ANALYTICAL

BIOCHEMISTRY

Micro BEATRIZ

92, 453-461

Determination M.

BRAGANCA,

(1979)

of Polyamines

with Snake Venom

ASHOK V. HOSPATTANKAR, SHABBIR AND NARENDRA R. VAIDYA

Received

May

Oxidase

E. ELECTRICWAL.LA

9. 1978

An accurate micromethod suitable for the assay of polyamines in concentrations of Z to 30 nmol is described. It is based on the oxidation of polyamines by purified fractions of crude L-amino acid oxidase from Russell’s viper venom. By a combination of two enzyme fractions. one which oxidizes polyamines and amino acids (AAP) and another which oxidizes only amino acids (AA). the techniqlue can accurately determine polyamine concentrations in extracts of sera which may not be free ofamino acids. Experiments described show 90 to 100% recovery of added polyamines in the presence of varying amounts of amino acids. Polyamines added to serum also showed recovl:ries ranging from 96 to 98%. The enzymes do not oxidize histamine and epinephrine and are very stable. The method does not require sophisticated equipment and is suitable for screening of large numher of clinical samples to assess the importance of polyamines as a diagnostic test or their prognostic value in diseases like cancer.

Polyamines have acquired importance in recent years as they are involved in growth regulation (l-3) and there is evidence to show that polyamine concentration of serum and urine is elevated in certain types of cancer (4-6), in pregnancy (7). and in regenerating liver (8,9). A variety of procedures have been described for the assay of polyamines. The more accurate methods involve thle use of the amino acid Autoanalyzer and the radioimmunoassay technique (10,ll). Among the enzymatic methods, bovine plasma amine oxidase has been used for assay of polyamines (12). The present paper describes an accurate micromethod for the assay of polyamines based on two partially purified fractions of I.-amino acid oxidase complex from Russell’s viper venom which requires no elaborate equipment. L-Amino acid oxidase from Russell’s viper venom is a flavoprotein that has been crystallized (13.14). This enzyme preparation catalyzes the oxidation of I.-amino acids according to the following reactions: 453

R-CH-NH,-COOH

+ H,O + 0, --;,

RCOCOOH + NH:, + H.,O*, [I] R-CO-COOH

+ H,O, -+ RCOOH + H,O + CO,.

[2]

Hayes and Wellner (15) have separated by electrofocusing 18 enzymatically active components from crystalline L-amino acid oxidase of Crotcllus cldarncrtlterls venom. Experiments described here demonstrate that the crude I.-amino acid oxidase also catalyzes the oxidation of putrescine, spermidine, and spermine. MATERIALS

AND METHODS

Russell’s viper venom was a lyophilized preparation purchased from Haffkine Institute, Bombay, India; spermine. N. B. Corporation; spermidine, L. Light and Company Ltd., England; putrescine and L-amino acids, Sigma Chemical Company; 2,4-dinitrophenylhydrazine, B. D. H.. England: catalase (EC 1.11.1.6). V. P. Chest Institute, Delhi, India; Dowex 50 W resin, Fluka AC, Buchs SG, Switzerland. 0003.2697/79/020453-09$02.00/O Co,qqht All right\

