Floyd J. Malveaux Approximately 90% of washed cells of PS 55 were lysostaphin-sensitive. Acid phosphatase was localized by controlled lysis of SpherOplasts that had been stabilized in 30% polyethylene glycol. Half the enzyme was removed with lysostaphin-solubilized material (cell wall). Follow- ing lysis of epheroplasts, the remaining half of enzymatic activity (48%) was associated with the cytOplasmio membrane. The initial step of the purification procedure for acid phosphatase was elution of the enzyme. Maximal elution of the loosely bound fraction, presumably from the surface of cells, occurred in the alkaline pH range. From log-phase cells, elution was maximally effected with buffered 1.0 M KCl (pH 8.5). The eluted fraction was dialyzed twice and passed through two cycles of molecular sieving (Sephadex 6-100). Specific activity of the purified product was 2350 which represented a BOO-fold purification. Approximately 17% of the initial activity (loosely and firmly bound) was recovered and the 280/260 ratio was 1.72. The final product was free of other enZymatic activities initially present (ooagulase, lipase, and deoxyribonuclease). Purified acid phosphatase appeared homogeneous after gel filtration, starch-block electrOphoresis, and analytical ultracentrifugation. Maximal enzymatic activity occurred at pH 5.2 between #5 and 50(3, but the enzyme was most stable in the alkaline pH range (8.5, 9.5) at temperatures below 50 C. Iodoacetate and EDTA were effective inhibitors Floyd J. Malveaux while meroaptoethanol and Cu++ proved to be stimulators. The purified enzyme which appeared basic in nature at pH 8.0, was most active against the substrates p-nitrOphenyl phosphate and glyceraldehyde 3-ph08phate. Km for the former substrate was 4.5 x 10")+ M and the energy of activation for the hydrolytic cleavage of the same substrate was 19.5 Kcal/ mole. Approximations of the molecular weight made by gel filtration and ultracentrifugation were 54,000 and 53,000 reSpectively. PURIFICATION, CHARACTERIZATION, AND LOCALIZATION OF STAPHYLOCOCCAL ACID PHOSPHATASE By Floyd J. Malveaux A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Microbiology and Public Health 1968 (3. /<Urunvfiubmvlfiu< cm COthDIUONI sCMVULnW-ni Percent Reduction in Absorboncy 49 Control (yin-KDIIIICM--()IIIK3Ill-(y-IIKDIIIIIII-I A. \A\ A. °O .-:r--Am--Am-IA_ 3()1/ A A. .:-.ulI-lall-lol-Iluu-llOl-IlnIII-II-IliL--:E:::f-- U 1. 60- ‘ \e\. ‘0 \ & A. 1. :75. I...!“i.. ‘\‘. “.-I‘, 7,5536 --.A. II... -IIIAflIIII-Inlmla-II-IIIIIIIIII .~.‘I- I I 0% 90- ' ‘00 I l 1 ' ' I O 10 20 3O 4O 50 60 Min u to: Fig. 7. Rate Of lysis of S. aureus PS 55 in the presence of lysostaphin (1.0 Enit7mI)-at 37 C and different concentrations (w/v) of polyethylene glycol (MW: 3000-3700). 1.3 LV§LL. 0.7- Absorbancy .0 '1" 0: C0 L 50 \40 O-Lysed Cells A-Lysed Spheroplasts O-lntact SpherOplOsts A-Intact Cells A ‘ A/‘\, ./ \ e\ \A \‘ __ \’ ’\ :\ o\° \Afi": ‘~l~::+: A Fig. 250 240 260 280 300 Wavelength,mp 8. Ultraviolet-absorption Spectra of supernatnu; fluids of intact cells, intact Spheroplasts, lysed cells, and lysed SpherOplasts. Table 10 51 Absorbancy of intact cells, osmotically protected (30% polyethylene glycol), and unprotected S. 333338 PS 55 in the pres- ence of lysostaphin (intact cells were not treated with lysostaphin) before and after resuspension in water following centrifugation at 7000 x g for 10 min. Before After Absorbancy at 610 mu Intact SpherOplasts Lysate cells (PEG + (Lysostaphin) (30% PEG) Lysost.) .420 .170 .070 .430 .080 -—- 52 lysis when treated in the same manner. Absorbancy Of sphero- plasts resuSpended in water was comparable to that Of unprotected cells. Socalization of the enzyme by an indirect method. Half the acid phosphatase activity was liberated when the cells were converted to spherOplasts (Fig. 9, Table 2). Following lysis Of these spherOplasts, most of the remaining half of activity was associated with the fraction containing the cytoplasmic membrane. In the case Of intact cells which were treated with PEG and dilute salt solution, most of the enzyma— tic activity remained bound to the cells. Sggification of Acid PhOSphatase Phage-propagating strain 3A was initially selected as the source of acid phosphatase because it had the highest enzymatic activity, and most (65%) of this activity was loosely bound (Fig. 1 and 2). During the course Of purification studies, however, the major portion (60%) Of the total enzyme activity shifted to the free (culture medium) fraction. This shift prompted the selection of another strain, S. aureus PS 55, as the source Of loosely bound acid phosphatase. A summary Of the stepwise purification procedure for loosely bound acid phosphatase is illustrated in Fig. 10. Cellular protein (0.39 mg/ml) extracted with 2 N NaOH represented 43% Of the dry weight of cells cultured in 16 liters of Trypticase Soy Broth. The whole culture usually 53 Cells (early stationary phase) Wash twice in water Suspend in 0.15 M NaCl—0.05 M Tris (pH 7.5) SpherOplast formation Centrifuge at 7000 x g for 10 min I I l SpherOplasts Supernatant fluid (A) ResuSpend in water Centrifuge at 20,000 x g for 20 min I l 1 Supernatant fluid (B) Membrane fraction (C) Fig. 9. Flow-sheet diagram for preparing different cel- lular fractions for the purpose of localizing acid phosphatase in S. aureus PS 55. 