- Email: [email protected]
[80] Chitin synthetase system from yeast
572
SYNTHESIS OF COMPLEX CARBOHYDRATES
[80]
[80] Chitin Synthetase System from Yeast 1
By
ENRICO CABIB
Chitin synthetase zymogen / activating factor . . . .
*Ix ....
l
activating factor inhibitor
active chitin synthetase
l
•
I
nUDP-N-acetylglucosamine -b partmle --~ n U D P -{- (N-acetylglucosamine),-particle
Yeast chitin synthetase, a particulate preparation, can be isolated in an inactive, or zymogen, form. By incubation either with trypsin or with an enzyme from yeast (activating factor) the zymogen is transformed into active forms. It is not known whether the active forms obtained with trypsin or activating factor are different or identical. The action of trypsin can be blocked with soybean trypsin inhibitor and that of activating factor with a heat-stable protein from yeast. In w~hat follows the preparation of chitin synthetase both in the zymogen and in the active form, of the activating factor and of the activating factor inhibitor will be described. I. Chitin Synthetase Zymogen Assay Method
Principle. Zymogen is first converted into an active form by incubation with trypsin• The incorporation of radioactivity from UDP-Nacetyl-n-glucosamine into insoluble particles is then measured: Reagents UDP-N-acetylglucosamine, 10 mM, labeled with 1'C in the acetylglucosamine moiety (specific activity 200,000 cpm//~mole), can be purchased commercially or prepared in the laboratory 2 Imidazole chloride, 0.5 M, pH 6.5 Crystalline trypsin, 2 mg/ml, in 0.05 M potassium phosphate, pH 7 Soybean trypsin inhibitor, 3 mg/ml, in 0.05 M potassium phosphate, pH 7 I F . A. Keller and E. Cabib, J. Biol. Chem. 246, 160 (1971); E. Cabib and F. A. Keller, J. Biol. Chem. 246, 167 (1971). Since the nature of the acceptor is not yet known, the noncomittal "chitin synthetase" is preferred to a more specific name. 2See Vol. 8 [18].
[80]
CHITIN SYNTHETASE SYSTEM FROM YEAST
573
N-Acetyl-D-glucosamine, 0.8 M Ethanol, absolute Ethanol, 66~o Ethanol, 66%, containing 0.1 M ammonium acetate Scintillation mixture: toluene, 1 liter; 2,5-diphenyloxazole, 5.35 g; 1,4-bis[2-(5-phenyloxazolyl)]benzene, 0.32 g, and CAB-O-SIL thixotropic gel (Packard Instrument Company), 43 g Procedure. ACTIVATION STEP. The incubation mixture contains 20 ~1 of zymogen suspension, 3 ~l of imidazole buffer, varying amounts of trypsin (see below), and water in a total volume of 43 ~l. After incubation for 15 minutes at 30 ° the reaction is stopped by adding a volume of trypsin inhibitor solution identical to the volume of trypsin used in the corresponding reaction mixture, and the tubes are immediately cooled in ice. The amount of trypsin necessary for maximal activation varies somewhat from preparation to preparation of zymogen. Also, with aged preparations, the optimal amount of trypsin is lower and an excess of the proteolytic enzyme leads to a substantial loss in chitin synthetase activity. Therefore, amounts of trypsin solution from 1 to 4 ~l are tried. With a fresh preparation of zymogen, the optimum amount of trypsin is usually about 2 #l. 3 AssAY STEP. To each incubation mixture from the activation step, 2 ~l of N-acetylglucosamine and 5 ~l of UDP-[14C]N-acetylglucosamine are added. Incubation is for 30 or 60 minutes at 30 ° . The reaction is stopped by adding 1 ml of 66% ethanol, and the tubes are centrifuged for 5 minutes at 1500 g. The pellets are washed twice with 1 ml of 66% ethanol containing 0.1 M ammonium acetate, and resuspended in 0.4 ml of absolute ethanol by stirring on a Vortex mixer at maximum speed. The suspension is poured into a scintillation vial, followed by a washing of the tube with 0.4 ml of absolute ethanol. Scintillation mixture (12 ml) is added, and the vials are vigorously stirred on the Vortex mixer and counted in a scintillation spectrometer. Note. I t should be noticed that the Mg 2÷ necessary for chitin synthetase action is contained in the enzyme preparation, which is suspended in buffer with 2 m M MgS04. If less enzyme is used, or the particles are suspended in Mg2÷-free buffer, the divalent cation should be added separately. The activation step can also be carried out with an excess of activating factor instead of trypsin. For details of the procedure see Assay of Activating Factor in Section III. The maximal activity obtained is usually about two-thirds of that induced by trypsin.
