Purification and enzymatic properties of gamma-glutamyltransferase from bovine colostrum.

/ . Biochem. 84, 1227-1236 (1978)

from Bovine Colostrum Kyoden YASUMOTO, Kimikazu IWAMI, Tom FUSHIKI, and Hisateru MITSUDA Department of Food Science and Technology, Faculty of Agriculture, Kyoto University, Sakyo-ku, Kyoto, Kyoto 606 Received for publication, May 29, 1978

7--Glutamyltransferase was purified 10,000-fold from bovine colostrum and was found to resemble the mammalian kidney enzymes with respect to molecular constitution and substrate specificity. The purification process involved separation of membrane material from colostral whey, treatment with papain, and column chromatography on DEAE-Sephadex and Sephadex G-150. The final preparation was apparently homogeneous on polyacrylamide gel electrophoresis, isoelectric focusing, and double immunodiffusion. It had a molecular weight (as determined by gel filtration) of about 80,000, a sedimentation coefficient of 5.0 s%),w, and an isoelectric point of pH 3.85, and was composed of two non-identical glycopeptides (with molecular weights of 55,000 and 25,000). Treatment with neuraminidase yielded a more negatively charged variant with intact enzymatic activity. The reaction with ^-glutamyl-/7-nitroanilide in the presence of glycylglycine as an acceptor was optimal at about pH 8.5 and at about pH 9.0 in its absence. Hydrolytic reaction, as assessed in terms of glutamate release, was practically absent at high pH. The activation profile by various amino acids and peptides was similar to that observed with the enzymes from other sources; glycylglycine was the best acceptor so far tested. The phosphate-independent glutaminase activity of the colostral enzyme was much lower than that of the human kidney enzyme either in the presence or absence of maleate; glutamate liberation from glutamine in the presence of maleate proceeded at only about 0.2% of the rate observed for transpeptidation between ^-glutamyl-/>-nitroanilide and glycylglycine. Initial velocity measurements at various substrate concentrations yielded results which were consistent with a pingpong mechanism modified by an autotranspeptidation shunt.

The enzyme ^-glutamyltransferase [EC 2.3.2.2] catalyzes the transfer of the f-glutamyl moiety from glutathione and other f-glutamyl compounds to a variety of acceptor amino acids and peptides, Abbreviations: SDS, sodium dodecyl sulfate; SIg A, secretory immunoglobulin A. Vol. 84, No. 5, 1978

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as well as to water. There is convincing evidence that this enzyme is largely membrane-bound in a variety of mammalian epithelial cells involved in transport and secretory processes; for example, those of the jejunum, proximal renal tubules, bile ducts, choroid plexus, ciliary body, and seminal vesicle (/). In addition, various lymphoid cells

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Purification and Enzymatic Properties of 7-Glutamyltransferase

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K. YASUMOTO, K. 1WAMI, T. FUSHIKI, and H. MITSUDA

Based on the co-localization of 7--gIutamyltransferase with secretory immunoglobulin A (SIg A) in human breast cyst fluid and colostral milk, co-purification of the transferase with SIg A, and the cross-reactivity of anti-SIg A with crude transferase preparations, it was suggested that the transferase was identical with the secretory component of SIg A (5). This hypothesis, which provoked considerable speculation on the physiological role of the transferase and the mechanism of protein secretion in these exudates, was rendered untenable by our finding (5) that in bovine colostrum the transferase is primarily membranebound and thus occurs separately from SIg A. Localization of the colostral transferase in those membranes involved in milk secretion, skim milk and fat globule membranes, is particularly marked, and these membranes represent a valuable source material for studies of this intrinsic membrane protein; they can be obtained in substantial quantities and high purity by fairly mild and simple processes; their evolutionary origin and secretion mechanism are also well documented in the literature (7, 8), though some controversy remains. The present study describes the isolation of an apparently homogeneous enzyme from bovine colostrum. The specific activity of the final