(c’ ,979 hy 4cadem~ be\\. Inc. of reprduct~m m any form rrxrved

454

BRAGANCA

Protein determitutiori. Protein was assayed by the absorbance method described by Kalckar (16). Preputwtion of cnzytne jkrctiotl AAP’ frotn Russell’s \,iper \-enom. Crude venom solution (10 mg/ml) was treated with ammonium sulfate (O-30% saturation) in cold and kept overnight at 4°C. The precipitated proteins were removed by centrifuging at 10,000 rpm for 30 min in the cold. Most of the L-amino acid oxidase activity was present in the supernatant and was reprecipitated by 30 to 60% saturation with ammonium sulfate. The precipitate so obtained was collected by centrifuging as above. It was dissolved in small amount of distilled water and the solution was dialyzed against 5 liters of water for 2 h in the cold. Further purification of this fraction was carried out by gel filtration through Sephadex G-200 column (3.5 x 60 cm) and elution in 3-ml fractions with Tris-HC1 buffer (0.05 M) at pH 7.4. All fractions collected were scanned for enzymatic activity as described later. The peak showing activity both for L-leucine and spermine is designated as AAP. The tubes comprising AAP were pooled and the protein was concentrated by precipitation with ammonium sulfate to 80% saturation. The precipitate was collected by centrifuging at 10,000 rpm for 30 min, dissolved in small amount of water, and dialyzed against 5 liters of water in cold for 2 h. The enzyme was more stable in the presence of ammonium sulfate and could be kept at 4°C for more than 6 months. Prepcrrrrtion oj- enzyme fraction AA from Russell’s bliper ~vtmm. The enzyme extract AA which catalyzes the oxidation of Lleucine but not of putrescine and the polyamines was prepared by further fractionation of AAP in DEAE-Sephadex A-50 column (2.5 x 25 cm) using gradient elution (TrisHCl buffer, 0.1 M, pH 9.5; NaCl gradient ’ Abbreviations used: AAP, Enzyme fraction which oxidizes amino acids and polyamines; AA, Enzyme fraction which oxidizes amino acids. DNPH, 2.4. dinitrophenylhydrarine.

E7 AI

0.1 to 0.5 M). Three-milliliter fractions were collected. Absorbance at 280 nm resulted in two peaks. The enzyme activity was scanned using L-leucine and spermine as substrate. The first peak was active on1 y for L-leucine. Fractions containing this peak were pooled. The protein was finally concentrated by 80% saturation with ammonium sulfate. The precipitate was collected as before by centrifuging and was freed from salt by dialysis against 5 liters of distilled water for 2 h in the cold. This enzyme extract was stable for more than 6 months at 4°C if stored in ammonium sulfate. Enzyme rrssrry for scmning the Iwrious frrrctions. Enzyme activity was assayed by trapping the carbonyl compound formed by enzymatic oxidation of an amino acid or a polyamine with 2,4-dinitrophenylhydrazine (DNPH). The intense reddish-brown color of the 2,4-dinitrophenylhydrazone derivatives in alkaline solution has an absorption maximum at 500 nm. The assay was carried out as follows: r-Leucine or spermine ( 1 Fmol in 0.1 ml) solution in distilled water was treated with 0.5 ml of Tris-HCI buffer, 0.1 M, at pH 9.5. To the mixture 0.1 ml of enzyme solution from each tube was added followed by 0.2 ml of catalase (10 pgiml) to prevent oxidation of the keto acid formed by H,O, generated in the reaction as in Eq. [ 11. The mixture was made to 1.0 ml with distilled water and incubated at 37°C for 30 min, then treated with 0.2 ml of 0.1% DNPH in 2 N HCl. It was heated at 70°C for 5 min and made alkaline by addition of 1.8 ml of 1 N NaOH. The total volume was 3 ml. The deep reddish-brown color produced was measured at 500 nm. Micromethod for nssc~y of polycittiitic~s using enzpnles AA and AAP. In this procedure the extracts containing mixtures of polyamines and amino acids are first treated with AA which oxidizes only the amino acids to the corresponding keto acid derivatives: by the addition of excess of H,O, the keto acids formed are quantitatively oxidized to the corresponding carboxylic acids as in

ENZYMIC

ASSAY

OF POLYAMINES

455

FIG. 1. Specific activities of AAP for various substrates. The reaction mixture contained substrate (1 ~mol). 0.1 ml. Tris-HCI buffer (0. I M, pH 8.5). 0.5 ml. AAP t 1 mg/ml). 0. I ml, catalase t 10 &ml). 0.2 ml. distilled water. 0.1 ml. and was incubated at 37°C for 30 min. Color development was as described under Methods. The keto derivative was quantitated by comparison with a standard graph drawn with varying concentrations ofoxaloacetic acid. Specific activity of the enzyme is defined as micromoles of keto acid formed per milligram of protein of the enzyme extract in the system.