574
SYNTHESIS OF COMPLEX CARBOHYDRATES
[80]
Definition o] Unit. One unit of zymogen is that amount which will yield 1 unit of active chitin synthetase (see Section II) when maximally activated. Preparation of Enzyme All operations are carried out at 0-2 ° , unless otherwise stated. Yeast Growth. Saccharomyces cerevisiae $288C, a haploid of the mating type (available from Dr. G. Fink, Department of Genetics, Cornell University, Ithaca, New York) is grown at 28-30 ° in a medium containing 1% yeast extract, 2% peptone, and 2% glucose. The cells are harvested by centrifugation during the logarithmic phase, at an absorbance of 0.3, as measured at 660 nm with the Coleman Junior spectrophotometer and 1-cm diameter cuvettes. The yeast is washed twice with distilled water and stored in the refrigerator until used. No decrease in enzyme activity was observed after 48 hours of storage. Spheroplast Preparation. Spheroplasts are obtained and washed as described in Vol. 22 [14]. The final spheroplast dilution is such that, for each gram of yeast (wet weight) used, 0.67 ml of spheroplast suspension is obtained. Lysis of Spheroplasts and Preparation of Particles. The spheroplast suspension is added dropwise, while stirring with a Vortex mixer, to 5 volumes of Buffer A~ containing 0.385 M mannitol. At brief intervals further additions are made of 3 and 5 volumes, respectively, of Buffer A.4 The suspension is then submitted to sonic oscillation in 15-ml portions, using the microtip of a Branson Sonifier Model $75 for three 10-second periods at setting 1 and two 5-second periods at setting 2, both at optimal tuning, while cooling in ice. If a different sonic oscillator is used, the criterion to be followed should be to use the minimal treatment, both in intensity and time, that will lead to almost complete disappearance of round forms of spheroplast size, as observed in the phase contrast microscope. The sonically treated suspension is centrifuged for 10 minutes at 20,000 g, and the pellet is washed twice with an amount of Buffer A4 equivalent to about 4 volumes of the original spheroplast suspension. If the zymogen is going to be used immediately the final pellet is resuspended, in Buffer A,~ to give a total volume equal to that of the original spheroplast suspension. If the zymogen must be stored, the final suspension, in the same buffer, should contain 33% glycerol. This suspension can be stored at - 1 0 ° without freezing. Before use, glycerol is eliminated by washing the particles twice with Buffer A~ by centrifugation and ' Imidazole chloride, 50 mM, at pH 6.5, containing 2 mM MgSO~.
[80]
CHITIN SYNTHETASE SYSTEM FROM YEAST
575
resuspending them in the same buffer. The preparation contains about 10 mg of protein per milliliter. Storage with glycerol results in a loss of about 30% of the activity in a week. Zymogen can also be prepared successfully from Saccharomyces carlsbergensis 74 S (National Collection of Yeast Cultures, England), using the above described technique. II. Active Chitin Synthetase
Assay Method The reagents and procedure are the same as used in the assay step of the measurement of zymogen (see Section I). The incubation mixture contains 20 ~l of active chitin synthetase, 3 ~l of 0.5 M imidazole chloride, pH 6.5, 5 ~l of 10 mM UDP-[14C]N-acetylglucosamine, 2 ~l of 0.8M N-acetylglucosamine, and water in a total volume of 52 ~l. Definition o] Unit. One unit of chitin synthetase is that amount which catalyzes the incorporation of 1 #mole of acetylglucosamine per minute into chitin. Enzyme Preparation Active forms of chitin synthetase can be obtained by incubating zymogen (see Section I) either with trypsin or with activating factor. The conditions for the former are given in Section I and for the latter in Section I I I ; the incubation mixtures can be scaled up as required. After incubating with trypsin or activating factor and stopping the reaction with soybean trypsin inhibitor or with activating factor inhibitor, respectively, the activated particles may be centrifuged, washed with Buffer A4 and resuspended in the same buffer. Properties Most properties were examined on preparations of zymogen contaminated with activating factor, 1,5 in which activation took place during the assay. Therefore, some of the kinetic data are subject to revision. Stability. Chitin synthetase has not been stored in the active form. Data are only available for the zymogen (see Section I). Kinetics. The pH optimum is around 6.2. The Km value for UDP-Nacetylglueosamine is between 0.6 and 0.9 mM. Activators. The enzyme has an absolute requirement for divalent cations. Mg 2÷ shows a broad optimum between I and 20 mM, whereas Mn 2÷ is maximally effective at 1 raM. Independently from the effect of E. Cabib and V. Farkas, Proc. Nat. Acad. Sci. U.S. 68, 2052 (1971).
576
SYNTHESIS OF COMPLEX CARBOHYDRATES
[80]
cations, free N-acetylglucosamine increases 3- to 5-fold the enzymatic activity, with a Km of 4.7 mM. Other polyhydric compounds, such as glucose and glycerol, are also effective, but at much higher concentrations. Inhibitors. The enzyme is strongly and competitively inhibited by the antibiotic Polyoxin A (K~ = 5 X 10-7 M). UDP, a reaction product, is also inhibitory, whereas other nucleotides are less effective. III. Chitin Synthetase Activating Factor
Assay Method
Principle. Chitin synthetase zymogen is incubated with different amounts of activating factor. After stopping the reaction with excess activating factor inhibitor, the amount of active chitin synthetase formed is measured. Reagents Chitin synthetase zymogen, see Section I Activating factor inhibitor, see Section IV Imidazole chloride, pH 6.5, 50 mM For other reagents see assay method for chitin synthetase zymogen (Section I). Procedure. ACTIVATION STEP. The incubation mixture contains 20 ~l of zymogen, 1 ~l of 40 mM MgS04, 1 /~l of 0.5 M imidazole chloride, pH 6.5, different amounts of activating factor (20 ~l maximum) and 0.05 M imidazole chloride, pH 6.5 to complete a total volume of 42 ~l. Controls without activating factor are included. Incubation is carried out for 30 minutes at 30 ° , and the reaction is stopped by adding an excess of activating factor inhibitor in 3 ~l (see Section IV), and cooling the tubes immediately in ice. AssAY STEP. This step is carried out exactly in the same way as the assay step for zymogen (see Section I above). Definition of Unit. One unit of activating factor is that amount which causes the formation of 1 unit of chitin synthetase activity (see Section II) in 30 minutes.