preparation was greater than that from any other source so far reported (9, 10). Its structural and catalytic properties resembled those of the kidney enzyme, particularly the human enzyme (10) in view of the low "phosphate-independent glutaminase" activity of our final preparation as well as its poor, but not completely absent, responsiveness toward maleate. MATERIALS AND METHODS Fresh colostrum (four days after calving) from the Holstein breed was kindly supplied by the owner of a local herd. L-7--Glutamyl-/>-nitroanilide, and Coomassie brilliant blue were obtained from Sigma Chemical Co. Neuraminidase (Streptococcus sp.), papain (Carica papaya), carrier ampholytes (Ampholine pH range 3.5 to 5), and Freund's complete adjuvant were obtained from Seikagaku Kogyo Co., Merck, LKB, and Miles Laboratories, respectively. A thimble-type protein bag used for ultrafiltration of protein solutions was from Sartorius Membranfilter GmbH. Protein standards for molecular weight determination on polyacrylamide gels were of commercial analytical grade and were used without further purification: bovine serum albumin, egg albumin, chymotrypsinogen (bovine pancreas), pepsin (hog stomach), cytochrome c (horse heart), trypsin (bovine pancreas), and hexokinase (yeast). Enzyme Assay—Unless otherwise stated, a mixture (1 ml) containing 3.5 ITIM 7"-glutamyl-/>nitroanilide, 10 mM glycylglycine, and an appropriate concentration of enzyme in 0.2 M Tris-HCI buffer (pH 8.5) was used to assay the enzyme. The enzymatic reaction was carried out at 37°C for an indicated period, and then terminated by adding 3 ml of 1.7 N acetic acid. The activity was estimated by measuring the absorbance at 410 nm. One unit of the enzyme was defined as the amount required for the release of one ^mol of p-nitroaniline per min under the standard conditions. Specific activity was expressed in terms of units per mg of protein as determined by the method of Lowry et al. (II) with human serum albumin as a standard. Phosphate-independent glutaminase activity was measured in a reaction mixture containing 10 mM glutamine (or other glutamyl compounds), 3.6 fig of transferase, 5 mM NAD, 50 fig of gluJ. Biochem.


have been investigated recently (2). This line of evidence, in conjunction with other experimental findings, has led Meister (3) to formulate a j - glutamyl cycle involving the transferase enzyme, and has also stimulated further studies on its physiological functions in different tissues. 7--GlutamyItransferase is not confined to mammalian tissues, but enjoys a broad distribution extending over plants and microorganisms. Previous studies (4) in this laboratory demonstrated a significantly high activity of the transferase in an edible mushroom, Lentinus edodes, and described its function in the first committed step in the sequence of lenthionine evolution from lentinic acid, the endogenous substrate occurring in the mushroom. The Lentinus enzyme exhibited a marked preference for lentinic acid as a substrate, compared with the transferase from animal tissues, which could hardly act on lentinic acid. These findings support the view that transferases from different sources may exhibit certain dissimilarities in their catalytic properties as well as in their physiological functions.

r-GLUTAMYLTRANSFERASE FROM BOVINE COLOSTRUM

For sodium dodecyl sulfate (SDS) electrophoresis, the sample proteins were previously incubated at 50°C for 2 h in 10 HIM phosphate buffer (pH 7.2) containing 10% 2-mercaptoethanol and 2 % SDS, and then applied to the separating gels (about 25 fig of protein per gel) consisting of 12 % acrylamide and 0.1 % SDS in phosphate buffer, pH 7.2. A constant current (6 mA per gel) was applied until the tracking dye, bromphenol blue, had moved about 7.5 cm into the gel. Gels were stained with Coomassie brilliant blue and, after destaining in 7 % acetic acid, were scanned in a densitometer. Isoelectric Focusing—The sample solution (25 /ig of protein in 50 fil of 5 % sucrose) was applied to 10% acrylamide gels containing 1 % Ampholine; 0.2% sulfuric acid and 1% ethanolamine were employed as anodic and cathodic electrolytes, respectively. A constant voltage (600 V) was maintained for 8 h at 4°C, then the gel ( 5 x l 5 x 150 mm) was cut lengthwise into two equal halves (2.5 mm in thickness). Both were then sliced into 5 mm slices; one sequence of slices was employed for enzyme assay and the other sequence for pH determination. Ultracetrifugation—Purified enzyme (4.6 mg/ ml) in 0.01 M Tris-HCl buffer, pH 7.2, containing 0.15 M NaCl was analyzed for sedimentation pattern with a Spinco E-type instrument. Schlieren pictures were taken at constant intervals after formation of the sedimenting boundary at 60,000 rpm at 20°C in a double sector cell at a 60° angle. Antiserum against y-Glutamyltransferase—The final preparation of colostral y-glutamyltransferase (4 mg protein) was emulsified with 2.5 ml of Freund's complete adjuvant. The emulsion was Vol. 84, No. 5, 1978

