Direct potentiometric study of the urea-urease system

PRELIMINARY NOTES

605

for methionine plus ATP in both the exchange reaction and in the net conversion of pyruvate (Figs. I and 2). Recent experiments with Dowex-5o (K ÷ form) show that the cofactor activity in freshly prepared cell extracts or enzyme preparations is mainly due to endogenous adenosylmethionine; 'methionine (and ATP) is present in only minor amounts. Filtration through the resin at pH 7 results in complete loss of activity, which can be restored to at least 80 % by addition of adenosylmethionine. Similarly, only adenosylmethionine need to be added to the inactive protein fraction obtained by Biogel P-2 filtration in order to restore the full initial enzymatic activity; this implies that no further low molecular weight compound is involved. The difficulties hitherto encountered in this and other laboratories in obtaining a more extensive purification of the clastic enzymes can now be readily understood. In particular, the various attempts to isolate the unknown factor were hampered by its great lability. Whether the adenosylmethionine (or a metabolite thereof) is directly involved in the as yet unknown elementary steps of the clastic reaction, or whether it might exert an allosteric action, has not yet been established. This work was supported by the Deutsche Forschungsgemeinschaft, Bad Godesberg. Organisch-Chemisches Institut der Universitdt Heidelberg, Heidelberg (Germany)

J. KNAPPE E. BOHNERT W. BRUMMER

I J. l~. QUAYLE, Ann. Rev. Microbiol., 15 (1961) 119. 2 ~N-.G. MCCORMICK,E. J. ORDALAND H. R. WHITELEY,J. Bacteriol., 83 (1962) 899. 3 G. G. MIDWINTER,H. NAKAYAMAAND L. O. KRAMPITZ,Federation Proc., 24 (1965) 531. 4 C. H. CHIN,L. O. KRAMPITZANDG. D. NOVELLI, Bacteriol. Proc., (1957) 1275 N. P. WOOD AND D. J. O't~[ANE, Bacteriol. Proc., 6 U. HENNING, Z. Vererbungslehre, 95 (1964) 236.

(196o)

155.

Received August 3rd, 1965 Biochim. Biophys. Acta, lO7 (1965) 603-605

PN 2I IO7

Direct potentiometric study of the urea-urease system Recently, a direct potentiometric method z which allowed the continuous measurement of ammonium ion was reported. The application of this method to study the effects of various ions on the urease-catalyzed hydrolysis of urea is the subject of this report. Urease (EC 3.5.1.5) solutions were prepared by suspending i g portions of urease powder (Arlington Chemical Co., Yonkers, N.Y., 480305) in 5° ml of o.I M Tris buffer (adjusted to pH 7.0 with hydrochloric acid) and stirring over-night at o °. The suspensions were centrifuged at low speed to remove suspended matter, and the clear liquid was filtered. Urease solutions were prepared fresh daily. Urea solutions were prepared by weight from the reagent grade solid. Solutions of the cations studied were prepared by weight from reagent grade metal chlorides. Solutions of the anions studied were prepared by titrating measured volumes of Biochim. Biophys. Acta, lO7 (1965) 6o5-6o8

606

PRELIMINARY NOTES

s t a n d a r d solutions of the corresponding acids to p H 7.0 w i t h a I M solution of Tris a n d diluting to an a p p r o p r i a t e volume. P o t e n t i o m e t r i c m e a s u r e m e n t s were m a d e w i t h a B e c k m a n 39137 cationic-sensitive glass electrode a n d a 3917 ° fiber j u n c t i o n calomel reference electrode in conj u n c t i o n w i t h a Corning m o d e l 12 p H m e t e r o p e r a t e d on the e x p a n d e d millivolt range. The m e t e r o u t p u t was r e c o r d e d on a L a b - L i n e G r a p h i c o r d e r - I o . All m e a s u r e m e n t s were m a d e at 25.0 ± o.I °, a n d t h e r e a c t i o n s y s t e m s were stirred during these measurements. To correlate the p o t e n t i o m e t r i c readings w i t h a m m o n i u m ion concentration, p o t e n t i o m e t r i c m e a s u r e m e n t s were m a d e on a series of solutions from I - I O - 4 to 1.1o -1 M in a m m o n i u m ion a n d m a i n t a i n e d at p H 7.0 b y a o.I M Tris buffer. A plot of the p o t e n t i a l against the l o g a r i t h m of the a m m o n i u m ion c o n c e n t r a t i o n gave a s t r a i g h t line of the form: E = 54.8 log (NH4+) + 229.4