Preparation of Enzyme Principle. Activating factor accompanies the particles which contain chitin synthetase, when these are obtained without making use of sonic oscillation. A mild sonic treatment then releases the activating factor in the soluble state. All operations are carried out at 0-2 °, except where otherwise stated.
[80]
CHITIN SYNTHETASE SYSTEM FROM YEAST
577
Yeast Growth. S. cerevisiae $288C is grown at 28-30 ° in a medium containing 2% glucose and 0.7% Bacto Yeast nitrogen base, up to the onset of the stationary phase (absorbancy at 660 nm between 0.4 and 0.45, as measured in a Coleman Junior spectrophotometer with 1-cm diameter cuvettes). The cells are harvested by centrifugation, washed twice with distilled water, and stored in the refrigerator until used. Spheroplast Preparation. See Section I. Since the cell wall of stationary phase cells is more resistant to the action of snail enzyme, the amount of the latter is doubled. It is also convenient to increase the concentration of mannitol to 1 M, both during incubation and washing, because of the fragility of spheroplasts from stationary phase yeast. Solubilization and Ammonium SulIate Precipitation. Spheroplast lysis, centrifugation and washing of the particulate fraction, and final resuspension in Buffer A,4 are carried out as for the preparation of zymogen, but omitting the sonic oscillation. The resuspended particles are submitted to two 5-second periods of sonic oscillation, using the microtip of the Branson sonifier at setting 1 and optimal tuning, and centrifuged for 10 minutes at 20,000 g. The supernatant fluid, including a layer of loose particles, is removed and incubated for 20 minutes at 37 ° . This incubation increases the yield of activating factor for unknown reasons. The suspension is then centrifuged for 30 minutes at 100,000 g. The clear supernatant is removed and frozen overnight. After thawing, an abundant precipitate of denatured protein is removed by centrifugation for 10 minutes at 20,000 g. Activating factor is precipitated from the supernatant fluid by adding 475 mg of solid ammounium sulfate per milliliter. The pellet is redissolved in 0.05 M imidazole, pH 6.5, containing 1 mM EDTA, in a total volume one-third that of the original, and dialyzed against two changes of the same buffer for 3 hours. Glycerol is added to the dialyzate to a final concentration of 33%. The yield in the ammonium sulfate step is around 70% and the specific activity of the product ranges between 15 and 35 munits per milligram of protein .6 Properties
Stability. The purified activating factor is stable for at least a month when stored at - 2 0 ° in the presence of glycerol. Specificity. Activating factor from S. cerevisiae is equally effective on zymogens from the same yeast or from S. carlsbergensis 74 S. Kinetics. The transformation of chitin synthetase zymogen into active enzyme produced by the activating factor shows a linear dependence on Attempts to prepare activating factor from S. carlsbergensis 74 S, using the method described for S. cerevisiae, have been unsuccessful so far.
578
SYNTHESIS OF COMPLEX CARBOHYDRATES
[80]
amount of activating factor added or time, until about two-thirds of zymogen has been converted. Whereas active chitin synthetase depends on a divalent cation, no such requirement was found for the activation of zymogen. Inhibitor. Activating factor is stoichiometrically inhibited by a heat-stable protein from yeast, the preparation of which is described in Section IV. In the same Section, directions for titrating activating factor against the inhibitor are given. IV. Activating Factor Inhibitor 1,5 Assay Method
Prhwiple. The inhibitor binds tightly to the activating factor for chitin synthetase zymogen. Addition of increasing amounts of inhibitor to an activation mixture produces a linear decrease in the amount of active chitin synthetase formed. Reagents Chitin synthetase zymogen, see Section Chitin synthetase activating factor, see Other reagents are the same used in the synthetase zymogen and for activating
I Section III assay methods for chitin factor.
Procedure. ACTIVATION STEP. The incubation mixture contains 20 tL1 of chitin synthetase zymogen, 1 t~l of 0.5 M imidazole chloride, pH 6.5, 1 tL1of 40 mM MgS04, 3 ~l of an appropriate dilution of activating factor, variable amounts of inhibitor in a volume not exceeding 15 #l, and 0.05 M imidazole chloride, pH 6.5, to complete a volume of 42 td. When dilutions of purified inhibitor are used, the diluting medium is 0.05M MES, ~ pH 6.1, containing 2 mg/ml of bovine serum albumin. Controls lacking inhibitor or both inhibitor and activating factor are included. Incubation is for 30 minutes at 30 ° . The reaction is stopped, by adding more inhibitor, in excess over the activating factor present and the tubes are immediately cooled in ice. AssAY STEP. This step is carried out as for the assay step for zymogen (see Section I above). The amount of activating factor used in the assay must be in the linear range of chitin synthetase activation, i.e., an amount which causes not more than 60-70% of the maximal activation. A plot of chitin synthetase activity as a function of added inhibitor should show a linear decrease2 The amount of inhibitor present is calculated from the linear portion of the graph. Since the determination involves the difference between relatively large numbers, the error is also YMES, 2-(N-morpholino)ethanesulfonic acid.