injected into the hind foot pads of a rabbit weighing about 3 kg. After 4 weeks, a booster shot, 4 mg protein, was given subcutaneously. The rabbit was fed a commercial diet ad libitum, and was bled six weeks after the first injection. Double immunodiffusion was performed in 1 % agarose gel in 0.05 M Tris-HCl buffer (pH 8) by the method of Ouchterlony (13). Purification of y-Glutamyltransftrase—All operations were carried out below 4°C unless otherwise noted. Stepl: Fresh colostrum (34 liters) was skimmed by centrifugation at 2,000 xg for 10 min. The casein of the skimmed milk (30 liters) was coagulated by incubation with rennin (3 g) plus calcium chloride (10 g) for 30 min at room temperature and was removed by straining through a layer of cheese cloth. Step 2: To the strained whey solid ammonium sulfate was added with stirring to 50% saturation. The precipitate was collected by centrifugation, suspended in one liter of 0.05 M Tris-HCl buffer (pH 7.6), and dialyzed against two changes of 10 liters each of the same buffer. Step 3: The dialyzed suspension was centrifuged at 75,000xg for 120min. The supernatant was discarded; the pellet was suspended in 380 ml of the above buffer, followed by centrifugation at 75,000 x g for 120 min. Step 4: The membrane preparation was resuspended in 200 ml of 0.1 M imidazole buffer (pH 7.0) containing papain (100 mg) and cysteine (160 mg), and the mixture was incubated at 37°C for 2 h. Solid ammonium sulfate was added to the supernatant to 50% saturation. The precipitate was removed by centrifugation, and additional ammonium sulfate was added to the supernatant to 70% saturation. The precipitate was collected by centrifugation, dissolved in 100 ml of 0.02 M Tris-HCl buffer (pH 7.6), and dialyzed overnight against 4 liters of the same buffer.


tamate dehydrogenase (cow liver) and 40 mM maleate in 1 ml of 0.2 M Tris-HCl buffer (pH 7.6). Incubation was at 3 T C for 1 h. The amount of glutamate released was determined by following the appearance of NADH as monitored at 340 nm. Electrophoresis—Polyacrylamide gel electrophoresis was carried out (72) in 7.5 % gel and 50 mM Tris-glycine buffer (pH 8.6). Immediately after electrophoresis, the gels were stained either with Commassie brilliant blue for protein or with periodic acid-Schiff reagent for carbohydrates. Unstained duplicate gels were cut into 2.5 mm slices for assay of the enzyme activity by the procedure described previously (6).

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Step 5: Volumes of one-sixth of the dialysate were separately applied to DEAE-Sephadex A-50 columns (2.5 X 35 cm) previously equilibrated with 0.03 M Tris-HCl buffer (pH 7.6). The column was washed successively with 0.03, 0.09, 0.15, and 0 . 1 8 M Tris-HCl buffers, all at pH 7.6, and the effluents were collected in 7 ml fractions. As shown in Fig. 1, the bulk of the enzyme activity was eluted with 0.18 M buffer. Fractions contain-

K. YASUMOTO, K. IWAMI, T. FUSHIKI, and H. MITSUDA

1230

TABLE I. Purification of r-glutamyltransferase from bovine colostrum

Skim milk 1. Whey 2. 0-50% (NH1),SO< 3. 75,000 x g pellet 4. Papain treatment & 50-75% (NHJtSO, 5. DEAE-Sephadex A-50 6. 1st Sephadex G-150 7. 2nd Sephadex G-150

Protein (mg)

Total enzyme units1

2,175,000 850,000 225,000 14,700

87,000 68,000 40,500 33,160

1,860

23,400

410

20,500 17,800 15,600

117 38

Specific activity (units/mg)

Purification (fold)

0.04 0.08 0.18 2.25

1

Yield (%) 100

2

78

5

47

55

38

12.6

310

27

50.0

1,250 3,800 10,170

24

152 407

20 18

» The enzyme activity was determined as described in " MATERIALS AND METHODS."

