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[71] Cytosine nucleoside deaminase from Escherichia coli
478
[71]
ENZYMES OF NUCLEIC ACID METABOLISM
K,~. A s u m m a r y of the affinities of various substrates for the deaminase is given in T a b l e I I . Distribution. As yet, the nonspecific deaminase described a b o v e has been found only in takadiastase. TABLE II AFFINITIES OF VARIOUS SUBSTRATES FOR DEAMINASE
Substance
Approximate Km
ATP DPN 5'-Adenylic acid ADP 3'-Adenylic acid ADPR Adenosine
M X 10-~ 1.2 1.8 0.8 0.7 1.7 1.5 0.6
[71] Cytosine Nucleoside Deaminase from Escherichia
coli
C R 1 ~ H20--~ U R 1 -~ NH3 C D R i ~ H 2 0 --* U D R 1 -t- NH3
(1) (2)
By T. P. WANG
Assay Method Principle. S p e c t r o p h o t o m e t r i c m e t h o d s are used in following the act i v i t y of this enzyme, since the deamination of the cytosine c o m p o u n d s is accompanied b y a decrease in absorption of 55 % at 282 m~. T h e molecular extinctions of C R and U R at 282 m~ are 6000 and 2700, respectively. 2 T h e absorption spectra of the corresponding deoxyribosides are not m a t e rially different from those of the ribosides. Reagents C R or C D R , a n y suitable concentration. E. coli extract, 1 ml., equivalent to 50 to 100 mg. of wet cells. Tris buffer (0.1 M), p H 7.5.
Procedure. Place in a 3-ml. silica B e c k m a n cuvette 1.5 ml. of Tris buffer and a solution of C R or C D R containing a b o u t 0.3 micromole of the 1 CR, UR, CDR, and UDR stand for cytidine, uridine, cytosine deoxyriboside, and uracil deoxyriboside, respectively. 2 T. P. Wang, H. Z. Sable, and J. O. Lampen, J. Biol. Chem. 184, 17 (1950).
[71]
CYTOSINE NUCLEOSIDE DEAMINASE FROM ESCHERICHI~_ COLI
479
nucleoside. Make up to 2.9 ml. with water, and take an initial reading at 282 m~. Then add 0.1 ml. of the enzyme. After a quick stirring, take readings at 30-second intervals. The reaction will be finished in about 30 minutes.
Preparation of Enzyme 2
Preparation of E. coli Cells. E. coli strain 15 (9723 of the American Type Culture Collection) is the source of this enzyme. Stock culture of E. coli is kept on agar slants containing 0.3% Difeo beef extract, 0.2% Difco yeast extract, 0.7 % Difco peptone, 0.4% glucose, and 1.5 % Difco agar. An inoculum is made by transferring a loopful of bacteria from the slant to 10 ml. of medium of the same composition as listed above except the agar. The inoculum is then incubated for 24 hours at 37 °. The cells are collected by centrifugation at 4500 r.p.m, for 15 minutes. Preparation of Cell-Free Extract. The packed wet cells of E. coli, washed once with 0.9 % NaC1, are ground in an ice-chilled mortar with two and one-half times their weight of alumina powder (A-303 or A-301 of the Aluminum Company of America) according to McIlwain. 3 The paste is then mixed with 10 to 20 vol. (with respect to the original cells) of cold 0.05 M Tris buffer, pH 7.5, allowed to stand at 2 ° for 30 minutes, and centrifuged at 20,000 X g for 15 minutes in a Servall centrifuge. The supernatant is slightly opaque and light yellow in color. Attempts have been made to purify the enzyme by alcohol and (NH4):SO4 fractionations. The efforts were unsuccessful. Properties
Specificity. The cell-free extract is specific for the cytosine nucleosides. No action is observed when adenine, adenosine, cytosine, isocytosine, cytidylic acid, guanine, and guanosine are tested. The deamination is faster with CDR than with CR. The Km for CR is 1.74 X 10-4 M, and that for CDR, 8.9 X 10-5 M. General Properties. The enzyme is not inactivated by prolonged dialysis or by freezing and thawing. Preparations kept for several months at - 2 0 ° retain their original activity. The enzyme has a broad pH oPtimum between 6.5 and 8.5. Products of the Reaction. The products of the reaction are uracil nucleosides and ammonia. The former can be identified by their absorption spectra in ultraviolet region and by paper chromatographic or ionophoretic methods. Ammonia can be easily demonstrated by any of the standard methods such as by use of Nessler's reagent. H. McIlwain, J. Gen. Microbiol. 2, 288 (1948).
