapidly associating and dissociating complex :
A+B*AB the reaction may be characterized dissociation constant &:
(1) by a
1 Scientific Journal Series No. 7160, Agricultural Experiment Station, University of Minnesota, St. Paul, Minnesota 55101.
(2) Furthermore, the total amount of A must be equal to that existing free in solution plus that bound in AB complex:
[AtI = [Al + WI
(3)
Equations (2) and (3) may be combined to eliminate [A] and rearranged to give the following result :
@I
[ABI
-@tl = & + [Bl
(4)
Since [AB] determines the velocity of lactose synthesis in an assay and [At] determines the maximal velocity that would be possible if all molecules of A were in the AB complex, Eq. (4) may be rewritten:
PI “‘,
= &
+ [B]
(5)
Therefore, the concentration of a-lactalbumin in the reaction mixture may be characterized by the experimentally measurable ratio, V/V, . The aims of this work were (1) to ascertain if saturation of A protein with B did proceed as predicted by simple adsorption theory, (2) to determine if the lactose synthetase
(u-LACTALBUMINS
AND
assay, using bovine A protein, could be used to investigate the homology of the cr-lactalbumins from various species and (3) to develop a procedure for determination of a-lactalbumin in the whey proteins of various milks. MATERIALS
AND
METHODS
Materials. The A and B proteins were prepared from a pooled bovine herd sample of milk. All centrifugations were done at 4” at 10,OOOg for 15 min. Five hundred milliliters of whole milk were centrifuged and the solid cream layer removed with a spatula. The casein was precipitated by the dropwise addition of 2 N HCl until the suspension reached pH 4.6. The suspension was centrifuged, and the precipitate was discarded. The supernatant fluid was made 40 mM in MnClz and 1 IILM. in mercaptoethanol. The suspension was centrifuged, and the precipitate was again discarded. The whey was fractionated with ammonium sulfate; the fraction precipitating between 35 and 75% saturation was saved and solubilized by brief dialysis against 0.02 N Tris-HCl buffer, pH 7.5, 5 nnvr in MgClt and 1 mu in mercaptoethanol. Ten milliliters of this crude preparation were applied to a Sephadex G-150 column, 3 X 160 cm, that had been previously equilibrated with the Tris buffer. Elution was carried out with the Tris buffer at the rate of 10 ml/hr. Three main peaks were obtained; the first consisted primarily of serum albumin and the proteins eluting in the void volume, the second was composed mainly of p-lactoglobulin, and the third was almost exclusively a-lactalbumin. The fractions eluting between the first and second peaks were assayed for A protein activity; those fractions showing high activity were subdivided and frozen under nitrogen at -20”. This preparation was stable .Eor several months. Once thawed, it could be stored in a refrigerator for about a week although it (continued to lose activity. For the preparation of a standard supply of bovine a-lactalbumin, the third peak from the rechromatochromatography was Sephadex graphed on a Sephadex G-150 column, 3 X 160 cm. The peak fractions from this second column were desalted with Sephadex G-25, using the centrifuge-basket technique, and freeze-dried. The whey proteins used in the study were prepared from the skim milks by acid precipitation of the casein followed by exhaustive dialysis of the whey and freeze-drying. ol-Lactalbumin was isolated from the white-tailed deer (Odocoileus virginianus), pig (Sus serofa), and rat (Rattus norvegicus) whey proteins by two successive Sephadex G-150 chromatography steps. Human (Homo
LACTOSE
SYNTHESIS
465