10

10 30 FRACTION NUMBER

40

Fig. 1. Chromatography of f-glutamyltransferase on DEAE-Sephadex A-50 (step 5 of the purification procedure). An aliquot of the 50-70% ammonium sulfate fraction (300 mg of protein) was added to the column (2.5 x 35 cm), and the adsorbed enzyme was then eluted with Tris-HCl buffer (pH 7.6); the stepwise increases in the buffer concentration are indicated by vertical arrows. Fractions (7 ml) were collected and analyzed for enzyme activity and for absorbance at 280 nm. The horizontal bar on the bottom represents the fractions pooled for subsequent processing. O, Enzyme activity; • , absorbance at 280 nm.

ing ^-glutamyltransferase were pooled. These operations were repeated to process the whole sample. The six pools of the active fractions were combined and concentrated to approximately 7 ml in an ultrafiltration device. Step 6: The concentrated sample was subjected in two separate portions to gel filtration on a Sephadex G-150 column (3.0x 110 cm) previously equilibrated with 0.05 M Tris-HCl buffer, pH 7.6, and eluted with the same buffer. Fractions of

high specific activity were pooled and concentrated by ultrafiltration to a volume of 3 ml. Step 7: Final purification of the enzyme was achieved by repeating gel filtration on Sephadex G-150 as in Step 6, followed by concentration by ultrafiltration. The results of a typical purification are summarized in Table I. As compared with the starting material (Table I, Fraction 1), the final preparation was purified approximately 10,000-fold with a recovery of 18% and a specific activity of 407 units per mg. RESULTS Enzyme Purification—Since we had previously located colostral 7--glutamyltransferase in skim milk fraction (6), the first procedure we followed was to collect the membrane materials. Cream was removed by centrifugation and casein was coagulated with rennin. The membrane materials were conveniently precipitated from the resulting whey by the addition of ammonium sulfate. After recovery, the membrane materials were washed by dispersion in buffer followed by low-speed centrifugation. While these washing procedures will remove any enzyme in solution or in loose association with the membrane materials, this process proved to be critically important for eliminating a large part of the entrained or absorbed contaminant proteins. The membrane preparation was then subjected to digestion with papain. The transferase J. Biochem.


Step


Isoelectric focusing: The homogeneity of the purified enzyme was further supported by isoelectric focusing on polyacrylamide gel. The enzyme activity was confined to a single segment of gel giving an isoelectric pH of 3.85 (Fig. 4). Immunodiffusion: Additional evidence for the purity of the transferase was provided by immunodiffusion experiments with antiserum developed against the purified enzyme (Fig. 5). A single precipitin line was formed between the antiserum and the purified enzyme. Thus, the purified enzyme preparation appears to be immunologically homogeneous and free from any significant protein contaminant. The enzyme fractions from DEAESephadex chromatography and the first gel filtration also gave a single precipitin line, indicating

Fig. 3. Polyacrylamide gel electrophoresis of colostral r-glutamyltransferase from step 7 of the purification procedure. The enzyme (25 fig) was subjected to electrophoresis before (A) and after (B) treatment with 0.02 activity units of neuraminidase at 37°C for 20 h in a solution (50 //I) containing 0.05 M Tns-HCI buffer (pH 7.3). The gels were stained with Coomassie brilliant blue. The direction of migration is from cathode to anode.

10

20

40

GO

80

FRACTION NUMBER Fig. 2. Gel filtration of r-glutamyltransferase on a column of Sephadex G-150 (step 6 of the purification procedure). A 3.5 ml aliquot of the enzyme pool in Fig. 1 was applied to the column (3.0x110 cm) in 0.05 M Tris-HCl buffer (pH 7.6). O, Enzyme activity; • , absorbance at 280 nm.

Vol. 84, No. 5, 1978

'o

io zo GEL SLICE NUMBER Fig. 4. Isoelectric focusing of colostral r-glutamyltransferase on polyacrylamide gel containing 1% Ampholine from pH 3.5 to 5.0. After electrofocusing, the gel was cut into two halves, and each half was sliced for the assay of enzyme activity ( O ) and pH determination ( • ) .