54 Table 2. Percent acid phosphatase activity of lysostaphin- solubilized and/or eluted material (A, see Fig. 9); intracellular contents Of spherOplasts or wash solutions of intact cells (B); intact cells or membrane fractions of lysed spherOplasts (C). Fraction ___n_ B C SpherOplasts 50.0 2.3 47.7 Cells in 30% PEG, pH 7.5 3.1 23.0 54.4 Cells in 0.15 M NaCl, pH 7.5 13.8 2.3 58.1 55 Cells (PS 55) washed with 0.1 M K01 in 0.05 M Tris-chloride buffer (pH 8.5) 1 l 1 Cells Wash Solution Elute enzyme with 1.0 M K01 in 0.05 M Tris-chloride buffer (pH 8.5) I I 1 Eluted Fraction Cells Dialyze against 0.01 M Tris-chloride buffer (pH 8.5) at 5 C l . J 1 D-I-P D-I-S Dissolve in 1.0 M K01-0.5 M Tris-chloride buffer (pH 8.5) Dialyze against 0.01 M Tris-chloride buffer (pH 8.5) at 5 C l l 1 D-II-P D-II-S Dissolve in 1.0 M KCl-0.5 M Tris-chloride buffer (pH 8.5) L~ Sephadex G-100 Combine tubes with maximal activity. Dialyze against 0.01 M Tris-chloride buffer (pH 8.5) l f l S-I-P S-I-S Dissolve in 1.0 M KCl-0.5 M Tris-chloride buffer (pH 8.5) l [Sephadex G—100 Certain tubes combined Fig. 10. Flow-sheet diagram for the purification Of staphylococcal CPS 55) acid phOSphatase. 56 contained 15-20 units of enzymatic activity/m1, and the cells normally accounted for half this activity. Slution of loosely bound phosphatase. Initial studies of elution of enzyme were carried out on PS 3A when the organism produced a significant amount of loosely bound enzyme. Maximal enzymatic activity was eluted in the alkaline pH range (Table 3). Acid phosphatase was equally eluted at 25 or 37 0. Of the enzyme associated with actively dividing cells grown in Trypticase Soy Broth, 70% was loosely bound to the cells (Fig. 11). The same figure shows that the enzyme ,was maximally eluted from the cells with 1.0 M K01 at pH 7.5, and that the amount eluted increased as a function of ionic strength up to 1.0 M. However, the elution pattern of acid phosphatase was different in the case Of stationary-phase cells grown in the casein acid-hydrolysate medium (Fig. 12). In this case, relatively higher enzymatic activity was found in the free fraction, and maximal elution Occurred at a final concentration of 2.0 M K01. In routine purification exper- iments, the elution step (Fig. 10) effected a seven-fold purification of enzyme. The 280/260 ratio of the eluted fraction was 0.71. Dialysis of the elgted fraction. During dialysis of the eluted fraction against dilute buffer (pH 8.5), the enzyme was precipitated. At salt concentrations less than 0.5 M, solubility Of the enzyme rapidly decreased (Fig. 13), and at 0.2 M, more than 90% of the enzymatic activity was precipitated. « The fraction D-I-P (Fig. 10) had a specific activity of 1300 57 Table 3. The pH dependence of acid phOSphatase elution from cells cultivated in shake cultures of S. aureus with 0.1 M buffer solutions at 25 and 37 0. Activity3 Fraction __§lution pH @ 37 C Elution pH @ 25 c 5.3 7.0 8.5 5.3 7.0 8.5 Eluted 2.82 1.90 8.11 0.10 1.50 9.80 Retained 25.60 32.00 23.00 25.50 31.60 27.60 aEXpressed as micromoles of p-nitIOphenol/ml/30 min at 37 Ce 58 .m.m ma pm How do maoaomapscosoo psmaom . wan Spas OopOOMLO was u smeSHo maznsm szoam how cmdoapaaaev mohdesO madam ca chopmsa mo: Odom bacon mHaHHa bad .Osson chmOOH .omaa Co mossosm c>HpmHOm .HH .maa 59 '/ umnmnm V . munnmnu IIIIIIIIIIIIIII E V . mg 3’. ummuum S llll i E mmuumn 7//////////////// C mnumnm o In at m cw '— 3$V1VHdSOHd 'lVlOl. :IO % Fig. 11 60 .m.a an no Hoa do odoaooaodcodoo odoaoeeao spas conceded was sodpsao meansm .Ampmsamosaoaoomaw Spas OonficEOHQQSM adapoa cadmmaoapzstpaom Sammmov MOHSpHsO madam ca oncomsmmona and OQSOD SHEHHM dam .OssOn SHOMOOH .0099 Mo mpssoam cbapmacm .NH .waa 61 Q N LO KCI (Final Molarity) l l u . I l 0.5 ImmmmumnmuwImnnummmmuumu . 7 OJ llllllllllmlllllllllllllllllllllllllllllllllllllllllllllllllflllllllllllll 7 O 2 at a o JSVIVHJSOHJ lVlOl :lO % - Loosely Bound N Firmly Bound g Free 401 Fig. 12. 62 .OHsHm esopmsHOQSM one wsaammmm an Oosfisacpco mm: hpabapom Odpmammso Houdamoa dam .Omfihoa mumpaaaocaa can as mmw pm wsa>hcmno an Omaaaampmp was manage one mo apHHHQOHOm wsammcaoma .soapcapaam How on Hoaaa L0H .wam com .muHHuQ sodoomamv Oncomsamona anm mo o>aso hpaaansaom .mH .mad 63 .ma .wfia 3:20.02 .05“: UV. .60 «.0 *0 0.0 £0 0.— . mink} _ . mNm we I. I/ u. .- lumuxwv o a. /J m L... w. I. an m. V w POT P flu 30.333me 0E.” N. S m or m d o .n uw D 0 WV? 60— m. o >uconcomn< by i ll... l. I n\U 64 which represented a 172-fold purification Of enzyme. The 280/260 ratio was 0.87, and there was unexpected increase in percent recovery over the previous purification step. Sel filtration. Figure 14 illustrates a typical elution pattern Of acid phosphatase and protein when the redissolved precipitate (D-II-P, Fig. 10) Obtained during dialysis was passed through a column of Sephadex G-100. One peak of enzymatic activity and two protein peaks were Observed. The partition coefficient (Kav) was 0.20 for the major protein peak and 0.61 for the minor protein peak. Figure 15 represents the elution pattern following gel filtration of the fraction S-I-P (Fig. 10). Four-m1 fractions were collected, and the Kav for the common protein and enzyme peak was 0.19. The elution constant (Ve/Vo), where Va is the elution volume and V0 is the void volume, for acid phosphatase was 1.46. Employing the data of Determan and Michel (1966) for “’globular proteins, a first approximation of the molecular weight (MW) of acid phosphatase passed through Sephadex G-100 was 54,000. Sfficiency Of the pgrification procedure. Efficiency Of the procedure is Shown in Table 4. As seen here, purified product obtained after the second cycle of gel filtration accounted for 17.1% of the enzyme bound to the cells. A Specific activity Of 2,350 represented approximately a 300- fold purification. The 280/260 ratio of this fraction was 1.72. The second dialysis step eliminated lipase activity r(Tab1e 5), and repeated gel filtration produced a higher 280/260 ratio and specific activity. 65 [Lu/uremia 6w oeaaoaao-oaae z no.0 ca HOS a o.H spas decade has osandm .m.m ma .Hmhhfin .o m on sssaoo r O 03 P So mm a m.m o mean: duHHrn doaoooae one do Looauo xoocsdomv coaocapaae Hoe .aa..maa LanSZ :OLLOOE an on mm on o." «a ON 3 o. 3 a. 1'3, — p P 0‘; U \O‘O'O’O’d’d \ \O O O / O o \ o ‘2‘. O ‘1’. £22.70 O 4/ 4 O 4 . 