Eq. [2]. The excess H,O, is then destroyed by addition of catalase. The extracts freed of amino acids are then incubated with AAP which oxidizes polyamines present in the extract to form the corresponding carbonyl derivatives. These are trapped with DNPH in 2 N HCI to form the corresponding 2,4-dinitrophenylhydrazone derivatives and the color intensity is determined at 500 nm as described earlier. The assay was carried out as follows: Sample extract, 0.1 ml, Tris-HCI buffer (0.1 M, pH 9.5). 0.01 ml, and AAP (250 pg). 0.01 ml, were incubated at 37°C for 4 h and I5 min. Preliminary experiments hald shown that for the range of concentration of amino acid used, this period was sufficient for complete conversion to their keto derivatives. The mixture was heated at 100°C for 10 min to destroy excess AA and treated with 0.08 ml of 30% H,O, and incubated for 30 min at 37°C. To the mixture Tris-HCI buffer (0. I M. pH 9.5). 0.01 ml. and catalase (4.8 mgiml). 0.01 ml, were added to destroy excess H,O,. The final volume was made to 0.24 ml with distilled water and incubated for 30 min at 37°C. At the end of incubation AAP (100 pgs) in 0.01 ml was added and the system again incubated overnight ;at 37°C.

The color was developed by treating with 0.1% DNPH in 2 N HCI (0.04 ml). It was heated at 70°C for 5 min in a water bath followed by addition of0.21 ml of I N NaOH. The final volume was 0.5 ml. Absorbance was determined at 500 nm. Pwtwtrtmcnt oj’ hiolo~~icrrl tuatetk1.s jiu pdyrtnint~ crsstr~. In experiments in which polyamines were extracted in the presence of serum (10 ml), the polyamines were first added to the serum which was then deproteinized by the addition of I g of sulfosalicylic acid in the cold and kept for 20 min. It was centrifuged at 10,000 rpm for 20 min. The clear supernatant obtained was saturated with 1 g of salt mixture (62.5 g of anhydrous sodium sulfate and 9 g of trisodium phosphate monohydrate) as described by Russell rt rrl. (9). The pH was adjusted to 11 with 5 N NaOH and extracted with n-butanol. Addition of an equal volume of n-butanol to the mixture with vigorous mechanical shaking for 30 min was repeated twice. The polyamines were extracted into the butanol layer and most of the amino acids were excluded. The organic layers were pooled together and passed through a column of cotton acid succinate prepared as described by McIntire rt trl. (17). Ethyl alcohol (3 ml)

456

FIG.

2. Specific

activities

BRAGANCA

ET AI.

substrates.

Experimental

of AA for various

was passed through the succinate column to remove the n-butanol followed by 3 ml of distilled water to remove the alcohol. The polyamines bound to cotton acid succinate were eluted with 3 ml of 1 N HCI and dried under vacuum. This step made the evaporation of the polyamine extract faster. The dried material was dissolved in water and

are the same as for Fig.

I

pH adjusted to 9.5 with 1 N NaOH. The total volume of the solution was 0.1 ml. With this procedure the average value of the polyamines present may be detected. Separrltion oj’ir~di~~iciurrl polycmin~~s. For separation of individual polyamines the procedure employed was as follows. Tabor rt ~1. (18) have reported the separation of poly-

IO n moles

conditions

20 SUBSTRATE

IO.5

30 ml SYSTEM

FIG. 3. Oxidation of standard polyamines with AAP. Reaction (400 nmoliml). Tris buffer (1 M. pH 9.5), 0.01 ml. catalase (50 &ml). volume was made to 0.25 ml with distilled water. Incubated overnight under Methods.

mixture contained polyamine solution 0.01 ml. AAP (1.5 mg/ml). 0.06 ml. and at 37°C. Color developed as described

ENZYMIC 1)

ASSAY

NHL(CH2)SNH(CH1),NH(Cti2),NH,t

457

OF POL.YAMINES Z02t2H20-

SPERMINE 0

\\

H

2)

NH,(CHL),

/

0 C-(CH21z

NH (CH,),

NH(CH,),

Ntt2tCtip),

2NH,

+

2H202

H

NH (CH,),

NH,+

O,+

Hz0

NH2

t 02

-

0 a /C(CH,),

NH (CH,),

NH,

+ NH,

+ HrOe

H

SPERM/DINE

3)

CT+

+

Hs!O

-

0 B C (CHr / H

),

NH,

+

NH3

-I- He 0.

PU TRESClh’E

FIG.