was reasonably resistant to papain treatment; very little of the enzyme activity was found to be lost after the treatment, and more than 70% of the transferase activity was solubilized by this procedure as judged from the enzyme activity remaining in the supernatant after centrifugation at 75,000 xg for 120 min. The solubilized enzyme was then subjected to ion-exchange chromatography on a DEAE-Sephadex column with stepwise elution. A typical elution profile is shown in Fig. 1. In another experiment, elution with a linear concentration gradient proved to take longer and did not significantly improve the total recovery of activity. The major portion of the enzyme eluted from the DEAE-Sephadex column was subjected to gel filtration on Sephadex G-150. A single peak of the enzyme activity emerged as the second protein peak (Fig. 2). Additional gel filtration was required to complete the purification. Homogeneity of the Purified Enzyme—Polyacrylamide gel electrophoresis: Electrophoresis of the purified enzyme on polyacrylamide gels indicated the presence of a single, diffuse protein band upon staining with Coomassie brilliant blue (Fig. 3A). When duplicate gels were cut into slices and one series of the slices was assayed for protein and the other for activity, the bands of protein and transferase activity corresponded well. Treatment with neuraminidase converted the purified enzyme into a single slow-migrating protein which was enzymatically active (Fig. 3B).

1231

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K. YASUMOTO, K. IWAMI, T. FUSHIKJ, and H. MITSUDA

(Ve/Vo)

Fig. 6. Molecular weight determination of colostral 7--glutamyltransferase by gel filtration on Sephadex G-150. The purified enzyme (2 mg) was applied to a Sephadex G-150 column (2.0x90 cm) previously equilibrated with 0.05 M Tns-HCl buffer, pH 7.6, and eluted with the same buffer at a flow rate of 11.5 ml/h. In separate runs, the elution volumes (Ve) of standard proteins of known molecular weight were determined relative to the exclusion volume (Vo) as measured by that skim milk membrane of bovine colostrum the use of Blue Dextran 2000. Effluents were continulacks proteins, or isozymes, which have immuno- ously monitored in terms of absorbance at 280 nm; logical cross-reactivity toward the tranferase anti- 3 ml fractions were collected and assayed for the enzyme activity. The standards were 1, yeast hexokinase; 2, serum. Molecular Weights of Native Transferase and bovine serum albumin; 3, egg albumin; 4, hog stomach pepsin; and 5, bovine pancreas trypsin.

Its Subunits—Sedimentation analysis: The purified transferase was subjected to ultracentrifugal sedimentation analysis in the presence and absence of both maleate and glutamine. In either case the enzyme sedimented as a single symmetric peak with S20,w of 5.0 consistent with other indications of its homogeneity. Gel filtration: The purified enzyme was applied to a Sephadex G-150 column (2.0x90 cm) which had been equilibrated with 0.05 M Tris-HCl buffer, pH 7.6. Figure 6 compares the relative elution volume of the enzyme with those of standard marker proteins; the molecular weight was estimated to be about 80,000 for the native enzyme. SDS-acrylamide gel.electrophoresis • On electrophoresis in SDS-polyacrylamide gels the purified enzyme after denaturation with 2% SDS gave two bands, both stainable for protein and for carbohydrates (Fig. 7). Comparison with standard proteins gave apparent molecular weights of 25,000 and 55,000 for the components; the sum of these values approximates to the molecular weight of the undissociated native form of the purified transferase. It thus follows that the colostra!

u ;-

(A) Fig. 7. SDS-polyacrylamide gel electrophoresis of colostral 7"-glutamyltransferase. The experimental details are given in " MATERIALS AND METHODS." Gels were stained with Coomassie brilliant blue (A), and with periodic acid-Schiff reagent (B). The direction of migration is from cathode to anode.

/. Biochem.


Fig. 5. Immunodiffusion of 7--glutamyltransferase antiserum against the transferase preparations at different stages of purification. The center well (Ab) contained approximately 2 ft\ of antiserum to the punfied transferase (through step 7 of Table I)- Peripheral wells contained approximately 2 p\ of the transferase preparation, the numbers given in the wells denote the purification step in Table I.


