TIZE JOURNAL OB B~omcic~r, C~rams~m' Vol.248, No.17,Issue of September 10, pp. 6029-6034, 1973

Printed in U.S.A.

The Proteolytic Enzymes of the IS-P Strain of StreptoPnyces

griseus Obtained from a Commercial

Preparation (Pronase)

PURIFICATION A31\j’D C:HiZlthCTEltIZhTION OF THE AMln’OI’I~:I’TIDASF:S*

(Received for publication, May 10, 1973)

KLAUS I). VOS~ECIC,$ &I-VU CHOW, AND WILLIAMS M. AWAD, JR.$

From the Deprtments oj Medicine and Biochemistry, l/niversity oj Miami ~School 01 Medicine, Biscayne Annex, Miami, Florida 33152

SUMMARY

We described earlier the purification of Sfrepfomyces griseus trypsin. This enzyme was demonstrated to bind diaminoalkanes and as a consequence was selectively re- tarded during CM-cellulose chromatography (AWAD, W. M., JR., SOTO, A. R., SIEGEL, S., SKIBA, W. E., BERNSTROM, 6. G. & OCHOA, M. S. (1972) J. BioL Chem. 247,4144-4154).

In order to facilitate this procedure an affinity matrix, 1,6- diaminohexane-agarose, was synthesized and demonstrated to bind the trypsin. More significant was the finding that this agarose derivative also bound the two components with aminopeptidase activity present in Pronase. The trypsin

was completely separated from the aminopeptidases by ini- tial passage through a CM-cellulose column. Thereafter, the trypsin was selectively retarded by the hexanediamine- agarose; all associated components were eluted with 1 M NaCl at pH 8, whereas the trypsin could be eluted only after application of an acidic (pH 3) buffer. This enzyme ap- peared to be homogeneous by other studies. Bovine trypsin was not retarded by this chromatographic procedure. The aminopeptidases were purified by passage through the same column. Their complete selective retardation was achieved only after prior treatment with sodium ethylenediamine- tetraacetate. Calcium ion at low concentrations eluted the two enzymes separately. Calcium ion was required for the activity of each enzyme and also stabilized each enzyme against heat denaturation. Strontium ion could restore about two-thirds of the activities to the metal-free proteins as compared to the activities noted with calcium; other di- valent cations provided much less activity. The maximum activity of each aminopeptidase lies between pH values of 7.5 and 10; each enzyme is stable between pH values of 6 and 11. These enzymes have been tentatively designated

* This investigation was supported by United States Public Health Service grants (NH-AM-09001, NIH-A&I-05472, and XIII-(X1-02011). This is the fifth paper in a series. Paper IV is Reference 1. A preliminary communication of portions of this work ha,s been published (2).

$ Physicisn-student, in the lIepart rncrlt of I<iochcrnistry. This work was carried out in partial fldfillment of the requirements for the degree of Doctor of Philosophy.

$ To whom all inquiriw sholdd Ix directed.

aminopeptidase 1 and aminopeptidase 2 after the order of their elution from hexanediamine-agarose; gel filtration re- vealed their approximate molecular weights to be 23,000 and 25,000, respectively. A single band was seen for each pro- tein after acrylamide gel electrophoresis.

WC have been interested in t,he purification and characterisa- tion of the many proteolytic components in Pronasc (1, 3-5). Endopcptidases, aminopeptidases, and a carboxypeptidase have been identified as contributing separate and significant activities (6). The commercial availability of this potent mixture serves as a ready source of possible new agents for the analysis of pro- tein structure. Previously we described the purification to homogeneity of four endopeptjidases which included a protease apparently homologous with subtilisin and also three other

smaller components homologous with the chymotrypsin family of serinc enzymes (3). One of the latter proteins has already been dcmonstratcd by us to be active and stable in 6 M guanidinium chloride (4, 5) ; these propcrtics may make this protease very useful for a variety of laboratory applications. Another of the latter three scrine cnzymcs was demonstrated to have marked homology with bovine l,rypsin (7, 8) and was selectively retarded during CM-cellulose chromatography after complexing with alkylamines (3). Our attention was directed to the possibility that the immobilization of one of these amines on an insoluble matrix would provide an affinity column for the facilitated puri- fication of the trypsin-like component. This proved to be the cast, as the following studies disclose. However, of greater importance was the finding, as described below, that this tech- niquc permit,tcd the parallel purification of the protein compo- nents containing the aminopcptidase activit,y.

