Interconversion of a-and y-Penicillinase from Bacillus cereus 569*

Extracellular penicilhnase from Bacillus cereus 569 was found to exist in two states, an iodine-sensitive state and an iodine-insensitive state. Iodine-sensitive penicillinase is inactivated by 2.5 X 10-a M iodine within 1 min, while the iodine-insensitive enzyme is inactivated at a much slower rate.


Interconversion
of a-and y-Penicillinase from Bacillus cereus 569* ( Extracellular penicilhnase from Bacillus cereus 569 was found to exist in two states, an iodine-sensitive state and an iodine-insensitive state. Iodine-sensitive penicillinase is inactivated by 2.5 X 10-a M iodine within 1 min, while the iodine-insensitive enzyme is inactivated at a much slower rate. Approximately 20% of the extracellular penicillinase activity found in crude or partially ptied preparations of penicillmase is iodine-sensitive. The two states of penicillinase were shown to be interconvertible. In the presence of saturated ammonium sulfate, penicillinase is converted quantitatively into the iodine-sensitive state, and, upon removal of the ammonium sulfate, it spontaneously reverts to the iodine-insensitive state.
It is concluded that o(-and y-penicillinase correspond to the iodine-insensitive and iodine-sensitive penicillinases, respectively, and that the two penicillinases are dBerent states of the same enzyme in dynamic equilibrium. The relative proportion of the two states depends upon the conditions imposed upon the enzyme preparation. Bacillus cereuS 569 is reported to produce two types of penicillinase: (Y, which represents approximately 95% of the penicillinase produced by the cell and is defined as totally extracellular and insensitive to iodine; and y, which represents approximately 5% of the total penicillinase produced and is defined as cell-bound, sensitive to iodine, and not neutralized by antiserum prepared against extracellular penicillinase (1, 2). A third form, 0, was also described; however, @ is neutralized by antiserum prepared against extracellular penicillmase and appears to be a small fraction of the ar-penicillinase that is superficially adsorbed onto the cell. Partial purification of y-penicillinase by ammonium sulfate precipitation was reported by Pollock (1). He compared various properties of L\I-and y-penicillinase and found * This investigation was supported in part by National Institutes of Health Grants ?ID 02168 and HD 04831 and is Journal Paper J-6480 of the Iowa Agriculture and Home Economics Experiment Station, Ames.
$ Predoctoral Fellow of the National Institutes of Health. 0 To whom inquiries should be directed, at the Department, of Genetics, Iowa State University, Ames, Iowa 50010. that, although the two enzymes differed in the properties indi,. cated above, their substrate specificities, Michaelis constantsand sedimentation rates were very similar. Studies by Citri (2) showed that when cr-penicillinase is treated with base or adsorbed onto glass it acquires many of the characteristics of y-penicillinase (i.e. it becomes iodine-sensitive and cannot be neutralized by antiserum prepared against extracellular penicillinase).
Therefore, we undertook studies to determine whether (Y-and y-penicillinase are the same protein or twos similar proteins.
Data presented in this communication show that the iodinesensitive and the iodine-insensitive penicillinase are readily interconvertible.
It is concluded that cy-and y-penicillinase correspond to the iodine-insensitive and the iodine-sensitive penicillinases, respectively, and that the two penicillinases are different forms of the same protein.