[80]
CHITIN SYNTHETASE SYSTEM FROM YEAST
579
large. It is recommended to include in the determination several amounts of inhibitor, each one in duplicate. Obviously, the method can also be used to assay activating factor, if the concentration of the inhibitor solution is known. Definition o] Unit. One unit of inhibitor is that amount which will neutralize 1 unit of activating factor (see Section III). Purification of Inhibitor from S. carlsbergensis 74 S All operations are carried out between 0 and 5 °, except where stated otherwise. A typical preparation is described below. Yeast Growth. S. earlsbergensis 74 S is grown in a medium containing, per liter, 3 g of Bacto malt extract, 3 g of Bacto yeast extract, 5 g of Bacto peptone, and 20 g of glucose, at 28-30 °, to the early stationary phase. From 9 liters of culture medium, about 144 g of yeast are obtained after centrifuging and washing three times with distilled water. The yeast is frozen overnight. Step 1. Extraction and Concentration. The thawed cells are suspended in 191 ml of boiling 50 mM imidazole chloride, pH 6.5, and boiled for 3 minutes. After rapid cooling in ice, the suspension is centrifuged for 10 minutes at 27,000 g. The pellet is reextracted once with 96 ml of the same buffer, and both supernatant liquids are combined. The extract, 317 ml, is concentrated to a final volume of 27 ml in a rotary evaporator at room temperature and dialyzed against two 3-liter changes of distilled water for 16 hours. The faintly turbid solution remaining in the dialysis bag is centrifuged for 10 minutes at 27,000 g and the pellet is discarded. Step 2. Treatment with DEAE-CeUulose. To the centrifuged dialyzate (34 ml) an equal volume of 50 mM imidazole chloride, pH 7.2, is added and the solution is passed, at room temperature, through a microgranular DEAE-cellulose (DE-52 from H. Reeve Angel Company, New York, New York) column (2.5 × 16 cm), which has been previously equilibrated with 25 mM imidazole chloride at pH 7.2. The column is washed with 152 ml of the same buffer, and the washing is combined with the first filtrate (total volume 220 ml). Step 3. Chromatography on CarboxymethylceUulose. The DEAE eluate is concentrated to 20 ml in a rotary evaporator at room temperature and applied to a Sephadex G-25 (fine) column (2.5 × 36 cm) previously equilibrated with 25 mM MES at pH 6.1. The same buffer is used for subsequent washing of the column. After the void volume, the next 40 ml are collecteds and directly applied to a microgranular carboxymethyl cellulose (CM-52, H. Reeve Angel Company, New York, 8In later experiments the Sephadex filtration, which sometimes results in low recoveries, has been substituted by dialysis against 25 mM MES, pH 6.1.
580
SYNTHESIS OF COMPLEX CARBOHYDRATES
[80]
New York) column (2.5 × 10 cm), previously equilibrated with 25 mM MES, pH 6.1. The column is then washed with 36 ml of the same buffer and a linear sodium chloride gradient is applied. The mixing chamber contains 250 ml of 25 mM MES, pH 6.1, and the reservoir 250 ml of the same buffer, to which NaC1 has been added to a final concentration of 0.4 M. Fractions of 10 ml are collected and assayed for inhibitor content. The fractions containing the bulk of the inhibiting activity (No. 21 tq 27 in the preparation described), are pooled and concentrated to 3.5 ml by ultrafiltration through an Amicon Diaflo filter, type UM-10. In the case described, the overall purification achieved was 65-fold and the protein concentration in the most purified fraction, 0.34 mg/ml. Purification of Inhibitor from S. cerevisiae 8288C The procedure is the same as for S. carlsbergens-is, with the following modifications: (1) The yeast is grown in a medium containing 1 ~ yeast extract, 2% peptone, and 2% glucose. (2) In the DEAE-cellulose step, the inhibitor remains adsorbed on the exchanger after passage and washing. It is then eluted with 150 ml of 25 mM imidazole chloride at pH 7.2, containing 0.1 M NaC1, and 'the purification is continued as described in the preceding section. Properties
Stability. The inhibitor appears to be quite stable when stored at - 2 0 ° . Even the purified preparations can be boiled without loss of activity. Nevertheless, activity is lost when the purified inhibitor is diluted in the absence of albumin. This inactivation appears to be due to adsorption to glass, because it is much less marked when polypropylene material is used. Purity. The purified preparations, both from S. carlsbergensis 74S and from S. cerevisiae $288C, when submitted to disc gel electrophoresis, ~ give a single major band which coincides with the inhibitory activity. On increasing the amount of protein applied to the gel, several minor bands appear. Specificity. An absolute quantitative comparison of the inhibitory activity of the preparations from S. carlsbergensis and S. cerevisiae has not yet been made. The inhibitor from each species is effective against the activating factors both of the same and of the other species.
oSee Vol. 22 [39].