b:XPERIMENTAI, PROCEDURES

Pronasc (grade 1%) was obtained from Calbiochem; several lots (K-OS. 101185, 200191, and 201305) were used for these studies. Dovine trypsin was obtained from Worthington Biochemical Corp. Sodium picrylsulfone.te dihydrate was oMained from

Aldrich Chcmicaal Co., Inc. Ovalbumin, soy bean trypsin in- hibilor, nra-acc:tyl-r,-tvrosirle ethyl ester, Not-benzoyl-D ,L-

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arginine-p-nitroanilide, and cadaverine dihydrochloride were obtained from Mann Research Laboratories. Casein was ob- ta.ined from Difco Laboratories and purified (3). Cyanogen bromide, 1 ,6-diaminohsxane, and I-ethyl-3-(3-dimethylamino- propyl)carbodiimide hydrochloride were obtained from Eastman Kodak Co. Ribonuclease and L-leucine-p-nitroanilide were purchased from Sigma Chemical Co. Sephadex G-25 and G-75 and also agarose beads (Sepharose 4B) were obtained from Pharmacia Fine Chemicals. Preswollen microgranular CM- cellulose (Whatman CM-52) was obtained from H. Reeve Ange & Co., Inc. Succinic anhydride was obtained from Matheson, Coleman and Bell. Human carbonic anhydrase C was a gift from Dr. Philip L. Whitney, Department of Biochemistry, Uni- versity of Miami School of Medicine, Miami, Florida; sperm whale myoglobin was obtained from Seravac Laboratories. All other chemicals were of reagent grade.

Measurement of Catalytic Activities-The extent of casein digestion was determined as described earlier (4). The activity against Ac-Tyr-OEtl was measured by previously described techniques using the pH-stat (9). The activities against Leu- NHNp and Bz-Arg-NHNp wcrc dct’ermined in the following manner. Leu-NIINp or Be-Arg-NHNp was prepared at 10 m&l concentration in 10 m&f HCl. Two milliliters of either of these stock solutions were mixed with 10 ml of 10 m&T Tris (pH 8.0) with 5 m&l CaC12; 10 to 50 ~1 of enzyme solut’ion were added to 2 ml of the substrate mixture for each assay, which was carried out at room temperature. The activities were followed by the change in absorbance at 405 nm. A molar extinction coefficient of 9620 M-I cm-l was used for p-nitroaniline at this wave length (10) ; this value was unchanged over the pH range of studies described in the present report.

Preparation of Afinity Columns-The affinity columns were synthesized by modification of previously described techniques (11, 12). Agarose beads (30 ml packed volume) were suspended in 265 ml of water after washing with water and also removal of fine particles by decantation. Cyanogen bromide solution (50 mg per ml of HzO), 210 ml, was added with gentle stirring under a hood. The pH was kept constant at 11 by addition of 5 M

NaOH. The temperature was maintained at 25” by addition of ice chips. When no further change in pH occurred (after 15 min), a large amoint of ice was added and the gel was transferred to a glass filter and washed with 1 liter of 0.1 M NaHC03 (pH 10.5). The washed beads were immediately mixed with 30 ml of 2 mM 1,6-diaminohexanc which had been adjusted to pH 10.5 with HCI. The solution was allowed to stand for 16 hours at 4” with gentle stirring. Thereafter, the beads were washed (through a glass filter) with several liters of water. The hexane- diamine-linked agarose beads were used to make the two other agarose derivatives: cadaverine-succinyl-hexanediamine-agarose and cadaverine-succinyl-cadaverine-succinyl-hexanediamine-aga- rose. The techniques utilized for the synthesis of these two derivatives involved standard procedures with the use of succinic anhydride and also ce.da,verine and the water-soluble coupling reagent 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (11, 12). The different states of bound amino groups were followed by reaction with sodium picrglsulfonate (11,13). No attempts were made to quantitate the degree of substitution for each gel.

Column Chromatography Procedures-All chromatographic steps were carried out at 4”. One gram of Pronase was dissolved in 2 ml of 5 mM Tris-HCl buffer (pH 8.0) containing 5 mM CaC&.