4 o onset—c.0574 / / olol O 4 c3 6 ‘f <3 m (‘9 u!Lu OE/lw/l°"°Hd°JI!N -d Wu 0 CW 5 <5 o 0 .000— 66 lm/ugeiou Btu .m.m ma .aoeaoc ooaaoasoumans z mo.o ca HOS : o.H sods oopsao mos masons .o n no asaaoo So mm a m.m o mean: danm concocsa on» to Locate aoodnaomL cofiponpafie How .mH .waa LOLEOZ c339...— Nm on ma 0N cu «N on a. 0— e.— ilclalfldl. - . /o d _ nu N T 0VN w. O mm id MW. 03 e u w. / our 0 To: m. III / S 08 cuOLOLQmosmuc O o um: 67 Sacpoaa wS\sfis om\HS\HOSOsaOHpHSua :5 mm commoaaxmm Loaomo Osooomv eon mc.H H.AH ommm ooa-c LOHOSO pmaamv mom ma.a S.Hm orma coauo maa om.o 6.:0 omma muHHun mea sw.o m.mc coma muHun a Ha.o m.mm a.em ooocam Ill! III! III: m.m maaoc cosmos aopodu anoboooa mhpabapom Soapomhh Scapcoamaasm owm\omm pacoaom camaocam Seduceahaasm .Loa .wam comL Ommpmnamosa Odom Hooooooahsampm sou pecan SHOSSSM sodpcoamaasa Oahmam .: canoe I + + + + + caom camaosz I I I I I + mpwhnhnonnmo I I I I I I Gamhaosannfim I I I I I I cflmhaosom I I + + + + amaze I I I H + + mmmeA I I I I + + ommaswwoo + + + + + + omdpmnmmosm oao4 A303 :3 338 mi mnmmum mnmnm mmmmmm mmmmw 33on oo Io ooaIw Hmoooooahnmwpm zodpomnm noapmoamansm .ommpmsmmosm oaow mo :oHpMoamHHsQ wcdndu mposoonq HmoooooahSQMpm meow mo monmmnm no monommnm .m capme 69 Table 5 illustrates the presence or absence of some staphylococcal products during different stages of purifica- tion. Acid phosphatase, coagulase, lipase, deoxyribonuclease, and nucleic acid were simultaneously eluted from the cells with 1.0 M KCl. Carbohydrate was not removed from the cells which were initially devoid of hemolysin and fibrinolysin activities. During dialysis against dilute buffer, phos- phatase, deoxyribonuclease, and nucleic acid were precipitated, but coagulase and lipase activities remained soluble. The first cycle of gel filtration eliminated nuclease, and the second cycle, increasing the 280/260 ratio to 1.72, essentially removed contaminating nucleic acid. Characterization of Acid Phosphatase gomogeneity ofpurified enzyme. Electrophoretic analysis on starch indicated that the preparation was homogeneous with reSpect to charge (Fig. 16). The basic nature of the phos- phatase was apparent from its movement (2 cm from the origin) toward the cathode at pH 8.0. Figure 17 illustrates the sedimentation pattern of acid phosphatase 8 min (tep) and 40 min (bottom) after the rotor reached maximal speed. In the top figure, the major peak represented acid phosphatase and the minor peaks represented high molecular weight components (probably contaminating ribosomal material). Interestingly, the 280/260 ratio of the sample put into the analytical centrifuge was 1.21 as - contrasted to 1.72, the characteristic value of highly purified .COapwamHa no soapomaav on» o» awHSQHowaHmQ xQOHD meadow on» Home poo mama nods: mucmammm we“: EoImno mo wadumamcoo macapomam Scam copsam Hmaampma on» Ca vmzfiaampmu mama moapa>fipow capwamNCo and caopohm .o 2 .o.m mo pm mammhocaoapomam xooap seamen ca ommpmsamono Uaom Umamahsa mo nodumHMa: mufiufiEzp—@U m 0 v N o N v o m Avj4uAuuu1wundunu4mudmnulwnlnmuuon11u ., n. II. |I__II IT. ll . Euto med. u. o 4 7 m. 4 u 2.0, w 6 / .wI II 9.? 5205-4 0N6. OnassamOPEIO m .wH .mfim o. c an M 4.. so m m u. So u 0| / as w / BO 0 acm— .w. _u 52 F18. 17o Sedimentation pattern of partially purified acid phOSphatase. The enzyme was in 0.6 M KC1-0.1 M Tris. pH 8.5. The upper photograph was taken after 8 min and the lower photograph was taken after #0 min. both at 56.100 rpm. The bar angle was 65°. The protein concentra- tion was 3.5 mg/ml. 72 material derived from the second cycle of gel filtration. Since only one major protein peak was attained, the prepara- tion probably contained a homogeneous species of acid phos- phatase. Calculation of the sedimentation coefficient of the enzyme is summarized in Table 6. An average S (svedberg) value of 2.36 at, the corrected temperature (“.3 C) in the given buffer system was attained by using the St,b values of pictures 3, 4, 7 and 8. The 320,w in this case was 3.68. An approximation of the molecular weight (MW) was made using the formula: 2/ i=mwa 3 ’ sb 1414,2373 where SE is the sedimentation coefficient of a given molecule and Sb is the sedimentation coefficient of a different molecule. Assuming a structure similar to the enzyme glyceraldehyde 3-phosphate dehydrogenase, which has a 320,w value of 7.h and MW of 150,000 (Constantinides, 1967, unpublished data), the MW of staphylococcal acid phoSphatase is approximately 53,000. Effect of pH on the activity of acid phosphatase. The range of optimal pH for the activity of acid phosphatase was 5.2-5.3 (Fig. 18). A decrease of one pH unit from the Optimal value resulted in about 50% reduction in enzymatic activity, and an increase of one unit caused a 30% reduction. The enzyme exhibited little or no activity in the alkaline range. Effect of ionic strength on the activity of acid phos- phatase. The Optimal ionic Strength for acid phosphatase activity was in the range 0.2—0.5M(F‘ig. 19). With 1.0 M KCl 73 mo.n u z.omm mm.m I p.pm .msa sm.m :Ioa x :oaa.m m: maoao.o mmmo.a Haz.o mom.o omn.ma m mm.m :Ioa a oomH.N o: ommoo.c oomo.H mmm.m Nmm.o emo.mm a o:.m :Ioa a mmma.m m msaoo.o o:oo.a mm~.m mmo.a o:m.mm : om.m :Ioa x onso.m : mmooo.o maoo.H mam.m mzo.H www.mm m NN.H ana a ooa.H m «mooo.o mooo.H omm.m :mo.a was.mm N III III 0 o oooo.a mom.o smo.a ems.mm H 95m 40$ cpIp “cpvn\3vn A0391»: on and amq and» woM\ a woa Ioam .omwpdSAmona caom Hmoooooahaompm no paoaoahmooc soapmpaoaauom 0:» mo scandaooaoo .0 «Home 70 .opwaumDSm mm opmcamosa stcnaoapasIo wadms mmmpmnamona paom mo moabapom on» :0 ma mo pommmm I a 0.0— 0.0 0.0 0% 0.0 0.m 0.? b h P n h b / o .mH .mHm AVIlllnv / 0 a 6 5 <5 v Aggagsav agsowkzug eAuolea / .06 75 .mpmpmow z Ioa N H was mmmdo Had a“ sodomapsmoaoo Am.m may ampmsm .ommpmsamosm Hod mo zpabauom esp no nowamapm canoH mo pommmm .mH .wam as: £9.23 :5. O." 3 3 mo o r\\ P h b H! 4 O 7* GD og/Iw/louoqdougN-d w If \\ N F _nvv.