4. Enzymatic

oxidation

amines using Dowex 50 H+ gel column by gradient elution with HCI. In the method described here individual polyamines present in a mixture were selectively eluted from Dowex 50 W resin column with different normalities of HCI. On passing an aqueous solution of each of the polyamines (20 nmol) through a Dowex 50 W resin column (0.6 x 2 cm), putrescine eluted with 2 N HCI whereas spermidine and spermine eluted with 3 and 4 N HCI. respectively. When a mixture of three polyamines was applied. the Dowex 50 W resin column was washed with 3.0 ml of 1 N HCI. Putrescine was then eluted with 3.0 ml of 2 N HCI over a period of 10 min followed by spermidine with 3 ml of 3 N HCI and spermine with 3 ml of 4 N HCI. Each of the extracts were dried and dissolved in distilled water and the pH adjusted to 9.5 with 1 N NaOH. Total volume of the solution was made tot 0.1 ml. For the separation of individual polyamines from serum or plasma the following procedure was employed. Serum or plasma was deproteinized with sulfosalicylic acid as described earlier. Protein-free supernatant thus obtained was applied directly on the Dowex 50 W resin column. It was washed with 5.0 ml of 0.1 M phosphate buffer, pH 8.0, containing 0.7 M NaCl to remove the bulk ofthe amino acids present in the extract as described by Inoue rt rrl. (19). The resin column was then washed with 3.13 ml of 1 N HCI. The rest of the procedure followed

products

of polyamines.

to separate the three polyamines scribed above.

was as de-

RESULTS Specific activities of AAP for different L-amino acids. amines, diamines, and polyamines are given in Fig. 1. It is seen that in addition to several amino acids and the amides, glutamine and asparagine. AAPalso oxidizes putrescine. spermidine, and spermine. Under the experimental conditions used the following r-amino acids were not oxidized: threonine. lysine. aspartic acid, glutamic acid, serine, proline. glycine, alanine, and hydroxyproline. Histamine and cadaverine are also not attacked by AAP. Similar experiments using AA are shown in Fig. 2. The enzyme fraction AA oxidizes the same amino acids as AAP but does not attack putrescine, spermidine. and spermine. Oxidation of varying quantities of putrestine, spermidine. and spermine by AAP as given in Fig. 3 show that the keto derivative formation is linear for the substrate concentrations ranging from 2 to 30 nmol. The molar absorbancy index of colored product calculated for spermine is 20 x 10:’ liter mall’ cm-‘. for spermidine the molar absorbancy index is 15 x IO:’ liter mall’ cm-‘, and for putrescine it is 12 X IO:’ liter mall’ cm-‘. The nature of the products formed on oxidation of polyamines by AAP was examined by the method of Lyttle ct trl. (20).

458

BRAGANCA

t7

TABLE ASSAY ot- MIXTURF Experiment No.

4 6 8 9 IO II 12 13 14 IS

16

OF SIANIIARD

Substrate” Leucine ( 10) Leucine (10) Leucine (20) Leucine (20) Spermine (IO) Spermine (IO) Spermine (20) Spermine (20) Spermidine (20) Spermidine (20) Putrescine (20) Putrescine (20) Leucine ( IO) + spermine (20) Leucine (10) + spermine (20) Spermine (IO) + 17 amino acids mixture (0.25 each) Spermine (20) + 17 amino acids mixture (0.5 each)

I

I -AMINO

ACIDS

AA (a)

AAP (PLg)

250 250 250 30 250 250 250 250 250 250 250 250

Al..

100 100 100 100 100 100

250

AND POLYALII~G~S Optical density at 500 nm

USING

AA *ND

AAP

Recovery (nmol)

Recovery (‘i)

0.28 0.02 0.54 0.02 0.02 0.21 0.02 0.39 0.02 0.28 0.02 0.23

10.0 nil 19.8 nil nil 9.8 nil 19.8 nil 19.6 nil 19.5

100 0 99 0 0 98 0 99 0 98 0 97

0.26

9.7

97”

250

100

0.39

19.8

99’

250

100

0.21

9.8

98”

250

100

0.39

19.8

99’

” Nanomoles of substrate indicated in parentheses. li Indicates percentage recovery of leucine added. (’ Indicates percentage recovery of spermine added.