1 The abbreviations used are: Ac-Tyr-OEt’, Nwscetyl-L-tyro- sine ethyl ester; Leu-NHNp, L-lencine-p-nitroanilide; Bz-Arg- NKNp, l~~a-benzoyl-n,L-arginine-p-nitroanilide.

The scant insoluble residue was removed by centrifugation (12,000 X g for 10 min) and the supcrnatant material was passed through a Sephadex G-25 column (1.6 x 27 cm) equilibrated with the above buffer. The fractions containing the excluded protein material were combined and applied to a CM-cellulose column (1.5 x 14 cm) equilibrated with the same buffer. After a period of flow with the buffer, retained protein material was eluted by stepwise application of the same Tris-HCl and CaClz buffer which also contained 0.3 RI NaCl.

The fractions containing the protein eluted with 0.3 M NaCl were combined and applied to a hexanediamine-agarose column (1 X 10 cm). This column was washed with 5 mM Tris-HCl (pH 8.0)-5 mM CaC&-1 sz NaCl. After a period of flow the column was washed by the stepwise application of 10 rnM glycine buffer (pH 3.0). The fractions containing protein eluted by the glycine buffer were concentrated by Diaflo ultrafiltration (Amicon Corp.) through a IX-2 membrane. Thereafter, this material was equilibrated with 5 mM sodium acetate (p1-I 5 0), containing 5 m&I calcium acetate, by passage through a Sephadex G-25 column (1.6 x 27 cm) equilibrated with t’hc same buffer. Further chromatography through CJI-cellulose at pH 5 was carried out by previously described techniques (3).

The fractions containing protein that was not retarded by C&cellulose in the Tris-HCl-CaCll buffer at pI1 8.0 were com- bined. To this solution was added 250 mM EDTA (pH 8.0) to give a final concentration of 25 mM. The solution was allowed to stand for 1 hour at 4”; lollgel- periods of incubation did not change the results of subsequent steps. Thereafter, the solu- tion was concentrated by Diaflo ultrafiltration through a URI-2 membrane. The retentate was passed through a Sephadex G-25 column (1.6 x 27 cm) equilibrated with 5 mM Tris (pH S.O)-0.5 mM EDTA. The fractions containing protein were combined and passed through the hesanediamine-agarose column (1 X 10 cm). The column was washed in order by the following salt solutions each containing 5 m&f Tris-HCl (pH 8.0): (a) 30 mlw. NaCl; (b) 5 mM CaCl,; (c) 20 mxl CaC&; and (d) i M NaCl.

Chromatographic components obtained from the affinity column were studied further for homogeneity by application to a Sephadex G-75 column (1.1 x 198 cm) eluted with 5 mM Tris- HCl (pH 8.0)-5 mM CaC12.

Activity and Stability 0.f the gnzinopeptidases-The pH de- pendence of the activity of the aminopeptidases was determined in the following manner. Two milliliters of 10 mM Leu-NHNp in 10 mM HCl were diluted with 10 ml of HzO; 3 ml of this solu- tion were placed in a pH-&at Tessel for each assay and the pH was adjusted to the desired value by addition of 10 mM NaOH. Ten microliters of enzyme solution in 10 mM Tris (pH 8.0)-5 rnM CaClz were used for each assay. The pH was maintained with addition where appropriate of either 10 mM NaOH or 10 mM

HCl during the reaction. The reaction was allowed to proceed at 30” for exactly 335 min when 2 ml of reaction mixture were mixed with 1.5 ml of 10 mM glycine buffer (pH 3.0) to stop further hydrolysis. Thereafter, the absorbance at 405 nm was determined against an assay solution blank. For studies at pH values above 10.5, corrections had to be made for base hydrolysis of substrate.