480
ENZYMES OF NUCLEIC ACID METABOLISM
[72]
When a demonstration of the f o r m a t i ~ of uracil nucleosides is desired, it is preferable to use a thoroughly dialyzed extract. Because of the presence of a pyrimidine nucleoside phosphorylase which requires inorganic phosphate for its activity in the extract, 2 it is essential to remove any inorganic phosphate present in the extract to prevent any splitting of the uracil nucleosides formed from the deamination of cytosine nucleosides.
[72] Guanase Guanine ~ H~O --* Xanthine ~ NH3
By Louis SHUSTER Assay Method Principle. When guanine is deaminated to xanthine, there is a shift in the ultraviolet absorption spectrum, the greatest change being a decrease of about 50% in the extinction at 245 m~. This change is the basis for the method of Roush and Norris. 1 The method of Kalckar, 2 which is more commonly used, involves measurement of the xanthine produced in the reaction by oxidation with xanthine oxidase. This oxidation is followed spectrophotometrically by measuring the increase in optical density at 290 m~ due to the formation of uric acid. The increase obtained is roughly sixfold, which makes this method more sensitive than that of Roush and Norris.
Reagents Guanine. A stock solution of 0.001 M can be made up by dissolving 15 rag. of free guanine in a few milliliters of 1 N N a O H and diluting up to 100 ml. 0.1 M glycylglycine or tris (hydroxymethyl)aminomethane buffer, pH 8.0. Xanthine oxidase, prepared from milk (see Vol. II [73]). An aliquot of 0.1 ml. should contain enough enzyme to oxidize 50 ~/of hypoxanthine per milliliter per hour. Guanase. The enzyme is diluted with glycylglycine or Tris buffer to contain 200 to 1000 units of enzyme per milliliter (see definitions below). i A. R o u s h a n d E. R. Norris, Arch. Biochem. 29, 124 (1950). z H. M. Kalckar, J. Biol. Chem. 167, 461 (1947).
[71]
ENZYMES OF NUCLEIC ACID METABOLISM
K,~. A s u m m a r y of the affinities of various substrates for the deaminase is given in T a b l e I I . Distribution. As yet, the nonspecific deaminase described a b o v e has been found only in takadiastase. TABLE II AFFINITIES OF VARIOUS SUBSTRATES FOR DEAMINASE
Substance
Approximate Km
ATP DPN 5'-Adenylic acid ADP 3'-Adenylic acid ADPR Adenosine
M X 10-~ 1.2 1.8 0.8 0.7 1.7 1.5 0.6
[71] Cytosine Nucleoside Deaminase from Escherichia
coli
C R 1 ~ H20--~ U R 1 -~ NH3 C D R i ~ H 2 0 --* U D R 1 -t- NH3
(1) (2)
By T. P. WANG
Assay Method Principle. S p e c t r o p h o t o m e t r i c m e t h o d s are used in following the act i v i t y of this enzyme, since the deamination of the cytosine c o m p o u n d s is accompanied b y a decrease in absorption of 55 % at 282 m~. T h e molecular extinctions of C R and U R at 282 m~ are 6000 and 2700, respectively. 2 T h e absorption spectra of the corresponding deoxyribosides are not m a t e rially different from those of the ribosides. Reagents C R or C D R , a n y suitable concentration. E. coli extract, 1 ml., equivalent to 50 to 100 mg. of wet cells. Tris buffer (0.1 M), p H 7.5.
Procedure. Place in a 3-ml. silica B e c k m a n cuvette 1.5 ml. of Tris buffer and a solution of C R or C D R containing a b o u t 0.3 micromole of the 1 CR, UR, CDR, and UDR stand for cytidine, uridine, cytosine deoxyriboside, and uracil deoxyriboside, respectively. 2 T. P. Wang, H. Z. Sable, and J. O. Lampen, J. Biol. Chem. 184, 17 (1950).
[71]
CYTOSINE NUCLEOSIDE DEAMINASE FROM ESCHERICHI~_ COLI
479
nucleoside. Make up to 2.9 ml. with water, and take an initial reading at 282 m~. Then add 0.1 ml. of the enzyme. After a quick stirring, take readings at 30-second intervals. The reaction will be finished in about 30 minutes.