SYNTHESIS OF COMPLEX CARBOHYDRATES
[80]
[80] Chitin Synthetase System from Yeast 1
By
ENRICO CABIB
Chitin synthetase zymogen / activating factor . . . .
*Ix ....
l
activating factor inhibitor
active chitin synthetase
l
•
I
nUDP-N-acetylglucosamine -b partmle --~ n U D P -{- (N-acetylglucosamine),-particle
Yeast chitin synthetase, a particulate preparation, can be isolated in an inactive, or zymogen, form. By incubation either with trypsin or with an enzyme from yeast (activating factor) the zymogen is transformed into active forms. It is not known whether the active forms obtained with trypsin or activating factor are different or identical. The action of trypsin can be blocked with soybean trypsin inhibitor and that of activating factor with a heat-stable protein from yeast. In w~hat follows the preparation of chitin synthetase both in the zymogen and in the active form, of the activating factor and of the activating factor inhibitor will be described. I. Chitin Synthetase Zymogen Assay Method
Principle. Zymogen is first converted into an active form by incubation with trypsin• The incorporation of radioactivity from UDP-Nacetyl-n-glucosamine into insoluble particles is then measured: Reagents UDP-N-acetylglucosamine, 10 mM, labeled with 1'C in the acetylglucosamine moiety (specific activity 200,000 cpm//~mole), can be purchased commercially or prepared in the laboratory 2 Imidazole chloride, 0.5 M, pH 6.5 Crystalline trypsin, 2 mg/ml, in 0.05 M potassium phosphate, pH 7 Soybean trypsin inhibitor, 3 mg/ml, in 0.05 M potassium phosphate, pH 7 I F . A. Keller and E. Cabib, J. Biol. Chem. 246, 160 (1971); E. Cabib and F. A. Keller, J. Biol. Chem. 246, 167 (1971). Since the nature of the acceptor is not yet known, the noncomittal "chitin synthetase" is preferred to a more specific name. 2See Vol. 8 [18].
[80]
CHITIN SYNTHETASE SYSTEM FROM YEAST
573
N-Acetyl-D-glucosamine, 0.8 M Ethanol, absolute Ethanol, 66~o Ethanol, 66%, containing 0.1 M ammonium acetate Scintillation mixture: toluene, 1 liter; 2,5-diphenyloxazole, 5.35 g; 1,4-bis[2-(5-phenyloxazolyl)]benzene, 0.32 g, and CAB-O-SIL thixotropic gel (Packard Instrument Company), 43 g Procedure. ACTIVATION STEP. The incubation mixture contains 20 ~1 of zymogen suspension, 3 ~l of imidazole buffer, varying amounts of trypsin (see below), and water in a total volume of 43 ~l. After incubation for 15 minutes at 30 ° the reaction is stopped by adding a volume of trypsin inhibitor solution identical to the volume of trypsin used in the corresponding reaction mixture, and the tubes are immediately cooled in ice. The amount of trypsin necessary for maximal activation varies somewhat from preparation to preparation of zymogen. Also, with aged preparations, the optimal amount of trypsin is lower and an excess of the proteolytic enzyme leads to a substantial loss in chitin synthetase activity. Therefore, amounts of trypsin solution from 1 to 4 ~l are tried. With a fresh preparation of zymogen, the optimum amount of trypsin is usually about 2 #l. 3 AssAY STEP. To each incubation mixture from the activation step, 2 ~l of N-acetylglucosamine and 5 ~l of UDP-[14C]N-acetylglucosamine are added. Incubation is for 30 or 60 minutes at 30 ° . The reaction is stopped by adding 1 ml of 66% ethanol, and the tubes are centrifuged for 5 minutes at 1500 g. The pellets are washed twice with 1 ml of 66% ethanol containing 0.1 M ammonium acetate, and resuspended in 0.4 ml of absolute ethanol by stirring on a Vortex mixer at maximum speed. The suspension is poured into a scintillation vial, followed by a washing of the tube with 0.4 ml of absolute ethanol. Scintillation mixture (12 ml) is added, and the vials are vigorously stirred on the Vortex mixer and counted in a scintillation spectrometer. Note. I t should be noticed that the Mg 2÷ necessary for chitin synthetase action is contained in the enzyme preparation, which is suspended in buffer with 2 m M MgS04. If less enzyme is used, or the particles are suspended in Mg2÷-free buffer, the divalent cation should be added separately. The activation step can also be carried out with an excess of activating factor instead of trypsin. For details of the procedure see Assay of Activating Factor in Section III. The maximal activity obtained is usually about two-thirds of that induced by trypsin.