The stability of the enzymes at various pH values was deter- mined by incubating 10 ~1 of enzyme solution in 350 ~1 of 10 m&f buffer at different pH values. The buffers used were: glycine (pH values of 2, 3, 9, 10, 11, and 12); sodium acetate (pH values of 4 and 5) ; imidazole-HCI (pH values of 6 and 7) ; and Tris-HCl (pH 8). No significant changes in pH were noted after incuba- t’ion in each buffer. Aliquots were removed after 1 hour and

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assayed against Leu-NHNp in the usual Tris-HCI-CaCls (pH 8.0) solution. The temperature stability of the aminopeptidase was determined by adding 10 ~1 of enzyme solution to 0.5 ml of Tris-HCl (pH 8.5) with either 5 mM CaC& or 10 mM EDTA. The solutions were heated at the desired temperature for 15 min; thereafter, the solutions were plunged into ice and after 10 min assayed against Leu-NHNp in the usual manner except that the solution contained 0.1 M CaC&.

The stability of the EDTA-treated enzyme was determined by incubating enzyme solutions at 4” in 10 mM Tris-HCl (pH 8) with 10 mM EDTA. Aliquots were removed and assayed against Leu-NHNp in 10 mM Tris-HCl (pH 8) with and without 0.1 111 CaC& to determine both the residual activity and the restorable activity, respectively.

The cation specificity for activity of the aminopeptidases was determined by incubating each enzyme in 10 mM Tris-HCl (pH 8.0) with 25 mM EDTA. After incubation for 1 hour at 4”, 30 ~1 of this solution were added at 4” to 470 ~1 of 10 mM Tris-HCI (pH 8.0) containing 10 mM metal chloride. Immediately there- after, lo-~1 aliquots were removed to be assayed against Leu- NHNp in the usual manner except that the same metal chloride used for incubation was substituted at 10 mM concentration for the CaC12. All results have been expressed as percentages of recovery of activity compared with those observed of the calcium- treated enzymes.

Other Studies-The apparent molecular weights of the amino- peptidases were determined by descending gel filtration (14) through a Sephadex G-75 column (1.1 x 198 cm) equilibrated in 10 mM Tris-HCl (pH 8.0) containing 5 mM CaC12. The column was calibrated by use of ovalbumin, ribonuclease, sperm whale myoglobin, human carbonic anhydrase C, and soy bean trypsin inhibit,or.

The homogeneity of the aminopeptidases was analyzed by acrylamide gel electrophoresis at pH 9.5 in a Canalco model 12 apparatus. The buffer composition was that designated by the Disc Electrophoresis bulletin of the Canal Industrial Corp., New York. The amount of protein applied was determined accord- ing to the method of Lowry et al. (15). Electrophoresis r\-as done at room temperature at 3 ma per tube for 3 hours. Gels were st,ained with Coornassie blue.

RESULTS

Fig. 1 depicts the chromatography of crude Pronase through CPI/I-cellulose at pH 8. The pattern of eluted proteins and ac- tivities is substantially different from those noted with earlier Pronase runs when carried out below pH 6 (6, 16-18). As can be seen, the act’ivities against Leu-NHNp and I(z-Arg-NHNp a.re completely separated. In contrast, the activities against casein and Ac-Tyr-OEt are present in all of the chromatographic components.

Fig. 2 demonstrates the elution pattern through hesanedi- amine-agarose of protein material obtained from fractions associated with Peak B in Fig. 1. There is complete separation of the activities against Ac-Tyr-OEt and Bz-Arg-NHNp. Pronase trypsin binds so tightly to the agarose derivative Ohat it is not eluted at pH 8 even with 1 M NaCl. In contrast, the change to the pH 3 buffer results in the quantitative release of the enzyme. The effect upon elution with acidification is not un- expected since binding undoubtedly is due in part to a charge pair between an amino group on the agarose and the carboxgl group of an aspartyl residue homologous with Asp-177 of bovine trypsin (8). In the latter enzyme, it has been demonstrated that the P-carboxyl group of Asp-177 binds to the positively

Fraction

FIG. 1. Chromatography of Pronase through CM-52 at pH 8. The vertical arrow indicates t)he change to elation with the addition of 0.3 M NaCl to the initial buffer. The protein elution nrofile was determined by absorbance at 280 nm&(O-a), an; activity against casein with lo-,~l aliquots is demonstrated (A,,,,, O---O). The activities against Ac-Tyr-OEt (a-- --a) expressed for 50-J aliquots; Bz-Arg-NHNp (A.-----A) expressed for 50.~1 ali- quots; and Leu-NHNp (0- -0) expressed for 2.~1 aliquots are indicated. Fractions of 6 ml were collected. See text for details.