|:< 4‘ nu _nv_99‘.lnv nu AV 0 o 4 "1“” é 76 or NaCl, 80% of the optimal activity was still present, but with 2.0 M less than half the Optimal enzymatic activity was apparent. Eggect of temperature on the activity of acid_phosphatase. Maximal enzymatic activity (Fig. 20) appeared above 50 C. However, using an Arrhenius plot (Fig. 21) for the same data, ‘maximal activity occurred at approximately #8 C. Between 15 C and 37 C there was a linear increase in initial activity, but below and above this range, the same relationship was not applicable. The slepe of the solid portion (between 15 C and 37 C) of the plot was used to determine the Arrhenius function A (energy of activation) by the equation _..._..é.._.. m “ 2.303 3’ where m is the negative 810pe, and R is the gas constant. The value for A was 19.5 Kcal/mole. Initial velocity of purified enzyme. Figure 22 is an Eadie-Hofstee plot of initial velocity of enzymatic activity. The slope of the line represents -Km for enzymatic activity against p—nitrOphenyl phosphate. The Km for the substrate was “.5 x 10'“ M, and the Vmax (intercept on Y axis) was #.4 x 10"2 uM P1 liberated/min. Using a double reciprocal plot (Fig. 23) of the initial velocity and substrate concentra- tion, values for Km and Vmax were the same. Egggpt of different_pompounds on acid phosphatase. Of the compounds tested, meroaptoethanol was the only agent that stimulated enzymatic activity (Table 7). Inorganic phosphate, IIIIII \IINI \II:\-°:°:°°Ii‘m2|c :ll 77 '1’ pM p-Nitrophenol /m|/3O min I a» 1 1 Y I o 10 20 3‘0 410 so so Temperotu re (°C) Fig. 20. Effect of temperature upon hydrolysis of p-nitrophenyl phOSphate by acid phosphatase. The solutions were buffered with 0.1 M acetate, PH 5.2. 78 1.2- :0- . O o. o o . .. l: o. . O o 1.0‘ .' o o : O I O 0.8‘ O J! 2 CD 3 0.6- 0‘ o ‘; 0.4‘ o, o O o o o o ‘b 0.2' 0. ‘t O 3.0 3.] 3.2 I 3.3 3.4 3.5 3.6 “/1 x 10"3 (°K“) Fig. 21. Arrhenius plot of the effect of temperature upon hydrolysis of p-nitrophenyl phosphate by acid phoSphatase. The slope of the solid portion (between 15 and 37 C) of the plot was used to determine the Arrhenius function A (energy of activation). 79 .0 mm one N.m ma ad :39 one: mcoapomoh one .Aoposamosa Hanona0HpasIov opoapmnom pass you open one omopmsamosa pace mo hpaooao> Hmfipfiad coozpop ownmonuoHoa on» we poaa oopmmomIoprm .NN .mfim E\> 00. On ow On ON 0— sad .0.— o 3.9M v. o o. 0 .Nd (3 o .06 lo 80 .0 mm one m.m ma pm :39 maoz macapommh one .Qoapmapcoozoo endgamosm Hanonaoapaslm paw omdpmsmmoca ofiom mo hpdooaob Hoapdza soozumn caanOapmHma can go poaa Hoooaadooa cannon .mm .wam EA 0.00m 005 00* 0 OVNNI 0 1n— .o o .3 .> \. nv nu a .on c .m? 81 Table 7. Effect of different compounds on staphylococcal acid phosphatase. The concentrations of these compounds given in the table refer to final concentra- tions. Buffer concentration in all cases was 0.1 M acetate, pH 5.2. Relative Compound activity None 100 0.003 M KHZPOH 104 0.034 M KHZPOu 79 0.032 M Mercaptoethanol 107 0.064 M Mercaptoethanol 132 0.128 M Mercaptoethanol 129 0.005 M Cysteine 86 0.005 M EDTA 62 0.005 M Sodium fluoride 79 0.005 M Sodium molybdate 87 0.005 M Tartaric acid 100 0.005 M Iodoacetate 55 8 M Urea 83 K“ () 'fl 82 cysteine, sodium fluoride, sodium molybdate, iodoacetate, and urea inhibited the enzyme, but tartaric acid had no apparent effect. Effect of divalent cations on acid phosphatase. The cations Co++, Mn++, Mg++, oa++, zn++, and Ba++ had little or no effect on acid phosphatase activity (Table 8). Hg++ and Pb++, on the other hand, reduced enzymatic activity significantly. Cu++ accelerated acid phOSphatase activity two fold at its given concentration. geat stability of the enzyme. In the absence of Cu++, stability of purified acid phosphatase under the conditions employed decreased rapidly between 40 C and 70 C, the tem- perature at which the enzyme was completely inactivated (Fig. 24). With ou++, the minimum temperature for complete inactivation was 80 C; thus the cation did afford some protec- tion of acid phosphatase. Stability_of the gnzyme_under different conditions. Purified acid phosphatase was completely inactivated in 1.0 M acetic acid and 1.0 N NaOH (Table 9). When suspended in water for 6 days, 55% of the initial activity was lost. The enzyme appeared most stable in the alkaline range (8.5 and 9.5). Agpivity of acid phosphatase with various substrates. Of the substrates tested, acid phosphatase was most active against p-nitrophenyl phosphate (Table 10). Substantial activity against glyceraldehyde 3-phosphate, and moderate activity against (I-glycerophosphate, fructose 6—ph03phate, and phenolphthalein diphOSphate were also observed. Acid 83 Table 8. Effect of different divalent cations on the activity of acid phosphatase. Final concentra- tion of the metal ions (used as chloride form) was 1 x 10'3 M. Metal Relative Activity None 100 Co++ 95-5 Mn++ 80.0 Ms++ 95-5 Ca++ 76.4 Zn++ 93.9 Cu++ 206.4 Hg++ 18.5 Pb++ 17.9 Ba++ 95-5 100 2 O-Without Cu++ rvityr % Residual Act A- With Cu”r + 80« 2 60- 4o A. 20- (3 £--- 0 1\n—¢>4 40 60 80 100 Tom porotu ro (°C) Fig. 24. Heat stability of staphylococcal acid hos- phatase with and without Cu++ (1 x 10' M). The enzyme was heated at each temperature for 5 min in 0.01 M Tris-chloride (pH 8.5) and cooled quickly in ice water. Assays were carried out at 37 C and pH 5.2. 