sapiens) cu-lactalbumin was prepared by Miss Nancy Phillips of this laboratory and the goat (Capra hircus) or-la&albumin was a gift from Dr. A. Sen, Bose Institute, Calcutta. Methods. The lactose synthetase assay reaction was carried out in 6 X 5C-mm culture tubes. The assay components, made up in 50 mM glycine buffer, pH 8.5, were added with a 56~1 syringe (Hamilton Instruments, Whittier, California). The 40-~1 of assay mixture contained 0.03 pmole uniformly labeled in the UDP-[%I -galactose, galactose moiety (New England Nuclear), containing approximately 6000 cpm, 3 rmoles n-glucose, 1.6 pmoles MnClz, 7 ~1 of the A protein preparation, and various amounts of B protein. The tubes were covered with Paralilm and incubated in a 37” water bath for 30 min. The reaction was stopped by the addition of 20 11 of 150 mM EDTA containing 1% lactose to act as a cold carrier in the subsequent steps. Twenty microliters of t,he reaction mixture were then spotted on an Eastman Chromagram Sheet No. 6965 (Distillation Products Industries, Rochester, New York) and developed by ascending technique with an ammonium acetate: ethanol solvent (1 vol 1 M ammonium acetate, pH 3.8, containing 0.5yo EDTA plus 2 vol 95yo ethanol). After development, the Chromagrams were oven-dried at lOO”, and the sections of the Chromagram corresponding to a lactose standard were cut out, and the cellulose powder from them was scraped into conical centrifuge tubes. Four milliliters of water were added and the tubes were allowed to stand for several hours with occasional mixing. The cellulose was then centrifuged down and 3.0 ml of the supernatant fluid were washed through an AG-1-formate column, 0.33 X 5 cm, that had been previously equilibrated with lactose. Eluate and wash were transferred to a planchet, evaporated on a hot and counted with a gas-flow planchet plate, counter (Nuclear Chicago Planchet Counting System No. 4338). A blank, containing all components except ol-lactalbumin, was used to correct for endogenous A protein activity. Under these conditions, the assay remained linear for at least 60 min. RESULTS
The effect of increasing concentrations of B protein on reaction velocity is shown in Fig. 1. To test if the data describes a true hyperbola, two methods of linear transformation were employed. Figure 2 shows the data plotted according to the method of Lineweaver-Burke, and Fig. 3 shows the same data plotted on a Hofstee plot. The four points on the linear portion of the
466
LEY
AND
JENNESS
Assays were also performed using the whey proteins of the six species as the source of B protein. The solutions of whey proteins were first heat-treated by holding in a 65” water bath for 3 min to inactivate any A protein activity they might have otherwise contributed to the assay. Final whey protein concentrations in the assay mixture ranged from 200-2000 pg/ml. The data from each experiment was plotted on either Lineweaver-Burke or Hofstee plots and the Kd values graphically obtained are tabulated in Table I. DISCUSSION 1. The effect of adding increasing amounts of B protein (~lactalbumin) to a constant amount of A protein in the lactose synthetase assay procedure. FIG.
The fact that the data from the hyperbolic plot (Fig. 1) could be satisfactorily applied to a Lineweaver-Burke or a Hofstee plot
60 h
40 > 20
01 0
8
*
8
0.20
*
’
0.40
8
0.60
(
8
0.80
“‘bl 0
0.04
0.08
0.12
“LB1 FIG. 2. Replot of the data from the hyperbolic plot (Fig. 1) according to the method of Lineweaver-Burke (l/V = l/V, + (Kd/Vm) (l/[BJ)). The Kd value is obtained from the r-intercept, which is equal to - 1/Kd.
hyperbola at low velocities were omitted from the Hofstee plot since the velocity per is approximately conunit protein (V/B) stant at these a-lactalbumin levels. The Kd values graphically obtained from the two plots are 62 and 66 pg per ml, respectively. Assays were then done using various concentrations (20-500 pg/ml) of the a-lactalbumins from other species as the source of B protein. The data from each of the experiments were plotted on either LineweaverBurke or Hofstee plots. Table I lists the & values graphically obtained with the a-lactalbumins of six different species.
FIG. 3. Replot of the data from the hyperbolic plot (Fig. 1) according to the method of Hofstee (V = V,(V/[Bl) (Kd)). The & value is obtained from the slope of the line.
TABLE Kd VALUES
Animal
cow Goat Deer Pig Rat Human
OF SIX HOMOLOGOUS
Kd Of or-lactalbumin bg/mO
64 70 74 103 111 118
I
a-LACTALBUMINS AND WHEY PROTEINS” Kc, Of whey protein GdmU
328 222 440 800 1330 148
THEIR
a-Lactalbumin in whey protein (%)
20 32 17 13 8 80
a The & values were determined in the lactose synthetase assay with bovine A protein. The a-lactalbumin content was calculated from the respective Kd values as outlined in the Discussion section.