I PH 8 I PH 3 I

::

10.0 - ::

. ; i . : : : : : :

8.0 - : : : : : : : :

. : : : :

- 1.2

a - I 1.0 ;

I I a - 0.8 :

.E

: - 6 0.6 -:

5 10 15 20 25

Fraction

FIG. 2. Chromatography through hexanediamine-agarose of protein components associated with Peak B of Ficr. 1. The vertical arrow indicates the change to elution with the p?I 3 buffer. The activities against Ac-Tyr-OEt (A- - -A) expressed for lo- ~1 aliquots and against, Rz-Arg-NHNp (A-----A) expressed for 5-J aliquots are indicated. Fractions of 6 ml were collected. See text for details.

charged side groups of a lysine or arginine residue in substrate molecules (19). Lowering of the pH should protonate this

homologous carboxyl group in Pronase trypsin and thus dis- charge the ion pair. Unfolding of the protein at pH 3 may also have contributed to the elution. Further purification of the trypsin component by chromatography through CM-cellulose or filtration through Sephadex G-75 gave no evidence of hetero- geneity. In contrast to these results with Xtreptomyces griseus

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trypsin, bovine trypsin did not bind to this column. Cadaverine- succinyl-hexanediamine-agarose and cadaverine-succinyl-cadav- erine-succinyl-hexanediamine-agarose were prepared in order to extend the free alkylamino group from the agarose. Each of these latter derivatives bound the microbial trypsiu as efficiently as hexanediamine-agarose. However, neither of the longer derivatives of agarose demonstrat.ed any affinity for the bovine enzyme.

The elution pattern through hesanediamine-agarose of pro- tein components obtained from fractions associated with Peak A of the CM-cellulose column (Fig. 1) resulted in the selective retention of all of the activity against Leu-NHNp (Fig. 3). As demonstrated, a large peak containing all of the activity against Ac-Tyr-OEt and most of the activity against casein elutes in the void volume. A second protein component is eluted following the addition of 30 mM NaCl to the buffer system. A long trail of protein material is eluted which demonstrates very low activity against Leu-NHNp. When the eluting buffer is changed so that CaC12 replaces the NaCI, a large amount of protein with high activity against Leu-NHNp is eluted. ‘This elution occurs despite the fact that the change in the eluting buffer is associated with a lowering of the ionic strength. A trail of protein with high activity against Leu-NHNp follows the peak. An increase in concentration of the CaClz to 20 mM results in the elution of a second peak with high activity against Leu-NHNp. No activity against casein or Ac-Tyr-OEt is demonstrated in these last two peaks. The column was washed further by the initial buffer containing also 1 M NaCl; however, no other protein components were eluted. Protein material obtained from fractions associ- ated with the peaks demonstrating activity against Leu-NHNp were analyzed by acrylamide gel electrophoresis (Fig. 4). The two different chromatographic peaks are associated with separate proteins possessing different electrophoretic mobilities. Al- though some smearing of stained material is apparent in each

tube, a single separate protein band is seen for each peak. We have tentatively designated the protein with activity against Leu-NHNp eluting earlier from the hesanediamine-agarose column as aminopeptidase 1 and the component eluting later as aminopeptidase 2.

Fig. 5 demonstrates the pH dependency of the activity against Leu-NHNp of the two proteins. The pH range of maximum activity lies between 7.5 and 10. The pH dependency of the stability of the two proteins is demonstrated in Fig. 6. Each protein demonstrates a sharp decrease in activity below pH 6 and above pH 11. The variations noted for each point may be partially attributed to the varying effects of different buffers during incubation. The pH ranges associated with the changes in stability are clearly different from the pI-I ranges associated with the changes in activity.

The effect of EDTA upon the activity of each enzyme is demon- strated in Fig. 7. Each enzyme rapidly becomes inactivated. However, the addition of calcium ion not only restores the ac- tivity but also, during the 1st day of incubation, yields activities which arc higher than those initially present. Not shown in Fig. 7 was the observation that 100% of the initial activity could be recovered even after 2 weeks of incubation in EDTA. The effects of different cations on the activity of the EDTA-treated aminopeptidases is shown in Table I. Calcium and strontium ions reactivate the enzyme significantly. Cobalt, magnesium, and manganese ions partially restore activity, whereas the rest of the metals provide little if any activity to the enzyme.