85 Table 9. Stability of staphylococcaleufid phos- phatase. Samples were stored at room temperature (25 C) for 6 days. The activities are relative to zero time. Relative Activity Conditions 1 day 3 days 6 days Water (no buffer) 100 100 45 0.1 M Acetate, pH 5.2 24 27 10 0.1 M Tris-chloride, pH 7.5 21 9 2 0.1 M Tris-chloride, pH 8.5 95 71 54 0.1 M Glycine-NaOH, pH 9.5 100 85 10 1.0 M KCl, pH 5.2 47 25 14 1.0 M KCl, pH 7.5 23 27 22 1.0 M KCl, pH 8.5 103 88 42 1.0 M KCl, pH 9.5 84 80 52 1.0 M Acetic Acid 0 0 0 1.0 N NaOH 0 O 0 86 Table 10. Relative activities of staphylococcal wfld.phosphatase against d fferent phosphate esters (1 x 10‘ M). Samples were incubated at 37 C for 30 min. A value of 100 was assigned to the activity of acid phosphatase against p-nitro- phenyl phosphate. Compound p-NitrOphenyl phOSphate Glucose 6-phosphate c-glycerOphosphate B-glycerophosphate Dihydroxyacetone phosphate Fructose 6-phosphate Ribose 5-phosphate Phosphoglyceric acid Glyceraldehyde 3-phosphate Fructose 1, 6-diphosphate Guanosine diphOSphate Cytidine 31 (2') phosphoric acid ATP DNA Sodium pyrophosphate Phenolphthalein diphosphate Casein ci-casein Lecithin Relative activity 100 3.5 34-5 14.1 0 38.0 0 0 82.8 13.8 17.3 10.4 10.4 3-5 24.1 4.1 3-5 87 phosphatase exerted little or no hydrolytic activity against the remaining substrates. DISCUSSION Acid phOSphatase has been implicated in the virulent nature of §paphylococcus aureus and has been screened in many strains of the organism; yet, to this writer's knowledge, there is no report on purification and localization studies of the enzyme in §. aureus, and only limited characterizations have been made on one enzyme preparation consisting of whole cells. There is a need, therefore, to purify, characterize, and localize the acid phosphatase of §. aureus. Experimental evidence indicates no restriction of high phosphatase activity to any particular group of phage- propagating strains of coagulase-positive staphylococci (Fig. l). Relatively little alkaline phosphatase activity is observed in the strains of S. aureus used in our studies. In this regard, it must be noted that the growth media (Brain Heart Infusion, Trypticase Soy Broth, and even the casein acid-hydrolysate medium) contain a considerable amount of inorganic phosphorus, and formation of alkaline phosphatase by §. aureus (Shah and Blobel, 1967) is subject to repression by inorganic phosphorus in the medium. Production of acid and alkaline phOSphatases by Eppherichia coli (Torriani, 1960) is similar to that of S, aureus; acid phosphatase is always present, but alkaline phosphatase is formed only when the concentration of inorganic phosphorus is limiting in the 88 89 medium. However, neither acid nor alkaline phOSphatase from E: 931i is found in the culture medium. Both Elek (1959) and Cannon and Hawn (1963) reported that staphylococcal phOSphatase appears to be an intracel— lular enzyme and cannot be detected in the culture medium. Our studies contradict such findings; in fact, every strain that we tested elaborated extracellular enzyme (Fig. 2). It is of interest that the relative amounts of acid phosphatase in the three fractions (free, loosely bound, and firmly bound) vary to such an extent in different strains of §. aureus. That such variation may occur even in the same strain is of greater interest. Initially, about 65-70% of the total phosphatase activity of phage-prOpagating strain (PS) 3A was loosely bound material. However, during later purification studies only 30% of the total activity was loosely bound, and therefore this strain was abandoned in favor of PS 55. The latter strain forming a greater percent of loosely bound acid phosphatase was retained as a routine source of enzyme for purification and characterization studies. When S. aureus is grown in a complex medium (Trypticase Soy Broth), the rate of whole culture acid phosphatase produc- tion is a function of cell number. Barnes and Morris (1957), as well as Cannon and Hawn (1963) made the same observation in certain strains of g. aureus. When cells are grown in the casein acid-hydrolysate medium, whole culture enzyme activity again increases with an increase in cell number; but in the 90 same medium supplemented with glycerOphosphate, there is an unexpected increase in enzymatic activity during the stationary phase of the growth cycle (Fig. 4). This latter increase can- not be due to increased cell number since cessation of cell division coincides with glucose depletion from the medium. Hofsten (1961) described a repressible effect of carbohydrates (glucose, glycerol, and glycerOphOSphate) on acid phosphatase production in E. 921i. However, similar repression is not observed in our system. Increased phosphatase activity in the stationary phase is probably the result of enzyme induc- tion, since a similar increase in activity is not observed in the absence of glycerophosphate (Fig. 5). Because acid phosphatase is easily eluted from the sur— face of §. aureus, we became interested in the cellular loca- tion of this enzyme. Our method for localizing acid phos- phatase in §. aureus depended upon the formation of sphero- plasts.. Unlike most gram-positive bacteria, cells of §. aureus are lysozyme-resistant (Mandelstam and Strominger, 1961 and Virglio et al., 1966). The prot0p1asts of §. aureus formed by an autolytic system described by Mitchell and Moyle (1957) were permeable to glycerol, but not to NaCl and sucrose. Hash and his coworkers (1964) stabilized protOplasts that were formed by a fungal N-acetylhexosaminidase with 0.5 M sucrose. And recently, Schuhardt et al. (1967) formed proto- plasts of S. aureus with 30% NaCl and lysostaphin. In our studies, 90% of the cells were lysostaphin-sensitive (Fig. 6). Polyethylene glycol (30%) as well as 30% NaCl were both 91 effective stabilizing