574
SYNTHESIS OF COMPLEX CARBOHYDRATES
[80]
Definition o] Unit. One unit of zymogen is that amount which will yield 1 unit of active chitin synthetase (see Section II) when maximally activated. Preparation of Enzyme All operations are carried out at 0-2 ° , unless otherwise stated. Yeast Growth. Saccharomyces cerevisiae $288C, a haploid of the mating type (available from Dr. G. Fink, Department of Genetics, Cornell University, Ithaca, New York) is grown at 28-30 ° in a medium containing 1% yeast extract, 2% peptone, and 2% glucose. The cells are harvested by centrifugation during the logarithmic phase, at an absorbance of 0.3, as measured at 660 nm with the Coleman Junior spectrophotometer and 1-cm diameter cuvettes. The yeast is washed twice with distilled water and stored in the refrigerator until used. No decrease in enzyme activity was observed after 48 hours of storage. Spheroplast Preparation. Spheroplasts are obtained and washed as described in Vol. 22 [14]. The final spheroplast dilution is such that, for each gram of yeast (wet weight) used, 0.67 ml of spheroplast suspension is obtained. Lysis of Spheroplasts and Preparation of Particles. The spheroplast suspension is added dropwise, while stirring with a Vortex mixer, to 5 volumes of Buffer A~ containing 0.385 M mannitol. At brief intervals further additions are made of 3 and 5 volumes, respectively, of Buffer A.4 The suspension is then submitted to sonic oscillation in 15-ml portions, using the microtip of a Branson Sonifier Model $75 for three 10-second periods at setting 1 and two 5-second periods at setting 2, both at optimal tuning, while cooling in ice. If a different sonic oscillator is used, the criterion to be followed should be to use the minimal treatment, both in intensity and time, that will lead to almost complete disappearance of round forms of spheroplast size, as observed in the phase contrast microscope. The sonically treated suspension is centrifuged for 10 minutes at 20,000 g, and the pellet is washed twice with an amount of Buffer A4 equivalent to about 4 volumes of the original spheroplast suspension. If the zymogen is going to be used immediately the final pellet is resuspended, in Buffer A,~ to give a total volume equal to that of the original spheroplast suspension. If the zymogen must be stored, the final suspension, in the same buffer, should contain 33% glycerol. This suspension can be stored at - 1 0 ° without freezing. Before use, glycerol is eliminated by washing the particles twice with Buffer A~ by centrifugation and ' Imidazole chloride, 50 mM, at pH 6.5, containing 2 mM MgSO~.
[80]
CHITIN SYNTHETASE SYSTEM FROM YEAST
575
resuspending them in the same buffer. The preparation contains about 10 mg of protein per milliliter. Storage with glycerol results in a loss of about 30% of the activity in a week. Zymogen can also be prepared successfully from Saccharomyces carlsbergensis 74 S (National Collection of Yeast Cultures, England), using the above described technique. II. Active Chitin Synthetase
Assay Method The reagents and procedure are the same as used in the assay step of the measurement of zymogen (see Section I). The incubation mixture contains 20 ~l of active chitin synthetase, 3 ~l of 0.5 M imidazole chloride, pH 6.5, 5 ~l of 10 mM UDP-[14C]N-acetylglucosamine, 2 ~l of 0.8M N-acetylglucosamine, and water in a total volume of 52 ~l. Definition o] Unit. One unit of chitin synthetase is that amount which catalyzes the incorporation of 1 #mole of acetylglucosamine per minute into chitin. Enzyme Preparation Active forms of chitin synthetase can be obtained by incubating zymogen (see Section I) either with trypsin or with activating factor. The conditions for the former are given in Section I and for the latter in Section I I I ; the incubation mixtures can be scaled up as required. After incubating with trypsin or activating factor and stopping the reaction with soybean trypsin inhibitor or with activating factor inhibitor, respectively, the activated particles may be centrifuged, washed with Buffer A4 and resuspended in the same buffer. Properties Most properties were examined on preparations of zymogen contaminated with activating factor, 1,5 in which activation took place during the assay. Therefore, some of the kinetic data are subject to revision. Stability. Chitin synthetase has not been stored in the active form. Data are only available for the zymogen (see Section I). Kinetics. The pH optimum is around 6.2. The Km value for UDP-Nacetylglueosamine is between 0.6 and 0.9 mM. Activators. The enzyme has an absolute requirement for divalent cations. Mg 2÷ shows a broad optimum between I and 20 mM, whereas Mn 2÷ is maximally effective at 1 raM. Independently from the effect of E. Cabib and V. Farkas, Proc. Nat. Acad. Sci. U.S. 68, 2052 (1971).
576
SYNTHESIS OF COMPLEX CARBOHYDRATES
[80]
cations, free N-acetylglucosamine increases 3- to 5-fold the enzymatic activity, with a Km of 4.7 mM. Other polyhydric compounds, such as glucose and glycerol, are also effective, but at much higher concentrations. Inhibitors. The enzyme is strongly and competitively inhibited by the antibiotic Polyoxin A (K~ = 5 X 10-7 M). UDP, a reaction product, is also inhibitory, whereas other nucleotides are less effective. III. Chitin Synthetase Activating Factor
Assay Method
Principle. Chitin synthetase zymogen is incubated with different amounts of activating factor. After stopping the reaction with excess activating factor inhibitor, the amount of active chitin synthetase formed is measured. Reagents Chitin synthetase zymogen, see Section I Activating factor inhibitor, see Section IV Imidazole chloride, pH 6.5, 50 mM For other reagents see assay method for chitin synthetase zymogen (Section I). Procedure. ACTIVATION STEP. The incubation mixture contains 20 ~l of zymogen, 1 ~l of 40 mM MgS04, 1 /~l of 0.5 M imidazole chloride, pH 6.5, different amounts of activating factor (20 ~l maximum) and 0.05 M imidazole chloride, pH 6.5 to complete a total volume of 42 ~l. Controls without activating factor are included. Incubation is carried out for 30 minutes at 30 ° , and the reaction is stopped by adding an excess of activating factor inhibitor in 3 ~l (see Section IV), and cooling the tubes immediately in ice. AssAY STEP. This step is carried out exactly in the same way as the assay step for zymogen (see Section I above). Definition of Unit. One unit of activating factor is that amount which causes the formation of 1 unit of chitin synthetase activity (see Section II) in 30 minutes.