The results indicate that the enzyme oxidizes spermine into a dialdehyde whereas spermidine and putrescine are converted to monoaldehydes (Fig. 4). rz-Butanol saturated with salt mixture (9, 21) has been commonly used to perferentially extract polyamines free from amino acids present in serum, urine, or other tissues. In the present study initial experiments showed that tz-butanol extracts from plasma or tissues did contain small quantities of amino acids. The two-enzyme assay method using the enzymes AA and AAP made it possible to determine the average polyamine concentration encountered in biological fluids in butanol extracts which were not completely free of amino acids. In Table 1 the results of the assay using mixtures of amino acids and polyamines are given. It is apparent (Experiments I and 3,

Table 1) that 10 and 20 nmol of L-leucine are completely oxidized when treated with AA for 4 h and 1.5 min and no leucine could be detected when subjected to both the enzymes (Experiments 2 and 4. Table 1) indicating that these concentrations of L-leucine do not interfere with the assay system. Spermine, 10 or 20 nmol was not oxidized in the presence of AA (Experiments 5 and 7, Table 1). however, when subjected to the second enzyme AAP the recovery of spermine was 98 to 99% (Experiments 6 and 8, Table 1). Similar results are obtained with 20 nmol each of spermidine and putrescine (Experiments 10 and 12, Table 1). When a mixture of r-Ieucine (10 nmol) and spermine (20 nmol) was treated with only AA. it was possible to recover 97% of the leucine added (Experiment 13, Table 1). In the complete system using AA and AAP. 99% spermine

ENZYMIC

ASSAY

OF

TABLE KECWVFRY

Experiment No.

Substrate

Hluman serum added

added”

(ml)

I. 2. 3 4. 5. ” Nanomoles

Spermidine Spermidine

(5) ( IO)

Spermidine

(20)

1 -Leucine

(20)

of substrate

indicated

2

ADDED

OF SPERMIDINE

TO NORMA]

Put]-excine

2

PUTRFSCINE,

Pulyamine eluted Putrescinr

+ spermidine + spermine 3

4

nm

(nmol)

0.09

IO IO

0.25 0.39

IO

0.09

0.16

4.8 9.8 19.8 nil

of Recovery (C-5’)

96 98 99 0

in parentheses.

OF INDIVIDU41

Normality of HCI

Recovery polyamine\

IO IO

was no increase as compared to the control with serum. Table 3 shows the recoveries of individual polyamines from the standard mixture of putrescine. spermidine, and spermine using Dowex 50 W resin column as described under Methods. The extracts of individual polyamines were assayed at 10 and 20 nmol levels in the micromethod. It is seen that recoveries of each of the three polyamines ranged from 91 to 97%. It is clear from Table 4 that the average polyamine levels in unhydrolyzed normal rat plasma and human serum ranged from 2.45 to 2.7 nmoliml and 0.46 to 0.58 nmol/ml. respectively. By applying the technique of Dowex 50 W resin for separation of individual polyamines, it was seen that unhydrolyzed normal rat plasma and human serum 3

POLYAhllNt

I-KO~~

SPFRMIDINE.

Level Polyaminc mixture

SERUM

density

at 500

TABLE

OF

HUMAN

Optical

added can be recovered (Experiment 14. Table I). Mixtures of spermine (IO and 20 nmol) in the presence of 17 L.-amino acids in concentrations of 0.25 and 0.5 nmol each, also showed a recovery of 98 to 99% of the added spermine in the assay system. Experiments in which the recovery of spermidine was tested in the presence of normal human serum subjected to l.he entire extraction procedure are shown in Table 2. Spermidine was added to the serum1 before it was deproteinized. Results show that the optical density for 10 ml of serum extract was 0.09. On addition of 5, 10, and 20 nmol of spermidine there was a progressive increase in the readings. The reclovery of added spermidine in all cases was more than 95%. When I_-leucine was added in place of spermidine (Experiment 5. Table 2) there

Ass,\u

459

POLYAMINES

Spermidine

Spermine

THE AND

STANDARD

of

extract (nmol)

MIXTURE

SPLRMINE

Optical density at 500 nm

IO

0.11

20

0.23

IO

0. I4

70

0.28

IO

0.18

20

0.38

Polyamine recovered (nmol)

Recovery (V)

9.2 19.4

92

9.4 19.6

94 98

9. I 19.2

91 96

97

460

BRAGANCA

E7‘AL.