2.4

1.8

0 z Q 1.2

0.6

0 10 20 -- 60 70 80 90

Frociion

FIG. 3. Chromatography through hexanediamine-agarose of protein components associated with Peak A of Fig. 1. The vertical arrows demonstrate the limits of fractions which were eluted by the addition of the indicated concentrations of each salt to the initial buffer. The protein elution profile was determined by absorbance at 280 nm (0-0 ), and activity against casein with 25-J aliquots is demonstrated (ASSO, O-O). The ac- tivities against Ac-Tyr-OEt (n-----n) expressed for 5-~1 aliquots and Leu-NHNp (0- -0) expressed for 40-~1 aliquots are indi- cated. Fractions of 6 ml were collected. See text for details.

FIG. 4. Acrylamide gel electrophoresis of Pronase aminopep- tidases. Left, aminopeptidase 2, 20 pg; cellter, aminopeptidase 1, 20 pg; right, mixture of aminopeptidases 1 and 2, 10 pg each. Migration from top to bottom, cat.hode at the top; 15% acrylamide gels were used.

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fl 0 6 8 10 12

PH

FIG. 5 (2&). The effect of pII on the activil)y of xminopep- tidascs 1 (0) and 2 (0) against Leu-NIINp. See t,ext for details.

FIG. G (right). The effect of pII on the stability of aminopep- tidases 1 (0) and 2 (0) after incubation in buffers at the indicated pII. The activities have been expressed as percentages of those act,ivit,ies not,ed after incubation at pII 8. See t’ext, for details.

HOLJE

FIG. 7. The effect of IWTA at pII on the activity against I~!rr- NIINp of aminopeptidases 1 (circles) and 2 (Iriangles). Follow- ing incubation at the indicated times in JGDTA, activit.ies were determined in the presence (solid symbols, --) and nhscnce (open symbols, - -) of C&12. See text for dctnils.

The effect of tcmpernture on the native and EIXR-t.rcatcd enzymes reveals that the metal-enzyme complex is stable to 70” whcrcas the metal-free cnzymc dcnaturcs irrcvcrsibly above 30” (Fig. 8). These results arc similar to those noted with the two smallest scrine endopeptidascs where treatment with EIX’A substantially reduced the temperature stability (1).

The results of gel filtration analysts of aminolqtidascs 1 and 2 gave approximate molecular n-eights of 23,000 and 25,000, respectively (Fig. 9).

The facilitated purification to homogeneity of the trypsin and the two aminopeptidascs contained in I’ronasc rcndcrs thcsc three enzymes readily available to all interested investigators. Earlier reports have described only the very preliminary purifica- tion of the latter two proteins (6, 20, 21). Hexancdiamine- agarose, which is the key to the present successful procedures, can be prepared without any difficulty. In our preliminary studies these purifications were carried out in one run through the hexanediamine-agarose column. However, in order to

6033

TAISLE I Reactivation of EDYA-treated aminopeptidases by metal ions

Cation

Caz+ &.2’

C02+

Mg2+ Mn2+ Ba2+ Zn2+ Cd*+ Ni2+ CP None

-/-

I

Aminopcptidase 1 Aminopeptidase 2

% YO 100 100 61 56 21 17 18 18.5 12 9 8 5.5 5 4 4.5 3

<l <I <1 <l <I <I

20 40 60 80 100

Temperature “C

FIG. 8. Temperature dependence of the stability of amino- pcptidascs 1 (circles) and 2 (/ka?zgZes) in the presence of CaClz (solid symbols, ---) or 1~1lWA (open symbols, - -). See text for details.

1”

18

1.7 -

\

Myoglob,n

1.6 -

-

soy Beon Trypi,,, Inh,t,to~

Ovolbum,n \

1 2 3 4 56

Molecular Weight x 1O-4

FIG. 0. Molecular weight analysis of Pronase aminopeptidases 1 (AR1) and 2 (AP-2) by gel filtration. V, represents the elution volume for each protein and I/o represents the void volume of the Sephadex G-75 column as dct,crmined with blue dextran.