1233

TABLE II. Activity of colostral 7-glutamyltransferase in the presence of various ammo acids and peptides. Acceptor

100% 102 159 115 116 165 106 106 101 101

Acceptor

Relative activity 106%

Glycine Glycylglycine (Gly), (Gly), (Gly). (Gly). Glycyl-L-alamne Glycyl-L-senne L-a-Glutamyl-L-alanine L-Serylglycine

350 105 155 97 97 228 128 126 192

» The enzyme was assayed as described m "MATERIALS AND METHODS" except that the acceptor (at 10 HIM) was varied as indicated. The activities are expressed relative to that obtained in the absence of added acceptor.

enzyme bears a close resemblance to the kidney enzymes in the number and size of its constituent subunits. Catalytic Properties of Bovine y-Glutamyltransferase—pH dependence: The pH-activity relationship was assessed using r-glutamyl-pnitroanilide as a glutamyl donor and with or without glycylglycine as an acceptor. In the presence of the acceptor, the rate of />-nitroaniline release exhibited a sharp maximum at around pH 8.5, whereas in its absence the rate fell over a wide pH range so as to give a broad optimum centered around pH 9.0. The pH value giving half-maximal activity, on the acidic side of the pH-activity curve, was about pH 7.3 irrespective of the presence or absence of the acceptor. These maxima and halfmaxima are similar to those observed with the kidney enzyme for the transpetidation reaction (14, 15). The release of glutamic acid as a measure of the hydrolysis reaction was slight within the pH range studied; therefore, the reaction assessed in terms of />-nitroaniline release represents the predominating transpeptidation. Activation by acceptors: Table II summarizes the effects of various L-amino acids and peptides on p-nitroaniline release with the colostral transferase. The effects are quite similar to those observed with the enzyme preparations obtained from other sources (14, 16). Among the common amino acids, glutamine and methionine activated the enzyme most strongly, whereas glutamic acid Vol. 84, No. 5, 1978


No addition L-Glutamic acid L-Glutamine L-Alanine L-Senne L-Methionine L-Proline a-Aminobutyric acid /9-Aminobutync acid ^-Aminobutync acid

Relative activity*

and its iV-acetylated derivative and a-, /9- and 7--aminobutyric acids activated it least. Among glycine homopolymers, glycylglycine was the strongest activator, while free glycine, and tri-, penta-, and hexa-glycine were much less effective; the activating function of tetra-glycine was weak but still significant. Glycylalanine and serylglycine followed glycylglycine as the best activators. Stimulation by maleate of substrate hydrolysis: It has been shown that maleate inhibits the transpeptidation reaction but enhances hydrolysis catalyzed by the transferase from various sources. Similar results were obtained with the colostral enzyme preparation described here for the hydrolysis of glutathione and glutamine, two of the most physiologically likely transferase substrates (Table III). Glutathione was much less effectively hydrolyzed under the standard assay conditions; in the TABLE III. Stimulation by maleate of glutamate release from various glutamyl compounds. Glutamate released (/imol/ml-h) Glutamyl compound

Glutathione Glutamine 7--Methylglutamic acid ar-Glutamylalanine

Without maleate

With maleate

0.17 0.02 0.00 0.00

0.82 0.16 0.12 0.00

1234

K. YASUMOTO, K. IWAMI, T. FUSHIKI, and H. MITSUDA

Double reciprocal plots of p-nitroaniline release versus substrate concentration at different fixed-eoneentrations-of the acceptor glycylglycine

resulted in a family of non-linear plots apparently converging at a point. This pattern is consistent with the prediction that transpeptidation and autotranspeptidation proceed simultaneously via a ping-pong mechanism of the type proposed by Tate and Meister (19) and London et al. (20). DISCUSSION Previous studies on ^-glutamyltransferase activity in bovine milk had suggested that the enzyme is primarily associated with the membrane materials, as is the case with other animal enzymes studies to date. This view has been confirmed by analysis of the purified skim milk membrane from the colostrum (6). In our purification procedure for the enzyme, we therefore included a conventional process to remove and collect the membrane material from the skimmed colostrum, i.e. ammonium sulfate fractionation followed by high-speed centrifugation. Attempts to purify the transferase of the purified membrane to homogeneity were unsuccessful unless the enzyme was first solubilized by the application of proteolytic enzymes. The use of papain for this purpose was justified inasmuch as the enzyme was released with little change in total enzymatic activity or catalytic functions. In several respects the colostral enzyme shows striking similarities with the kidney enzymes, which have so far been most extensively studied with two general types of preparations: a "light" form with a molecular weight of 68,000-90,000 and a "heavy" form of much higher molecular weight. The "light" form can be obtained by treatment with proteolytic enzymes of either purified "heavy" form or a washed microsome fraction (21). A quite similar conversion accompanied the purification of the colostral enzyme. The molecular weight estimated for the purified colostral enzyme, 80,000, is within the range of values reported for the "light" form of the kidney enzymes. Dissociation into two heterogeneous subunits on SDS treatment of the colostral enzyme parallels that established for the kidney enzymes (15, 22). These findings support the view that conversion of the "heavy" into the "light" form by proteolytic enzymes and heterogeneous dissociation of the "light" form on SDS treatment are properties shared by membraneassociated f-glutamyltransferase.