MATERIALS AND METHODS
Organism-B. cereuS 569 was obtained from Dr. Martin

Pollock.
A variant strain of B. cereus 569, which is used in the commercial production of penicillinase, was obtained from Riker Laboratories, Northridge, California, and is referred to here as. the Riker strain.
The doubling time and amino acid requirements of the two strains are the same; however, they produce a slightly different penicillinase as shown by disc gel electrophoresis. ' Preparation of Penicill&ase-For these studies both a commercial preparation of penicillinase and enzyme partially purified. in our laboratory were used. The commercial preparation (Neutrapen) was obtained from Riker Laboratories, Northridge, California, and taken up in distilled water and used directly (3). Partial purification of penicillinase was accomplished as follows: Two l-liter cultures of B. cereus 569 were grown overnight in media which contained per liter: trisodium citrate 2Hz0, 5.9 g; (NH&Sod, 2 g; K%HPOJ, 3 g; MgS04.7H20, 0.52 g; casamino acids, 10 g; glucose, 2 g. These cultures, in log phase, were used to inoculate 13.5 liters of previously warmed medium in a carboy.
The culture was aerated by passage of air and in early log phase penicillinase synthesis was induced with 2 units per ml of benzylpenicillin.
When exponential growth ceased the cells were harvested with a Szent-Gyorgyi and Blum continuous flow apparatus.
To the spent medium was added 2 volumes of acetone and, after stirring, the solution was allowed to stand overnight at 4". The liquid was decanted and the white precipitate was taken up in as little water as possible (usually 500 ml for a la-liter culture). To remove any acetone that was carried over the solution was placed in an aspirator flask and maintained under reduced pressure. The crude preparation was flash-evaporated to about 70 ml. After dialysis for 3 hours against 2 volumes of 2 X lo-* M EDTA, pH 7.0, the preparation was centrifuged and passed over a phosphocellulose column.  3)). All steps were carried out in the cold. The specific activity of crystalline penicillinase is approximately 2 X lo6 units per mg of protein (4). Enzyme Assay-Enzyme preparations were diluted with 0.1 M phosphate buffer, pH 7.0, to a concentration of 10 to 100 units of enzyme per ml. At zero time g-ml fractions of the diluted enzyme preparations were incubated at 30". At 60 see, 1 ml of iodine solution (2.5 X 10m2 M iodine and 1.25 M KI) was added to the enzyme solution (2). Enzyme samples were removed at 1-min intervals for 6 min and penicillinase activity was assayed (5). A control was always run simultaneously in which water instead of iodine was added to the previously incubated enzyme preparation.
A unit of penicillinase activity is defined as the amount of enzyme required to hydrolyze 1 pmole of penicillin in 0.05 M phosphate (pH 7.0) per hour at 30" (4).
Preparation of Washed Cells-Cultures of B. cereus 569 or the Riker strain were grown to log phase, induced with 2 units per ml of penicillin, and harvested 1 hour later while still in log phase. Cell growth was stopped by adding 1.6 x 10-* M hydroxyquinoline. An aliquot was kept on ice and the remainder of the culture was centrifuged. The supernatant was kept on ice and the cells were washed with cold 0.3 M phosphate buffer, pH 7.0. The 0.3 M phosphate wash was kept on ice and the cells were resuspended in 0.1 volume of 0.3 M phosphate. Whole culture, spent medium, wash, and washed cells were assayed for both iodine-sensitive and iodine-insensitive penicillinase activity. Treatment of Penicillinase with Ammonium Sulfate-Partially purified extracellular penicillinase was diluted 1: 1000 with 0.1 M cold phosphate, pH 7.0, or with cold 0.1 M phosphate saturated with ammonium sulfate. These diluted enzyme preparations were kept on ice for 2 hours after which an aliquot of each was assayed for both iodine-insensitive and iodine-sensitive penicillinase. The remainder of the enzyme preparation was dialyzed against two changes of 200 volumes of the 2 X 10-* M EDTA, pH 7.0. After 3 hours of dialysis the volumes of the dialyzed enzyme samples were measured to correct for any volume increase during dialysis. Each sample was again assayed for both iodine-insensitive and iodine-sensitive penicillinase.