Preparation of Enzyme Principle. Activating factor accompanies the particles which contain chitin synthetase, when these are obtained without making use of sonic oscillation. A mild sonic treatment then releases the activating factor in the soluble state. All operations are carried out at 0-2 °, except where otherwise stated.
[80]
CHITIN SYNTHETASE SYSTEM FROM YEAST
577
Yeast Growth. S. cerevisiae $288C is grown at 28-30 ° in a medium containing 2% glucose and 0.7% Bacto Yeast nitrogen base, up to the onset of the stationary phase (absorbancy at 660 nm between 0.4 and 0.45, as measured in a Coleman Junior spectrophotometer with 1-cm diameter cuvettes). The cells are harvested by centrifugation, washed twice with distilled water, and stored in the refrigerator until used. Spheroplast Preparation. See Section I. Since the cell wall of stationary phase cells is more resistant to the action of snail enzyme, the amount of the latter is doubled. It is also convenient to increase the concentration of mannitol to 1 M, both during incubation and washing, because of the fragility of spheroplasts from stationary phase yeast. Solubilization and Ammonium SulIate Precipitation. Spheroplast lysis, centrifugation and washing of the particulate fraction, and final resuspension in Buffer A,4 are carried out as for the preparation of zymogen, but omitting the sonic oscillation. The resuspended particles are submitted to two 5-second periods of sonic oscillation, using the microtip of the Branson sonifier at setting 1 and optimal tuning, and centrifuged for 10 minutes at 20,000 g. The supernatant fluid, including a layer of loose particles, is removed and incubated for 20 minutes at 37 ° . This incubation increases the yield of activating factor for unknown reasons. The suspension is then centrifuged for 30 minutes at 100,000 g. The clear supernatant is removed and frozen overnight. After thawing, an abundant precipitate of denatured protein is removed by centrifugation for 10 minutes at 20,000 g. Activating factor is precipitated from the supernatant fluid by adding 475 mg of solid ammounium sulfate per milliliter. The pellet is redissolved in 0.05 M imidazole, pH 6.5, containing 1 mM EDTA, in a total volume one-third that of the original, and dialyzed against two changes of the same buffer for 3 hours. Glycerol is added to the dialyzate to a final concentration of 33%. The yield in the ammonium sulfate step is around 70% and the specific activity of the product ranges between 15 and 35 munits per milligram of protein .6 Properties
Stability. The purified activating factor is stable for at least a month when stored at - 2 0 ° in the presence of glycerol. Specificity. Activating factor from S. cerevisiae is equally effective on zymogens from the same yeast or from S. carlsbergensis 74 S. Kinetics. The transformation of chitin synthetase zymogen into active enzyme produced by the activating factor shows a linear dependence on Attempts to prepare activating factor from S. carlsbergensis 74 S, using the method described for S. cerevisiae, have been unsuccessful so far.
578
SYNTHESIS OF COMPLEX CARBOHYDRATES
[80]
amount of activating factor added or time, until about two-thirds of zymogen has been converted. Whereas active chitin synthetase depends on a divalent cation, no such requirement was found for the activation of zymogen. Inhibitor. Activating factor is stoichiometrically inhibited by a heat-stable protein from yeast, the preparation of which is described in Section IV. In the same Section, directions for titrating activating factor against the inhibitor are given. IV. Activating Factor Inhibitor 1,5 Assay Method
Prhwiple. The inhibitor binds tightly to the activating factor for chitin synthetase zymogen. Addition of increasing amounts of inhibitor to an activation mixture produces a linear decrease in the amount of active chitin synthetase formed. Reagents Chitin synthetase zymogen, see Section Chitin synthetase activating factor, see Other reagents are the same used in the synthetase zymogen and for activating
I Section III assay methods for chitin factor.
Procedure. ACTIVATION STEP. The incubation mixture contains 20 tL1 of chitin synthetase zymogen, 1 t~l of 0.5 M imidazole chloride, pH 6.5, 1 tL1of 40 mM MgS04, 3 ~l of an appropriate dilution of activating factor, variable amounts of inhibitor in a volume not exceeding 15 #l, and 0.05 M imidazole chloride, pH 6.5, to complete a volume of 42 td. When dilutions of purified inhibitor are used, the diluting medium is 0.05M MES, ~ pH 6.1, containing 2 mg/ml of bovine serum albumin. Controls lacking inhibitor or both inhibitor and activating factor are included. Incubation is for 30 minutes at 30 ° . The reaction is stopped, by adding more inhibitor, in excess over the activating factor present and the tubes are immediately cooled in ice. AssAY STEP. This step is carried out as for the assay step for zymogen (see Section I above). The amount of activating factor used in the assay must be in the linear range of chitin synthetase activation, i.e., an amount which causes not more than 60-70% of the maximal activation. A plot of chitin synthetase activity as a function of added inhibitor should show a linear decrease2 The amount of inhibitor present is calculated from the linear portion of the graph. Since the determination involves the difference between relatively large numbers, the error is also YMES, 2-(N-morpholino)ethanesulfonic acid.