J. Biochem.


presence of 40 ITIM maleate the hydrolysis reaction increased by a factor of 5 as judged in terms of glutamate liberation. In comparable studies with 10 mM glutamine, glutamate liberation occurred to an almost immeasurable degree in the absence of maleate, but to a significant extent in its presence at 40 mM. A similar but less marked effect of added maleate was observed for the hydrolysis of 7--methylglutamic acid. L-cr-Glutamyl-L-alanine was inactive as a substrate regardless of the presence or absence of maleate. The activating effect of maleate on the hydrolysis function was reduced by the addition of increasing concentrations of alanine. This inhibitory effect is also consistent with the previously established view that alanine and maleate act reciprocally in dissociating the catalytic functions of the transferase {17). The transpeptidase activity of the colostral transferase was 400 times greater than its maleatestimulated glutaminase activity, when assayed with 7--glutamyl-p-nitroanilide and glutamine, respectively, under the standard assay conditions. This activity ratio is similar to that reported for human kidney (10), but significantly lower than the ratio observed with rat kidney enzyme (17,18), indicating a substantial difference in maleate susceptibility with species as well as organ. Kinetic constants: Previous studies of the specificity of the transferase reaction have shown that the synthetic substrate ^-glutamyl-p-nitroanilide functions not only as a f-glutamyl donor but also as its acceptor (14). We also found by means of ascending paper chromatography in propanol/pyridine/water ( 1 : 1 : 1 ) that the major product formed by the colostral transferase from 7'-glutamyl-/>-nitroanilide was, in the absence of added acceptor, ;--glutamyl-^-gIutamyl-/>-nitroanilide (an autotranspeptidation reaction product), and that free glutamic acid was essentially absent; this indicates that no measurable hydrolysis reaction occurred under the experimental conditions used. Addition of an acceptor, glycylglycine, to the incubation mixture led to the formation of yglutamylglycylglycine, which could be readily identified by amino acid analysis after paper chromatographic separation.


Besides these membrane-associated forms, however, a small but not negligible amount of the transferase (about 8% of total enzymatic activity in the colostrum) was also found to occur in a soluble form in milk serum. It exhibited gel filtration and chromatographic behavior and catalytic properties similar to those of the solubilized "light" form of the colostral enzyme. Although this soluble form might reflect either incomplete processing of this presumably integral material into the membrane structure during fat globule secretion, or such incidental post-secretory desquamation from the membranes as would be effected by the action of endogenous protease, its origin and physiological significance warrant further investigation. The carbohydrate content of the colostral transferase, as estimated using orcinol and H,SO4, amounted to 20%. An extensive treatment of the transferase with neuraminidase decreased its mobility on polyacrylamide gel electrophoresis. These findings support the view that the colostral transferase is a sialoglycoprotein organized with other proteins into the membrane structure, and hence raise the possibility that the colostral enzyme exhibits a charge heterogeneity arising from variation in sialic acid content; Tate and Meister have resolved at least 12 charged variants of the rat kidney transferase by isoelectric focusing on Vol. 84, No. 5, 1978

polyacrylamide gel {23). However, few or no charged variants were found to occur with the colostral enzyme; the chromatogram shown in Fig. 1 provides circumstantial evidence for the occurrence of a minor variant emerging from the column earlier than the major component, but it became indistinguishable in the later stages of purification. However, we cannot completely exclude the possibility that the isolated enzyme is a partially desialylated variant of the native enzyme formed during milk secretion or enzyme purification by the action of neuraminidase, which has been found at higher levels in lactating mammary gland than in any other mammalian organs (24). There is an interesting report that "phosphateindependent glutarrunase" of rat kidney, previously shown by Katsunuma et al. (25) to be activated by maleate, is a potential catalytic function of the transferase. There is little evidence that the enzyme contributes significantly to the physiological formation of ammonia in the kidney, however, and this attractive possibility requires further study. The ability of the colostral enzyme to act on glutamine was evidently low even in the presence of maleate; this observation may rule out the possibility of a glutaminase function for the colostral transferase.