a-LACTALBUMINS
AND
(Figs. 2 and 3) is evidence that the saturation of A protein with B could be adequately described by a Langmuir-type adsorption isotherm. Thus a particular a-lactalbumin concentration may be characterized by a particular ratio of V/V, . V, is determined graphically from the Lineweaver-Burke or Hofstee plot of velocities obtained with various concentrations of or-lactalbumin. Equation (5) shows that when the concentration of I3 protein is equal to the constant & , then the ratio V/V, will be 34. Furthermore, since V/y, is a ratio, it does not depend on absolute reaction velocities and hence is not sensitive to day-to-day fluctuations in the potency of the A protein. Thus when a given amount of bovine A protein is present in an assay mixture, one-half maximal velocity will be observed when 64 fig/ml of bovine a-la&albumin is present. This concept of the interaction of the A and B proteins conflicts somewhat with the observations of other workers. Brodbeck and Ebner (1) reported being able to titrate either the A or B protein in the presence of a constant amount of the other. Although they did not publish any saturation curves, it would not be possible from our model to truly saturate B protein with A. However, it would be possible to assay for low concentrations of a-la&albumin in the presence of a constant amount of A protein or to assay units of A protein activity in the presence of saturating amounts of a-la&albumin. Likewise, Palmiter (4) reported a linear response in his assay system when increasing amounts of B protein were added, up to a point at which he felt the A subunit became limiting. At this point the increase in reaction velocity abruptly leveled off. Recently, the existence of a third component of lactose synthetase has been proposed (5). The author has suggested that in vivo and in crude preparations, a Z component acts to prevent the dissociation of the AB complex. However, when the preparations were further purified, the Z component was presumably lost, so that the system then obeyed a simple chemical equilibrium:
LW” I-L = [A]“[B]”
(6)
LACTOSE
SYNTHESIS
467
When increasing concentrations of a crude B protein preparation were added to several different concentrations of crude A protein preparation, a family of curves, roughly approximating hyperbolas, was obtained. The author ascribed this to the saturation of A protein with B protein in the presence of the Z component which prevented dissociation of the AB complex. The curves flattened when the concentration of B protein reached saturation levels. When the same experiment was repeated with purified A and B proteins, a family of straight lines was obtained. The author attributed this to behavior according to Eq. (6), where increasing the concentration of B protein in the presence of a constant amount of A results in a linear increase in [AB]. The fallacy in thii reasoning lies in the fact that the A term in Eq. (6) denotes the concentration of A existing free in solution. [At], not the concentration of free A, remains constant when increasing concentrations of B are added to a constant amount of A protein. Thus Eq. (6), developed as outlined in the opening paragraphs of the paper, would predict that if the experiment were carried to sufficiently high concentrations of B protein, a family of hyperbolas, not straight lines, would be obtained. Similarly, the results obtained with the crude preparations of A and B would not require invoking of the Z component to explain the flattening of the curves. If the assay procedure outlined above, utilizing bovine A protein, is used to assay for the a-lactalbumins of other species, it is first necessary to ascertain if the B proteins of various species all exhibit the same affinity for bovine galactosyltransferase. Brodbeck, Denton, Tanahashi, and Ebner (3) had concluded that the B proteins of the eight species that they investigated were at least qualitatively equivalent, based on the measurement of reaction velocity obtained with a single low concentration of B protein and a constant amount of bovine A protein. Apparently the authors did not take into consideration the hyperbolic nature of the saturation curve. Thus their measurements of reaction velocity at a single cy-lactalbumin concentration would be influenced both by the affinity of the various B proteins for bovine A protein and by the maximal
46s
LEY
AND