agents, but spherOplasts could not be formed even in 2 M sucrose. Since any stabilizing agent must be unable to penetrate the cytOplasmic membrane, it is likely that sucrose, but not polyethylene glycol (PEG) and NaCl, did penetrate §. aureus PS 55. Ultraviolet-absorption spectra of the supernatant fluids of intact cells, intact spherOplasts, lysed cells, and lysed spherOplasts indicate preservation of the osmotic barriers of spherOplasts (in 30% PEG). There is a great deal more 260 mu- absorbing material liberated when these spherOplasts are lysed in water. Apparently, increased absorption at 260 mu results from freed nucleic acid. Osmotic fragility is again demonstrated by measuring cell turbidity when SpherOplasts are suspended in water. Reduction in turbidity (at 610 mu) then approaches a value comparable to that of cells broken down by lysostaphin. } When certain cellular fractions (cell wall, cytOplasmic membrane, and intracellular contents) of §. aureus were prepared after spheroplast formation, the fractions were analyzed for acid phosphatase activity. Half the activity was liberated with the lysostaphin-solubilized material (cell wall). Most of the remaining half of phosphatase activity was associated with the cyt0plasmic membrane. Thus, the enzyme is not intracellular, but is located at the level of the cytOplasmic membrane. Since half the enzyme is liberated with the cell wall fraction, that half may well be combined with the cell wall, or at least be located outside the 92 the cytoplasmic membrane. The conclusions drawn from our studies are in agreement with those of Mitchell and Moyle (1956) who concluded that most of the acid phosphatase of E. aureus is found in the cytoplasmic membrane. A similar location for the same enzyme has been found for E. 99;; (Hofsten, 1961 and Dvorak et al., 1967). Neu and Heppel (1965) suggested the existence of a family of degradative enzymes on the cell surface of E. ggEi. Even alkaline phos- phatase of E. 99;; is located at the cell surface (Kushnarev and Smirnova, 1966). Since the cytoplasmic membrane is considered impermeable tgfgfibsphate esters, it is reasonable that phosphatase be located at the cellular surface (Malamy- and Horecker, 1961). The same investigators noted that such a location for alkaline phosphatase of E. QQEi would account for the ability of the cell to preserve its intracellular pool of phosphate ester intermediates. This is a type of compartmentalization which has sometimes been suggested to account for coexistence of enzymes and substrates in bacterial cells. As noted by Cedar and Schwartz (1967), all of the bacte— rial enzymes so far reported to be situated in the periplasmic region are degradative, and would probably inhibit cellular function if they were not separated from the cytOplasm. Elution of acid phosphatase from E. aureus is a function of pH and ionic strength. Salt concentrations of 1.0 to 2.0 M at alkaline pH values are optimal for elution of the enzyme. Our system of elution necessitates a higher salt concentration than 0.5 M KCl which is reported for acid phosphatase elution 93 from Saccharomyces mellis (Weimberg and Orton, 1965). The enzyme of yeast cells could not be eluted at higher salt concentrations unless a thiol compound were added to the eluting menstruum. The same authors (Weimberg and Orton, 1966) found great variation for conditions of acid phos- phatase elution among related species of yeast cells. Elution of acid phosphatase with a salt solution indicates the enzyme is associated at least in part with the cells through electrostatic interactions. Weimberg and Orton (1965) noted that a requirement of ionic compounds for elution of acid phosphatase from yeast cells meant the enzyme was held to the cell wall by electrostatic forces. The fraction which we eluted from E. aureus with salt solution is probably loosely bound to the cell surface and gives rise to the free fraction found in the culture medium. Rogers (1956) suggests that extracellular enzymes of E. aureus are extruded into the growth medium as a "capsular-like" material that later dis- solves. The ionic binding prOperties of the surface of E. aureus were studied by Cutinelli and Galdiero (1967). They showed that divalent cations combined with the cell wall of E. aureus more readily than monovalent ions. In fact, the cell wall of E. aureus behaved like a weak ion-exchange resin. It appears then, the surface of E. aureus bears a negative charge as does most bacteria. Since phosphatase is eluted with potas— sium and sodium ions, the enzyme is expected to have basic prOperties at pH 7.5. Our studies indicate that this is 94 indeed the case; because the purified enzyme migrates toward the cathode at pH 8.0 in a starch block (Fig. 16). There- fore, since the enzyme is associated with the cells by elec— trostatic interaction, it probably undergoes ionic exchange with the cations of the eluting menstruum. Also employing ionic elution, Takeda and Tsugita (1967) liberated alkaline phosphatase from Bacillus subtilis with . Mg++. The one-step elution caused an increase in Specific activity of the enzyme which subsequently required high salt concentration for its dissolution. The eluted enzyme which we obtained from E. aureus required at least 0.5 M KCl for its dissolution (Fig. 13). Kidwai and Murti (1965) also noted that certain oxidative enzymes of E. ggEE which were associated with the cytoplasmic membrane could not be sol- ubilized by conventional means. Thus, it appears that some enzymes that are part of the cytOplasmic membrane have unu- sual solubility properties once they are liberated from their native site. Solubilization of acid phosphatase in a protein-free ionic solution provides us with a major step in enzyme