In other experiments (unpublished) we confirmed that fat globule membrane prepared from colostral cream also contains f-glutamyltransferase at a level comparable to that in skim milk membrane on a protein basis. It thus follows that the major loci of the milk transferase are the skim milk and fat globule membranes. There is considerable evidence that dunng milk secretion the milk fat globule gains a membrane which is derived with structural rearrangment and some compositional changes from the plasma membrane of the mammary cell and possibly also from Golgi vesicle membrane; at the same time, excess cell membrane material is sloughed off, appearing apparently unaltered in the skim milk (7). The demonstration that ^-glutamyltransferase, recognized generally as a plasma membrane component, constitutes a substantial portion of the proteins in both skim milk and fat globule membranes supports the view that both membranes have close evolutionary links to the plasma membrane.

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REFERENCES 1. Meister, A., Tate, S.S., & Ross, L.L. (1975) in The Enzyme of Biological Membranes (Martonosi, A., ed.) Vol. 3, pp. 315-348, Plenum Press, New York 2. Novogrodsky, A., Tate, S.S., & Meister, A. (1976) Proc. Natl. Acad. Sci. U.S. 73, 2414-2418 3. Orlowski, M. & Meister, A. (1970) Proc. Nail. Acad. Sci. U.S. 67, 1248-1255 4. Iwami, K., Yasumoto, K., Nakamura, K., & Mitsuda, H. (1975) Agric. Biol. Chem. 39, 1933 5. BmkJey, F. & Wiseman, M.L. (1976) Life Sci. 17, 1359-1362 6. Yasumoto, K., Iwami, K., Fushiki, T., & Mitsuda, H. (1976) FEBS Lett. 67, 328-330 7. Patton, S. & Keenan, T.W. (1975) Biochim. Biophys. Ada 415, 273-309 8. Kitchen, B.J. (1974) Biochim. Biophys. Ada 356, 257-269 9. Tate, S S. & Ross, E. (1977) / . Biol. Chem. 252, 6042-6045 10. Miller, S.P., Awasthi, Y.C., & Satish, K.S. (1976) /. Biol. Chem. 251, 2271-2278 11. Lowry, O.H., Rosebrough, N.J., Farr, A.L., & Randall, R J . (1951) /. Biol. Chem. 193, 265-275

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Cardiac myosin from pig heart ventricle. Purification and enzymatic properties.

Dihydrofolate reductase from bovine liver. Enzymatic and structural properties.

Purification and properties of urease from bovine rumen.

Purine nucleoside phosphorylase from bovine lens: purification and properties.

Purification and properties of bovine pituitary follitropin.

Purification and enzymatic properties of lysyl hydroxylase from fetal porcine skin.

Gamma-glutamyl transpeptidase of bovine ciliary body: purification and properties.

Purification and some physicochemical properties of bovine kappa-casein.

Bovine liver dihydrofolate reductase: purification and properties of the enzyme.

Bovine colostrum: an emerging nutraceutical.

Post-proline dipeptidyl aminopeptidase (dipeptidyl aminopeptidase IV) from lamb kidney. Purification and some enzymatic properties.

Purification and properties of four species of lysyl oxidase from bovine aorta.

Purification and properties of the cGMP-inhibited cAMP phosphodiesterase from bovine aortic smooth muscle.

Purification and properties of a protamine kinase from bovine kidney microsomes.

Purification and properties of UDP-glucose galactosylhydroxylysine collagen glucosyltransferase (EC 2.4.1.?) from bovine arterial tissue.

Purification and properties of 4-hydroxybiphenyl UDP-glucuronyltransferase from bovine liver microsomes.

Purification and some properties of a phospholipase A2 from bovine platelets.

Purification and properties of an acid phosphatase from lactating bovine mammary gland.

The purification and properties of an aryl beta-hexosidase from bovine liver.

Specific gravity of bovine colostrum immunoglobulins as affected by temperature and colostrum components.

Purification of prokallikrein from bovine pancreas.

Enzymatic properties of neuraminidases from Arthrobacter ureafaciens.

Tryptase from rat skin: purification and properties.

Purification and characterization of phospholipase A2 from bovine prostate.