Preparation of Enzyme 2
Preparation of E. coli Cells. E. coli strain 15 (9723 of the American Type Culture Collection) is the source of this enzyme. Stock culture of E. coli is kept on agar slants containing 0.3% Difeo beef extract, 0.2% Difco yeast extract, 0.7 % Difco peptone, 0.4% glucose, and 1.5 % Difco agar. An inoculum is made by transferring a loopful of bacteria from the slant to 10 ml. of medium of the same composition as listed above except the agar. The inoculum is then incubated for 24 hours at 37 °. The cells are collected by centrifugation at 4500 r.p.m, for 15 minutes. Preparation of Cell-Free Extract. The packed wet cells of E. coli, washed once with 0.9 % NaC1, are ground in an ice-chilled mortar with two and one-half times their weight of alumina powder (A-303 or A-301 of the Aluminum Company of America) according to McIlwain. 3 The paste is then mixed with 10 to 20 vol. (with respect to the original cells) of cold 0.05 M Tris buffer, pH 7.5, allowed to stand at 2 ° for 30 minutes, and centrifuged at 20,000 X g for 15 minutes in a Servall centrifuge. The supernatant is slightly opaque and light yellow in color. Attempts have been made to purify the enzyme by alcohol and (NH4):SO4 fractionations. The efforts were unsuccessful. Properties
Specificity. The cell-free extract is specific for the cytosine nucleosides. No action is observed when adenine, adenosine, cytosine, isocytosine, cytidylic acid, guanine, and guanosine are tested. The deamination is faster with CDR than with CR. The Km for CR is 1.74 X 10-4 M, and that for CDR, 8.9 X 10-5 M. General Properties. The enzyme is not inactivated by prolonged dialysis or by freezing and thawing. Preparations kept for several months at - 2 0 ° retain their original activity. The enzyme has a broad pH oPtimum between 6.5 and 8.5. Products of the Reaction. The products of the reaction are uracil nucleosides and ammonia. The former can be identified by their absorption spectra in ultraviolet region and by paper chromatographic or ionophoretic methods. Ammonia can be easily demonstrated by any of the standard methods such as by use of Nessler's reagent. H. McIlwain, J. Gen. Microbiol. 2, 288 (1948).
480
ENZYMES OF NUCLEIC ACID METABOLISM
[72]
When a demonstration of the f o r m a t i ~ of uracil nucleosides is desired, it is preferable to use a thoroughly dialyzed extract. Because of the presence of a pyrimidine nucleoside phosphorylase which requires inorganic phosphate for its activity in the extract, 2 it is essential to remove any inorganic phosphate present in the extract to prevent any splitting of the uracil nucleosides formed from the deamination of cytosine nucleosides.
[72] Guanase Guanine ~ H~O --* Xanthine ~ NH3
By Louis SHUSTER Assay Method Principle. When guanine is deaminated to xanthine, there is a shift in the ultraviolet absorption spectrum, the greatest change being a decrease of about 50% in the extinction at 245 m~. This change is the basis for the method of Roush and Norris. 1 The method of Kalckar, 2 which is more commonly used, involves measurement of the xanthine produced in the reaction by oxidation with xanthine oxidase. This oxidation is followed spectrophotometrically by measuring the increase in optical density at 290 m~ due to the formation of uric acid. The increase obtained is roughly sixfold, which makes this method more sensitive than that of Roush and Norris.
Reagents Guanine. A stock solution of 0.001 M can be made up by dissolving 15 rag. of free guanine in a few milliliters of 1 N N a O H and diluting up to 100 ml. 0.1 M glycylglycine or tris (hydroxymethyl)aminomethane buffer, pH 8.0. Xanthine oxidase, prepared from milk (see Vol. II [73]). An aliquot of 0.1 ml. should contain enough enzyme to oxidize 50 ~/of hypoxanthine per milliliter per hour. Guanase. The enzyme is diluted with glycylglycine or Tris buffer to contain 200 to 1000 units of enzyme per milliliter (see definitions below). i A. R o u s h a n d E. R. Norris, Arch. Biochem. 29, 124 (1950). z H. M. Kalckar, J. Biol. Chem. 167, 461 (1947).