purification. Neu and Heppel (1965) recognized that a good first step in purification of an enzyme is its selective removal from the cells. They were able to elute certain enzymes from E. EQEE by their "osmotic Shock" procedure. Hofsten and Porath (1962) demonstrated limited effective- ness of precipitation procedures for purification of acid phosphatase of E. coli, but they were able to purify the 95 enzyme ZOO-fold by gel filtration and zone electrOphoresis. Dvorak et a1. (1967) observed two acid phosphatases in E. ggEE, one (hexose phOSphatase) which was purified 870-fold by column chromatography, and the second (nonSpecific phosphatase) which resisted purification. We were able to purify staphy- lococcal acid phOSphatase 300-fold by employing the mild procedures of elution, dialysis, and gel filtration (Fig. 10, Table 4). A 280/260 ratio of 1.72 indicated the purified material is essentially free from contaminating nucleic acid, and 17% of the original enzymatic activity associated with washed cells was recovered. It is noteworthy that the fraction D-I-P has a higher percent recovery (62.5%) of enzyme than the previous fraction (33.3%). Thus, some small molecular weight inhibitor(s) may be removed during dialysis. Another possibility is the disaggregation of acid phosphatase from other macromolecules that remain soluble during dialysis against dilute buffer while phOSphatase is precipitated. Such aggregates could function by masking the active sites of acid phosphatase. Purified staphylococcal acid phosphatase appeared as a homogeneous protein by gel filtration (second cycle on Sephadex G-lOO), starch-block electrOphoresis, and analytical ultracentrifugation. Attempts to characterize purified enzyme by disc-gel electrOphoresis at pH 8.3 and 7.5 were unsuccess- ful because the sample failed to migrate. Most of the purified material which displayed a high 280/260 ratio ()I1.70) was 96 soluble in dilute buffer (0.05 M Tris-chloride, pH 8.0), but material with a lower 280/260 ratio (< 1.70) required a solvent having an ionic strength of at least 0.5 M for dissolution. Thus, it appears that contaminating 260 mu- absorbing material affects the solubility of acid phosphatase by requiring solutions of high ionic strength. We did obtain some evidence concerning the nature of the contaminating 260 mu-absorbing material. When a sample of the purified enzyme (dissolved in 0.6 M K01-0.1 M Tris, pH 8.5) which had a 280/260 ratio of 1.21 was analyzed by the sedimentation velocity method in the ultracentrifuge, contaminating material was of high molecular weight, and acid phosphatase appeared as one sharp symmetrical schlieren pattern (top of Fig. 17). The contaminating fraction was probably ribosomal material. The presence of ribosomes is reasonable because in order to obtain sufficient protein (3.5 mg/ml) for adequate resolution in the ultracentrifuge, we combined chromatographic fractions (second cycle) which had high enzymatic activity and a low 280/260 ratio (1.10) with those that had high enzymatic activity and a high 280/260 ratio (1.72). However, it is unlikely that the ribosomal material is present in purified fractions having a 280/260 ratio of 1.72. Approximations of the molecular weight (MW) of purified acid phosphatase by two different methods were comparable: 54,000 by gel filtration and 53,000 by analytical ultra- centrifugation. The acid phOSphatase purified by Hofsten and Porath (1962) from E. 99;; has a MW of 13,000 (estimated 97 by amino acid analysis). Their enzyme is quite different because it is unstable in dilute solutions, stable in l M acetic acid, and denatured in the presence Of neutral salts. Staphylococcal acid phosphatase, on the other hand, is relatively stable in water and l M KCl, but inactivated in the presence of 1 M acetic acid (Table 9). Dvorak et a1. (1967) isolated two acid phosphatases from E. coli: hexose phosphatase and nonspecific phosphatase. The latter enzyme more closely resembles the staphylococcal acid phosphatase. Like the enzyme from E. EQll: acid phos- phatase from E. aureus has a pH optimum near pH 5 (Fig. 18), is inhibited by EDTA (Table 7), and readily hydrolyzes p-nitrOphenyl phosphate (Table 10). The two enzymes differ in that activity of the staphylococcal enzyme is stimulated 2-fold by Cu'”+ (Table 8), whereas metals have no effect on the enzyme of p. ggEE. Cu++ not only stimulated enzymatic activity, but also gave greater stability to the enzyme between 50 and 80 C (Fig. 24). The native staphylococcal enzyme may contain Cu++, just as alkaline phosphatase of E. 93;; contains Zn++ (Plocke and Vallee, 1962). Ionic strength has little effect on staphylococcal acid phosphatase activity up to 1.0 M KCl or NaCl (Fig. 19). How- ever, progressive loss of enzymatic activity does occur at higher salt concentrations. There is a rather narrow pH (range (5.2 to 5.3) for optimal activity (Fig. 18). Though Optimal activity occurs at acidic pH values, the enzyme is more stable in a slightly alkaline menstruum (Table 9). Both 98 extremes of the pH scale completely inactivate staphylococcal acid phosphatase. As noted by Dixon and Webb (1964), the effects of tem- perature on enzyme reactions are very complex. Deleterious effects due to instability of the enzyme itself can be studied by first exposing the enzyme to various temperatures for a definite period of time and then measuring its activity at a temperature in which it is stable. Discontinuity of the slope of an Arrhenius plot (Fig. 21) at 48 C suggests irreversible inactivation of the enzyme since it loses its stability rapidly at temperatures above 50 C (Fig. 24). The Arrhenius function A (energy of activation) for staphylococcal acid phOSphatase is 19,500 cal/mole. This value is reasonable, because