velocities possible with the various AB complexes. Table I shows that the six B proteins tested in our procedure did not all have the same affinity for bovine A protein. Rather, the range of affinities for the bovine galactosyltransferase roughly correspond to the phylogenetic divergence of the organisms from the bovine. Goat and deer, both ruminants, have the lowest dissociation constants, while pig, rat, and human have higher & values. It is of interest at this point to compare the enzymic activities of the various B proteins with their immunological cross-reaction. If the B proteins of different species are tested with rabbit antisera prepared in response to crystallized bovine cr-lactalbumin, cross-reaction is observed only with the ruminant cu-lactalbumins (3, 6). This contrasts with the continuum of enzymic reaction observed when both ruminant and nonruminant B proteins are tested with bovine A protein. The fact that ar-lactalbumin has a functional role as a “specifier protein” makes it unique for the study of evolutionary homology. The non-ruminant a-lactalbumins have undergone sufficient amino acid substitution so that their conformation is no longer recognized by antisera made in response to bovine or-lactalbumin. However, they are still capable of interacting with bovine A protein, although with considerably less affinity than the ruminant cr-lactalbumins. From the Kd values of the various a-lactalbumins and the Kd values of their homologous whey proteins, the percentage of a-lactalbumin in each of the whey proteins could be calculated. For example, the Kd value of bovine whey protein, or the concentration of bovine whey proteins that resulted in one-half maximal velocity, is 328 pg/ml. This concentration of whey protein must then contain 64 pg/ml of &a&albumin, since that is the concentration of or-lactalbumin that produced one-half max-’ imal velocity in the experiments with Lu-lactalbumin alone. Thus the concentration of a-lactalbumin in this sample of bovine whey is 64/328 or 20 %. The validity of this
JENNESS TABLE LACTOSE
Rat
CONTENT CONTENT
2.8 4.8 4.6 4.6 5.5 7.0
cow Goat Deer Pig Human a Our
II
AND a-LACT~LBUMIN OF SIX MILKS
1.3 0.6 0.7 1.0 2.0 0.6
8 9 10 (L a 9
8 20 32 17 13 80
0.10 0.12 0.22 0.17 0.26 0.48
analyses. 1 E
human
LACTOSE,
and
FIG. 4. Correlation the a-lactalbumin
g/l00
ml milk
between the lactose levels of six milks.
levels
approach was checked by the following experiment. Various concentrations of the purified bovine ol-lactalbumin (20-150 pg/ ml) were again assayed, but this time each assay mixture also contained 191 pg/ml of bovine whey protein. The whey protein was assumed to contribute 38 pg/ml of a-lactalbumin (20% of its weight) to the assay. If this percentage were correct, and if there were no activators or inhibitors of the AB complex in the whey protein, then the Kd value generated by this experiment should be the same as that obtained in the experiment with bovine cy-lactalbumin alone. When the data from the experiment were plotted on a Hofstee plot, the Kd value graphically obtained was 65 wg/ml, in good agreement with values obtained previously. Recently, a model has been proposed whereby the flux of cr-lactalbumin through the secretory cell of the mammary gland is responsible for the regulation of lactose synthesis at various stages of lactation (7).
LU-LACTALBUMINS
AND
Thus it wa,s of interest to investigate the possibility that a-lactalbumin might play a role in determining the lactose levels of the milks of various species. The lactose content and the w’hey protein content of several milks are listed in Table II. From the percentage of a-lactalbumin in the whey protein, obtained as outlined above, and the whey protein content of the milk, the (Ylactalbumin. content of the milk was calculated. The correlation between the lactose levels and the whey protein levels in the milks is diagrammed in Fig. 4. The linear correlation coefficient between the two sets of measurements is +0.95, indicating that perhaps cu-lactalbumin does have a role in determining the lactose levels of the milks of these six species. ACKNOWLEDGMENTS The authors thank H.kR. Warner for throughout the course
Dr. J. E. Gander and Dr. their helpful discussions of this investigation.
LACTOSE
SYNTHESIS
469
REFERENCES 1. BRODBECK, U., AND EBNER, K. E., J. Biol. Chem. 241, 762 (1966). 2. BREW, K., VANAMAN, T. C., AND HILL, R. L., Proc. Nat. Acad. Sci. U.S.A. 60, 491 (1968). 3. BRODBECK, U., DENTON, W. L., TANAHASHI, N., AND EBNER, K. E., J. Biol. Chem. 242, 1391 (1967). 4. PALMITER, R. D., Biochem. J. 113,409 (1969). 5. PALMITER, R. D., Biochim. Biophys. Acta 178, 35 (1969). 6. LYSTER, R. L. J., JENNESS, R., PHILLIPS, N. I., AND SLOAN, R. E., Comp. Biochem. Physiol. 17, 967 (1966). 7. BREW, K., Nature London 222, 671 (1969). 8. Cox, W. M., AND MUELLER, A. J., J. Nutr. 13, 249 (1937). 9. MACY, I. G., KELLY, H. J., AND SLOAN, R. E., “The Composition of Milks,” Nat. Res. Council Pub. No. 254 (1953). 19. GAMBLE, J. A., ELLIS, N. R., AND BESLAY, A. K., “The Composition and Properties of Goat’s Milk as Compared with Cow’s Milk.” U.S. Dept. Agr. Tech. Bull. 671 (1939).