TABLE POLYAMINE

CONCENTRATION

IN UNHYDROLYZED

4 NORMAL

Polyamine Sample No. 1 2

Extract Rat plasma Human serum

Putrescine

PLASMA

2.6 0.54

of experiments.

contained 2.6 and 0.54 nmol/ml of spermidine, respectively. Putrescine and spermine were not detectable. DISCUSSION Elevated concentrations of polyamines have been clearly demonstrated in sera and urine of cancer patients (22). This has led to a great deal of interest as to their potential importance as diagnostic markers or in following the efficacy of drug treatment in cancer patients (23,24). Polyamines can be detected by separation in high voltage electrophoresis and thin layer chromatography prior to calorimetric or fluorimetric assay (9,21,25). Among various enzymic methods, hog kidney diamine oxidase has been used to oxidize putrescine into A-pyrroline which is detected colorimetrically. However, this enzyme also oxidizes histamine normally present in sera and also cadaverine (26,27). The molar absorbancy index of the colored product formed was 1.86 x 10:’ liter mol-’ cm-‘. An enzyme from dried Serrrrtitr tm~rce.sc~~n.s cells oxidizes spermidine (28). This enzyme does not attack spermine and putrescine. The method is suitable for concentrations ranging from 0.05 to 0.4 pmol of spermidine. Bovine plasma amine oxidase (12) which oxidizes spermine and spermidine but not putrescine is sensitive to 0.01 pm01 of polyamines. The molar absorbancy index for the colored product for spermine and spermidine was 12.5 x 10% and 6.25 x 10:’ liter mall’

AND

HUMAN

SERUM

(nmoliml)

Spermidine

ND’ ND

” Amine was not detected. ’ Number in parentheses indicates number consisted of pooled blood from eight rats.

RAT

Spermine ND ND

In the case

Average polyamines (nmoliml)

of rat plasma

2.45-2.7 0.46-0.58

each

(3)” (5)

experiment

cm-‘, respectively. The most accurate methods employ amino acid Autoanalyzer with special columns to separate the polyamines (5,lO) and have been used to study serum and urine concentration of polyamines in normal and under diseased conditions (29). This technique requires specialized and expensive equipment. Recently a radioimmunoassay technique has been described for assay of polyamines in sera ( 11). Though the method is very sensitive it is dependent on specific antibodies for each of the polyamines (30). The procedure described here provides a simple and accurate enzymatic assay which is more sensitive than other reported enzymatic methods. It does not require sophisticated equipment and is thus very suitable for screening of polyamines in body fluids in a clinical laboratory. The enzymes once prepared are highly stable and can be kept at 4°C for over 6 months. The technique can be applied to determine the average value for the polyamines present in serum and also for determination of individual polyamine concentrations with the help of Dowex 50 W resin. The enzyme (AAP) purified from Russell’s viper venom which oxidizes amino acids and polyamines did not oxidize histamine and epinephrine normally present in sera and was not active on cadaverine. The relationship of activity to concentration was linear for concentration of polyamines ranging from 2 to 30 nmol. Further purification (AA)

ENZYMIC

ASSAY

resulted in elimination of components active for oxidation of polyamines. Study of the oxidation products of the three polyamines show that spermidine and putrescine form monoaldehyde derivatives whereas spermidine is converted to a dialdehyde. In this respect it may be recalled that bovine plasma amine oxidase also oxidizes spermidine to monoaldehyde derivative and spermine to a dialdehyde (3 1). By using the two enzymes it was possible to (eliminate interference of trace amounts of amino acids which find their way both into butanol extracts of serum and also into exlracts obtained by fractionation through Dowex 50 W resin. With standard mixtures of polyamines and amino acids the recovery of added polyamines ranged from 90 to 100%. Addition of polyamines to serum from the start before deproteinization also showed good recoveries. With a stock of prepared enzymes this technique permits the screening of a large number of clinical samples of pathological sera. The values obtained for unhydrolyzed normal human sera which contain mainly spermidine are comparable to those obtained by using the amino acid Autoanalyzer. radioimmunoassay, or gas chromatographymass spectrometric method (30). Other observations (unpublished results) have shown considerable elevation in plasma concentration of polyamines in cases of human lymphoma and acute myeloid leukemia as well as in animals bearing tumors.