(i)

The liberation of a m m o n i a b y the action of urease on u r e a was s t u d i e d as follows. A m e a s u r e d volume of urease solution, u s u a l l y 5.0o ml, was t r e a t e d with a m e a s u r e d volume of w a t e r such t h a t the t o t a l volume of the reaction s y s t e m would be 70.0 ml. Ten ml of I M Tris buffer was added, a n d a m e a s u r e d volume of solution containing a known a m o u n t of the cation or anion u n d e r investigation was added. The electrodes were i m m e r s e d in the solution, a n d the strip c h a r t recorded was started. A m e a s u r e d volume of s t a n d a r d u r e a solution was added, a n d the liberation of a m m o n i a was recorded on the strip chart. Velocity values were c a l c u l a t e d from the change in a m m o n i u m ion c o n c e n t r a t i o n between 0. 9 a n d I . I min after the a d d i t i o n of urea. The a m m o n i u m ion c o n c e n t r a t i o n s were c a l c u l a t e d from Eqn. I using t h e p o t e n t i a l s recorded on the strip c h a r t at 0. 9 a n d I . I min. These velocity values then represent the rate of the reaction at I rain after it started. The " d o u b l e reciprocal" plot of LINEWEAVER AND BURK 2 (Fig. I) shows t h a t copper a n d zinc b o t h inhibit the action of urease. B o t h a p p e a r to be i n v o l v e d in n o n - c o m p e t i t i v e inhibitions. The n i t r a t e ion d e m o n s t r a t e s an inhibition effect on the action of urease, a n d the inhibition a p p e a r s to be competitive. The p h o s p h a t e ion, u n d e r the conditions of this e x p e r i m e n t , exhibits an a c t i v a t i o n effect on the system. The inhibition of urease b y copper (II) has been p r e v i o u s l y r e p o r t e d b y BAHABUR AND CHANDRA3 in 1962 , b u t LEUTHARDT AND KOLLER~ r e p o r t e d t h a t copper h a d no effect on urease in the absence of ascorbic acid. The i n h i b i t o r y effects of zinc have not been r e p o r t e d to date. PITNER 5 a t t r i b u t e s the inhibition of urease b y m e t a l s to t h e irreversible b i n d i n g of the m e t a l at the active sites of the enzyme. GORIN 6 a n d his co-workers r e p o r t t h a t the active sites of urease are s u l f h y d r y l groups. I t is interesting to note t h a t copper is a stronger urease i n h i b i t o r t h a n zinc a n d t h a t copper sulfide is more insoluble t h a n zinc sulfide. Current studies in our l a b o r a t o r y are d i r e c t e d to the correlation of t h e e x t e n t of enzyme inhibition b y m e t a l s with the solubility of the corresponding m e t a l sulfides. The c o m p e t i t i v e inhibition of urease b y n i t r a t e is slight. I t is possible t h a t the n i t r a t e ion has no effect on urease a c t i v i t y at p H 7.0. The a c t i v a t i o n of urease b y p h o s p h a t e (HPO42-) has been r e p o r t e d b y FASMAN Biochim. Biophys. Acta, lO7 (1965) 605-608

PRELIMINARY NOTES

607

AND NIEMAN7 and others s. It is possible that carbamyl phosphate is an intermediate in the reaction mechanism 9. The ease with which the urease-catalyzed hydrolysis of urea can be studied by the direct potentiometric technique will permit the investigation to be carried out under a wide variety of experimental conditions. The effects of pH, temperature, enzyme concentration, substrate concentration, various inhibitors and activators are currently under investigation in our laboratory.

15C lOOj~

J

0--0,

Fig. I. Double reciprocal plot. no i n h i b i t o r & - - & , 1 . 4 7 - I o - 4 M copper (II); t l ) - - C 1.85" lO -3 M zinc (II); 0 - - O , 1.43" IO-z M n i t r a t e ; [ ] - - E 2 , 1.43" IO-z M p h o s p h a t e .