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minimize the time of binding of these proteins and also to permit the later resolution of the other serine endopeptidases, the purifi- cations were divided into the two separate procedures as de- scribed. The studies of the pH dependence of stability of the aminopeptidases readily explain the past failures of attempts at complete purification in the presence of acidic buffers.

The separation of the trypsin component may well be based upon the principle of affinity chromatography; the alkylamino groups resemble very much the lysine side chains which are part of specific substrate residues, and therefore the tight binding of this protein to the agarose derivative was to be expected. It is difficult to understand why bovine trypsin does not bind in a manner similar to the microbial enzyme. These two enzymes have marked homology of primary structure and of activity suggesting very similar tertiary structures (8, 9). The failure to bind to the agarose derivatives with longer alkylamine arms suggests strongly that steric hindrance is probably not the feature preventing binding of the bovine enzyme.

In contrast to the microbial trypsin, the basis for the selective binding of the aminopeptidase components is not immediately apparent. It would be attractive to postulate that the alkyl- amino groups, which are attached to the agarose, sufficiently resemble the amino ends of polypeptide chains leading to the selective binding of these enzymes. As noted, the affinity of these proteins for hexanediamine-agarose is much less than that of the trypsin component. At the same time that a preliminary report of the present work appeared (2) a more extensive de- scription was published of the chromatographic separation of proteins by use of alkylamine derivatives of agarose (22). The conclusions of this latter report were that these agarose deriva- tives provided a form of resolution based on hydrophobic inter- actions; the authors pointed out also that elution of certain macromolecules could be dependent upon specific ion effects. The elution of the aminopeptidases by calcium ions when sub- stituted for higher sodium ion concentrations may be an example of such an effect. The more favorable binding of the metal-free aminopeptidases may reflect some conformational relaxations permitting stronger hydrophobic interactions. The elution with calcium ion, which stabilizes these proteins, may reflect a shift in equilibrium from tight binding and relaxed conformations to poorly binding, but tight conformations.

The structure and function of known aminopeptidases have been reviewed recently (23) and will not be considered in detail here. The calcium ion requirement of the Pronase proteins is unusual. Only in the related microorganism Streptomyces fradiae has an aminopcptidase been described with calcium as the preferred metal (24). In all other cases, zinc (25, 26), cobalt (27), magnesium (23, 25), and manganese (23, 25) have been found to be the required metals for this family of enzymes. An earlier report on partially purified Pronase aminopeptidase described cobaltous ions as providing activity similar to that noted with calcium (28). The present report reveals that cobaltous ion restores only about 20% of the activity to the purified metal-free enzymes as compared with calcium. Calcium was described initially as the metal required for Pronase stability (29) ; we recently described the requirement of calcium for stability of two Pronase endopeptidases (1).

These aminopeptidases appear to be the smallest such enzymes yet described. Other defined aminopeptidases, with few excep- tions, are much larger and are characterized by subunit struc- ture (23, 25, 27). The possibility that the present enzymes are related to a subunit of the larger enzymes is of some interest.

To date, the only differences that can be seen between the two

aminopeptidases are the volumes of elution from hexanediamine- agarose, their molecular weights, and their electrophoretic mobilities. Otherwise, their heat stabilities, pH ranges for activity and stability, and also their metal requirements appear to be identical. The possibility exists that there is only one gene product present but that heterogeneity is present because of limited proteolysis. The large number of potent endopeptidases in Pronase renders such an explanation not unlikely. How- ever, it must be pointed out that we have carried out these puri- fication procedures several times with the use of different Pro- nase lots; the ratio of the two aminopeptidases in the different runs varies only slightly. With the present purification scheme, six proteases from Pronase have now been purified to homogeneity in this laboratory. Each can be prepared rapidly by readily available techniques (3, 5).

Acknowledgment-We wish to express our appreciation for the encouragement and support that Mr. B. 13. Sigelbaum has ex- tended to the efforts of this laboratory.

1.

2.

3.

4.

5.

G. 7.

8.

9.

10. Il. 12. 13.

14. 15.

IG.

17. 18. 19.

20.

21. 22.

23.

24.

25.

26.

27.

28.

29.