the values of A for ordinary chemical reactions (including catalytic ones) range from a few thousand to 40,000 cal/mole, with the majority in the neighborhood Of 15,000 to 25,000 cal/mole (White, Handler, and Smith, 1964). Values for either the Michelis constant (Km) or maximal velocity (V ) were the same on both Eadie—Hofstee and double max reciprocal plots of initial velocities of acid phOSphatase against p-nitrophenyl phosphate. The Km was 4.5 x 10'“ M, and vmax was 4.4 x 10‘2 uM P1 liberated/min (Fig. 22 and 23). Barnes and Morris (1957) reported the Km for p-nitrOphenyl phosphate was 2.0 x 10'“ M. However, as previously noted, the enzyme preparation used in their studies was a suspension of whole cells. Statistical methods for obtaining Km and Vmax were not applied to our data since the graphical methods proved 99 adequate. In this regard, Dixon and Webb (1964) state that for nearly all purposes, graphical methods suffice for deter- mining Km and Vmax' These authors prefer the double reciprocal plot to the Eadie-Hofstee plot. Staphylococcal acid phOSphatase probably requires a free sulfhydryl (-SH) group for maximal activity. Iodoacetate which usually reacts with thiol groups to give alkylated derivatives (Dixon and Webb, 1964) proved to be the most effective inhibitor (Table 7), and meroaptoethanol which usually preserves -SH groups had a stimulatory effect. Since EDTA (a metal chelator) also inhibited enzymatic activity, the enzyme may be more active in the presence of certain metals. As noted earlier (Table 8), acid phosphatase is twice as active in the presence of Cu++ than in its absence. The Law of Mass Action could explain the inhibition of inorganic phosphate in high concentration. Other compounds tested had little or no inhibitory effect on staphylococcal acid phOSphatase. Relative activity of the enzyme with the different sub- strates tested (Table 10) gives little insight into the natural substrate and role of the enzyme in vivo. Acid phos- phatase was most reactive against p-nitrOphenyl phosphate and glyceraldehyde 3-phOSphate. This writer is aware of no biological system where the latter compound is a normal sub- strate for phosphatase. The role of acid phosphatase in microorganisms is still a matter of conjecture. The enzyme in E. aureus may play some 100 important biological and ecological role, since some strains produce significant amounts of the enzyme. The enzyme may render some compounds more readily available to the cells, for many phOSphorylated esters cannot cross the cytOplasmic membrane. The presence of enzyme in the cytoplasmic membrane and in the culture medium suggests that it may degrade organic phOSphates present in the growth medium. A similar function has been prOposed for acid phOSphatase of E. ggEE. Yet, the cytoplasmic membrane of the same organism is permeable to glucose 6-phosphate, and alkaline phosphatase is not related to the entry of the phosphorylated ester into the cell (Fraenkel, Kelly and Horecker, 1964). Staphylococcal acid phosphatase may even be a part of the ”translocase” mechanism involved in phosphate transfer across the cell membrane as prOposed by Mitchell (1957). Heppel (1967) recently noted that proteins of E. 22;; found at the ”surface of the cell are capable of binding with substances in the medium, and these proteins may be components of active tranSport systems reaponsible for the concentrative uptake of these nutrients.“ We believe, as does Kedzia and his coworkers (1966), that staphylococcal acid phOSphatase plays an important role in aiding the penetration Of certain phosphorylated compounds into the bacterial cell by hydrolyzing the esteric bond, thereby making the products more readily available for uptake by the cells. SUMMARY Strains of Staphylococcus aureus from the International- Blair and the Seto-Wilson Series of phage—prOpagating strains (PS) were examined for acid phosphatase activity. All selected strains showed enzyme production after 24 hr of growth, and when all samples were adjusted to the same optical density (0.5), PS 3A surpassed all others in enzyme activity. Acid phosphatase occurred in varying amounts in three different fractions: free (6 to 60%), loosely bound (25 to 82%), and firmly bound (0 to 40%). Rate of whole culture enzyme production paralleled cell growth in Trypticase Soy Broth. In the casein acid-hydrolysate medium supplemented with glycerOphOSphate, initial production of enzyme during logarithmic growth was followed by increased production during the stationary phase of the growth cycle. This biphasic pattern was not observed in the absence of glycerOphosphate. SpherOplasts of PS 55 were made in the presence of lysostaphin and 30% polyethylene glycol. Following spheroplast formation and controlled lysis of the spherOplasts, half the total phosphatase was located in the cell wall fraction, and most of the remaining enzyme (48%) was associated with the cytOplasmic membrane. Loosely bound acid phosphatase was purified 300-fold by elution, dialysis, and two passages through Sephadex G-100. lOl 102 The specific activity of purified enzyme was 2350 and approximately 17% of the initial activity (loosely and firmly bound) was recovered. Purified acid phosphatase appeared homogeneous by gel filtration, starch-block elec- trophoresis, and analytical ultracentrifugation. Maximal enzymatic activity occurred at pH 5.2, between 45 and 50 C. The enzyme was most stable in the alkaline pH range and at temperatures below 50 C. Iodoacetate and EDTA were potent inhibitors, but meroaptoethanol and Cu++ stim- ulated enzymatic activity. Purified enzyme was basic in nature since it migrated toward the cathode at pH 8.0 in a starch block. 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