Partial financial support from the Rutgers University Research Council is greatfully acknowledged. Special thanks go to Professor W. W. UMBREIT for supplying the urease powder.

Department o/Chemistry, Rutgers - the State University, Camden, N.J. (U.S.A.) i 2 3 4 5

S. H. K. F. T.

SIDNEY A.

KATZ

J O S E P H A . COWANS

A. KATZ, Anal. Chem., 36 (1964) 2500. LINEWEAVER AND D. BURK, J. Am. Chem. Soc., 56 (1934) 658. BAHABUR AND V. CHANDRA, Proc. Natl. Acad. Sci. India, A32 Pt. I (1962) 72. LEUTHARDT AND F. KOLL~R, Helv. Chim. Aeta, 17 (I934) IO3O. PITNER, Biochem. Z., 325 (1953) 239.

Biochim. Biophys. Acta, lO 7 (1965) 605-608

608 6 7 8 9

PRELIMINARY NOTES

G. G. I{. I~.

GORIN, Biochemistry, I (1962) 911. FASMAN AND C. NIEMAN, dr. Am. Chem. Soc., 73 (1951) 1646I'qATH AND T. PRADHAM, Bull. Calcutta School Trop. Med., 9 (1961) 63F. KOUBA, personal communication.

Received July 2Ist, 1965 Biochim. Biophys. Acta, lO 7 (1965) 605-608

P~,*

21 lO8

Spontaneous incorporation of biogenic amines into purified proteolipid Experiments demonstrating the ability of purified proteolipids to incorporate amino acids spontaneously z and irreversibly led to similar experiments on the binding of amines to proteolipids. Alkaloids and other amines are known to be bound reversibly to serum proteins2, 3 and nerve proteins 4. Work leading to the experiments reported here revealed that, while amines could be bound reversibly to proteolipids as to other proteins, there was a fraction of the bound amine which could not be dissociated except by destruction of the proteolipid. Bovine white matter proteolipid, prepared according to FOLCH, LEES AND SLOANE STANLEY5 and LEES, CARR AND FOLCHs, was precipitated from CHCls-CH30H solution by 4 vol, of diethyl ether at o °. The precipitate was washed once with ethanol (o °) and the centrifugal residue was drained of free ethanol. The residue was then emulsified in distilled water (o °) to a conch, of io mg/ml. In a typical incubation, 0.25 ml of this emulsion was added to 0.25 ml of a solution containing 0.02 M Tris-HC1 (pH 7.5) and 0. 4 mM Iz4Clamine at o °. The incorporation reaction was begun by immersing the tubes in a 39 ° bath. The reaction was stopped after 30 rain by the addition of i ml of a IO mM solution of the nonisotopic amine and 5 ml of CHC13 and CFI30H (i : I, v/v). The lower phase from this mixture was then washed at least five times according to FOLCHv with an upper phase mixture 5 mM in the appropriate non-isotopic amine. These proteolipid solutions were chilled to o °, and precipitated with 4 vol. of cold ether. After centrifugation the proteolipids were resuspended in cold ethanol and centrifuged. Finally the proteolipids were emulsified in cold distilled water, or dissolved in CHC13-CHsOH mixtures or dissolved in 88 % HCO2H, depending upon the subsequent treatment intended. The [z4Clamines were purified within a week of their use by elution from Amberlite IRC-5o by a formic acid gradient, and the purity of the eluted product was checked by paper chromatography in two or more solvent systems. When spots appeared having as much as 1 % of the radioactivity of the main amine spot, the amine was repurified. The biphasic washing procedure and the subsequent solvent precipitations of the proteolipid freed it of all the reversibly bound isotope. Starch column filtration s revealed no trace of unreacted or dissociable [z4C]amines although CHC13-CH3OH solutions of proteolipid and of the [14C]amines used are completely separable by this technique in any of the variations of eluants previously tried 1. More vigorous methods were next tested to separate the [14C]amine from re-isolated proteolipid. Table I illustrates the effect of some mild and some drastic reagent treatments Biochim. Biophys. Acta, lO 7 (1965) 6o8-61o