7. Russell, D. H.. Levy. C. C. Schimpff, S. C.. and Hawk, I. A. (1971) Cuncer Res. 31, 1555-1558. 8. Raina, A.. Janne, J.. and Simes, M. I 1966) Bioc.him. Bioplzy.s. Actcr 123. 197-201. 9. Russell, D. H.. Medina. V. J., and Snyder. S. H. ( 1970) J. Rio/. Clrc,ru. 245, 6732-6738. 10. Marton. L. J.. and Lee. P. L. Y. ( 1975) C/h. Chum. 21, 1721-1724. II. Bartos, D.. Campbell. R., Bartos. F.. and Grettie, D. P. (1975) Cnnc,c,r Rc~s. 35, 2056-2060. 12. Bacharach. U.. and Reches. B. (1966) Antrl. Biochrm. 17, 38-48. 13. Singer, T. P., and Kearney, E. B. (195llArc11. Bioc~hrnr.

2. 3.

4. 5. 6.

Raina. A., and Janne, J. (1975) ,211cd. Biol. 53. 121147. Tabor, C. W.. and Tabor. H. (19761 /111/1/l. Kc\,. Rir~c~lfr~rJl. 45, 785-306. Bacharach. U. (1973) Function of Naturally Occurring Polyamines. pp. l-175, Academic Press. New York. Russell. D. H. (1971) Nrrrrrr-cd Ncn, Viol. 233. 144145. Mar-ton. L. J.. Russell. D. H.. and Levy, C. C. (1973) Clirl. C/w/u. 19, 973-976. Nishioka. K.. and Romsdahl. M. M. (1974) C/i!l. C/ii/~.

Ac,/tr

57.

155-161.

Bioph~.\.

33,

377-396.

14. Wellner, D.. and Meister, A. ( 196015. BLJI. C‘hcrtr. 235, 2013-2018. IS. Hayes, M. B.. and Wellner. D. (1969) J. Bid. Chrm. 244. 6636-6644. 16. Kalckar, H. M. (1947) 1. Bid. Chcwz. 167, 46l476. 17. Mclntire. F. C.. Roth. L. W.. and Shaw, J. L. 11947) J. Bicd. Chr,m. 170. 537-544. 18. Tabor. H.. Rosenthal. S. M.. and Tabor. C. W. (1958) J. Biol. Chem. 233, 907-914. 19. Inoue. H.. and Mizutani. A. ( 1973)Ancrl. Biocht~rr~. 56, 408-416. 20. Lyttle, D. A., Jensen, E. H., and Struck. W. A. ( 1952) Autrl. Chc,rn. 24, 184% 1844. 21. Dreyfuss. G.. Dvir, R.. Harell, A., and Chayen. R. (1973) Clirr. C‘hinr. Ar,ftr 49, 65-71. 22. Russell, D. H. (1973) Polyamines in Normal and Neoplastic Growth. pp. l-429, Raven Press. New York. 23.

24. 25. 26. 27.

REFERENCES I

461

OF POLYAMINES

28. 29.

30.

31.

Russell, D. H.. and Russell. S. D. (1975) C‘lie. c‘hem. 21. 860-863. Russell. D. H. (1977) C/in. Chum. 23, 22-27. Raina. A. ( 1963) Ac,/cl Plly.tio/. Scuud. 60 (SuppI. 218). 7-81. Holmstedt, B.. and Tham. R. (1959) Acrrr Phy.vid. Sctrrd. 45. 152- 163. Kappler-Adler. R. (1970) Amine Oxidases and Methods for Their Study. Wiley-Interscience. New York. Bacharach. U.. and Oser, I. S. (1963) ./. Bid. C.hrru. 238. 2098-7101. Marton. L. J.. Vaughn, J. G.. Hawak, I. A.. Levy, C. C.. and Russell. D. H. (1973) irf Polyamines in Normal and Neoplastic Growth (Russell. D. H.. ed.). pp. 367-372. Raven Press. New York. Barton, F.. Bartos. D.. Grettie, D. P.. Campbell, R. A., Marton, L. J.. Smith, R. G.. and Doyle, D. G. (1977) Bic~hcm. Biop/q\. Kc,.\. C‘c~n~rriro,. 75,915-919. Tabor, C. W.. Tabor. H., and Bacharach. U. (1964) .I. Hicd. C‘hrr~. 239, 2194-2703.