REFERENCES SIEGEL, S. & Awan, W. M., JR. (1973) J. Biol. Ch,em. 248,

32333240 V~~BE~K, K., CHO\~, K.-F. & A\~.AD, W. M., JR. (1973) Feel.

Proc. 32, 504 A~AD, W. M., Jx., SOTO, A. R., SII~X;L, s., &IIBA, W. E.,

BKILNSTROM, G. G. & OCHOA, h/I. S. (1972) J. Biol. Chem. 247, 41444154

SIEGEL, S., BRADY, A. H. & A\T-~I~, W. M., JR. (1972) J. Biol. Chem. 24’7, 4155-4159

Aw‘lu, W. M., JR., OCHOA, M. S. & TOOMEY, T. P. (1972) Proc. #al. Acad. Sci. U. S. A. 69. 2561-2565

NARAHASHI, Y. (1970) Methods Enz&oZ. 19, 651-GG4 JUR&K, L., F~CXRX, D. 8: SMII,LIX, L. B. (1969) Biochem.

Biophys. ILes. Commun. 37, 99-105 OLSON, M. 0. J., NAGABHUSHAN, N., DZWNIEL, M., SMILLIE,

L. B. & WHIT.UCEIZ, D. 1:. (1970) Ayat?cre 228, 438-442 ATTAD, W. M., JR. & WILCOX, P. >:. (1!163) B&him. Biophys.

Actu 73, 285-292 PFLEIDF:RER, G. (1970) Methods Enzymol. 19, 514-521 CUATI~ECASAS, P. (1970) J. Biol. Chem. 246, 3059-3085 AXON, It. 8: ~XRKBXK, S. (1971) Eur. J. Biochem. 18, 351-36’0 INMAN, J. K. R: DINTZIG, H. M. (1969) Uiochemislr~~ 8, 4074-

4082 ANDKINS, P. (1965) Biochem. J. 96, 595-GO6 LOWRY, 0. H., I~OSICHROUGH, N. J., FARR, A. 1,. & ~:AxDBLL,

It. J. (1951) J. Biol. Chem. 193. 205-275 Nonxo&, M., NaltsH.4~~1, Y., ‘OUCHI, T. & HIRAMATSU, A.

(1964) 1%~. 1nt. Congr. Biochem., Abstrucls, p. 325 W~HI,HY, S. (1968) Hiochim. Bioph,us. Acfa 161, 394-401 TROP, M. & BIKK, Y. (1968) Biochem. J. 109,475-476 STlLOUl), 11. M., K.~Y, L. M. & DICI~JXSO~V, It. 15. (1971) Colcl

Spring Harbor Synp. Qz~ant. Biol. 36, 125-140 JUR&:IC, L., JOHNSOX, P., OLAFSON, 11. W. RE S~IILLIE, L. B.

(1971) Can. J. Biochem. 49, 1195-1201 Tnor, M. B BIRI~, Y. (1970) B&hem. J. 116, 19-25 SHALTIEL, S. & .ER-EL, Z. (1973) Proc. ~\‘cct. Acad. ,%i. u. 8. A.

70, 778-781 I~ICLANGE, It. J. & SMITH, E. L. (1971) in The Enzymes (BOYER,

P. I)., ed) 3rd Ed, Vol. 111, pp. 81-118, Academic Press, New York

MORIHARA, K., OKA, T. & TSUZUIZ, H. (1967) Biochim. Bio- phys. Acta 139, 382-397

CAIWENTER, F. H. & VAHL, J. M. (1973) J. Biol. Chem. 248, 294-304

HJMMWHOCH, S. R. (1969) Arch. Xochem. Biophys. 134, 597- 602

STOLL, E, HERMODSON, M., ERICSSOX, 1,. 11. & ZUBER, H. (1972) Biochemistry 11, 4731 4735

NARAH.\SHI, Y. & YAXAGITI, M. (1967) J. Uiochem. (Tokyo) 62, G33-641

NOMOTO, M., NARAHASHI, Y. & ~VUI~.~KAMI, M. (1960) J. Biochem. (Tokyo) 48,453-463

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Klaus D. Vosbeck, Kai-Fu Chow and William M. Awad, Jr.CHARACTERIZATION OF THE AMINOPEPTIDASES

Commercial Preparation (Pronase) : PURIFICATION AND Obtained from aStreptomyces griseusThe Proteolytic Enzymes of the K-1 Strain of

1973, 248:6029-6034.J. Biol. Chem. 

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