[80]
CHITIN SYNTHETASE SYSTEM FROM YEAST
579
large. It is recommended to include in the determination several amounts of inhibitor, each one in duplicate. Obviously, the method can also be used to assay activating factor, if the concentration of the inhibitor solution is known. Definition o] Unit. One unit of inhibitor is that amount which will neutralize 1 unit of activating factor (see Section III). Purification of Inhibitor from S. carlsbergensis 74 S All operations are carried out between 0 and 5 °, except where stated otherwise. A typical preparation is described below. Yeast Growth. S. earlsbergensis 74 S is grown in a medium containing, per liter, 3 g of Bacto malt extract, 3 g of Bacto yeast extract, 5 g of Bacto peptone, and 20 g of glucose, at 28-30 °, to the early stationary phase. From 9 liters of culture medium, about 144 g of yeast are obtained after centrifuging and washing three times with distilled water. The yeast is frozen overnight. Step 1. Extraction and Concentration. The thawed cells are suspended in 191 ml of boiling 50 mM imidazole chloride, pH 6.5, and boiled for 3 minutes. After rapid cooling in ice, the suspension is centrifuged for 10 minutes at 27,000 g. The pellet is reextracted once with 96 ml of the same buffer, and both supernatant liquids are combined. The extract, 317 ml, is concentrated to a final volume of 27 ml in a rotary evaporator at room temperature and dialyzed against two 3-liter changes of distilled water for 16 hours. The faintly turbid solution remaining in the dialysis bag is centrifuged for 10 minutes at 27,000 g and the pellet is discarded. Step 2. Treatment with DEAE-CeUulose. To the centrifuged dialyzate (34 ml) an equal volume of 50 mM imidazole chloride, pH 7.2, is added and the solution is passed, at room temperature, through a microgranular DEAE-cellulose (DE-52 from H. Reeve Angel Company, New York, New York) column (2.5 × 16 cm), which has been previously equilibrated with 25 mM imidazole chloride at pH 7.2. The column is washed with 152 ml of the same buffer, and the washing is combined with the first filtrate (total volume 220 ml). Step 3. Chromatography on CarboxymethylceUulose. The DEAE eluate is concentrated to 20 ml in a rotary evaporator at room temperature and applied to a Sephadex G-25 (fine) column (2.5 × 36 cm) previously equilibrated with 25 mM MES at pH 6.1. The same buffer is used for subsequent washing of the column. After the void volume, the next 40 ml are collecteds and directly applied to a microgranular carboxymethyl cellulose (CM-52, H. Reeve Angel Company, New York, 8In later experiments the Sephadex filtration, which sometimes results in low recoveries, has been substituted by dialysis against 25 mM MES, pH 6.1.
580
SYNTHESIS OF COMPLEX CARBOHYDRATES
[80]
New York) column (2.5 × 10 cm), previously equilibrated with 25 mM MES, pH 6.1. The column is then washed with 36 ml of the same buffer and a linear sodium chloride gradient is applied. The mixing chamber contains 250 ml of 25 mM MES, pH 6.1, and the reservoir 250 ml of the same buffer, to which NaC1 has been added to a final concentration of 0.4 M. Fractions of 10 ml are collected and assayed for inhibitor content. The fractions containing the bulk of the inhibiting activity (No. 21 tq 27 in the preparation described), are pooled and concentrated to 3.5 ml by ultrafiltration through an Amicon Diaflo filter, type UM-10. In the case described, the overall purification achieved was 65-fold and the protein concentration in the most purified fraction, 0.34 mg/ml. Purification of Inhibitor from S. cerevisiae 8288C The procedure is the same as for S. carlsbergens-is, with the following modifications: (1) The yeast is grown in a medium containing 1 ~ yeast extract, 2% peptone, and 2% glucose. (2) In the DEAE-cellulose step, the inhibitor remains adsorbed on the exchanger after passage and washing. It is then eluted with 150 ml of 25 mM imidazole chloride at pH 7.2, containing 0.1 M NaC1, and 'the purification is continued as described in the preceding section. Properties
Stability. The inhibitor appears to be quite stable when stored at - 2 0 ° . Even the purified preparations can be boiled without loss of activity. Nevertheless, activity is lost when the purified inhibitor is diluted in the absence of albumin. This inactivation appears to be due to adsorption to glass, because it is much less marked when polypropylene material is used. Purity. The purified preparations, both from S. carlsbergensis 74S and from S. cerevisiae $288C, when submitted to disc gel electrophoresis, ~ give a single major band which coincides with the inhibitory activity. On increasing the amount of protein applied to the gel, several minor bands appear. Specificity. An absolute quantitative comparison of the inhibitory activity of the preparations from S. carlsbergensis and S. cerevisiae has not yet been made. The inhibitor from each species is effective against the activating factors both of the same and of the other species.
oSee Vol. 22 [39].