Iodine-sensitive
Extracellular Petillinase-Under normal growth conditions approximately 90% of the penicillinase activity produced by B. cerew 569 is secreted by the cells into the culture medium. In an attempt to determine whether the extracellular penicillinase is composed only of Lu-penicillinase activity (i.e. is insensitive to 2.5 X lOTa M 1~) or whether it also contains y-penicillinase activity (i.e. is partially sensitive to 2.5 x 10ea M Iz), the penicillinase activity present in spent medium of either B. ce-reus 569 or the variant "Riker strain" of B. cereus 569 was assayed in the absence or presence of 2.5 x 10sa M iodine. As may be seen in Fig. 1, A and B, approximately 50% of the extracellular penicillinase activity secreted by either strain is inactivated immediately by 2.5 X 10ea M iodine. Partially purified preparations of extracellular penicillmase that were obtained by two different purification procedures were also assayed for iodine-sensitive penicillmase activity. Approximately 20% of the penicillinase activity in either the commercial preparations of extracellular penicillinase ( Fig. 2A) or in the partially purified preparations prepared in our laboratory (Fig.  2B) was found to be inactivated immediately by 2.5 x 1OW M  the various components of a log phase culture of B. cereus 569. Data presented in Table I show that approximately 18% of the penicillinase activity associated with a "whole" log phase culture is iodine-sensitive while approximately 46% of the penicillinase activity associated with the various components of the culture is iodine-sensitive.
These data also show that while 90% of the total penicillinase activity was recovered only 60% of the iodineinsensitive penicillinase activity was recovered. On the other hand, 225% of the iodine-sensitive penicillinase activity was recovered.
These data suggest that the iodine-insensitive penicillinase activity can be converted into an iodine-sensitive penicillinase activity.
Conversion of Iodine-insensitive Penicillinase to Iodine-sensitive Penicillinase-Since iodine-sensitive penicillinase was found to be a normal component of extracellular penicillinase and since the relative amount of iodine-sensitive penicillinase appeared to increase when the cells were collected and washed, an attempt was made to show directly the conversion of iodine-insensitive penicillinase to an iodine-sensitive penicillinase.
It should be noted that the first iodine-sensitive penicillinase to be characterized had been obtained by precipitation with 90% saturated ammonium sulfate (1). Therefore, it was decided to examine the effect of saturated ammonium sulfate on extracellular penicillinase. Enzyme preparations were treated as described under "Materials and Methods." As shown in Fig. 3, preliminary incubation of penicillinase in saturated ammonium sulfate causes some inac-  tivation of the enzyme; however, more striking and more interesting is the observation that the ammonium sulfate-treated enzyme preparation contains much more iodine-sensitive penicillinase activity than does the control sample that was treated identica.lly except for the omission of ammonium sulfate.
In fact, the amount of iodine-sensitive penicillinase activity in the ammonium sulfate-treated preparation increased by 62 units while the amount of iodine-insensitive penicilliiase activity in the same preparation had decreased by approximately 100 units (see Fig. 3 and Table II).
Similar results were obtained with commercially prepared penicillinase (Table II).
Thus a direct conversion of iodine-insensitive penicillinase into iodine-sensitive penicillinase could be shown.
Conversion of Iodine-sensitive Penicillinase to Iodine-insensitive Penicillinuse-Since iodine-insensitive penicillinase activity can be converted into an iodine-sensitive penicillinase activity, an attempt was made to determine whether or not the conversion is reversible.
Data presented in Fig. 3 and Table II show that the newly formed iodine-sensitive penicillinase activity is spontaneously converted back into an iodine-insensitive state when the ammonium sulfate is removed by dialysis.
Thus a reversible interconversion between the iodine-insensitive and the iodinesensitive penicillinase activities can be easily shown. DISCUSSION

Early investigations
(1, 2) on the properties and induction of penicillinase showed that there are two distinct types of penicillinase activity associated with B. cereus 569: a-penicillinase, which is insensitive to 2.5 X 10V3 M iodine, and y-penicilliiase, which is rapidly inactivated by 2.5 x lOWa M iodine. It was reported that all of the y-penicillinase activity remained associated with the bacterial cell while all of the extracellular penicillinase was iodine-insensitive (1). Other data suggested that cr-and y-penicillinase activities may or may not represent different conformations of the same protein molecule (2,6,7). Since penicillmase is known to undergo reversible denaturation readily (3)) experimentation on the distribution of y-penicillinase and on the possible interrelationships, if any, between cy-and y-penicillinase was undertaken.
Data presented in Fig. 1, A and B, show that approximately 50% of the crude extracellular penicillinase activity is iodinesensitive.
Partially purified preparations of extracellular penicillinase that ranged from 10 to 50% pure were also examined for iodine-sensitive penicillinase activity. As shown in Fig. 2, A and B, approximately 20% of the penicillinase activity found in these preparations is inhibited within 1 min by 2.5 X lop3 M iodine.
Thus, contrary to a previous report (I), both crude spent medium and partially purified preparations of extracellular penicillinase contain iodine-sensitive penicillinase activity. This finding is substantiated in part by a recent report (8). Analysis of the various components of exponential cultures showed, consistent with an earlier report (11, that more than 50% of the penicillinase activity associated with washed cells is iodinesensitive (Table I). These data suggest also that the iodineinsensitive activity can be converted to iodine-sensitive penicillinase activity.
This statement is based on the fact that the total number of units of iodine-sensitive penicillinase activity increased during the fractionation procedure while the number of units of iodine-insensitive activity decreased. In an attempt to show directly an interconversion between iodine-insensitive and iodine-sensitive penicillmase activities, partially purified preparations of penicillinase were diluted into saturated ammonium sulfate.
Data presented in Fig. 3 show that this treatment causes approximately 70% of the penicillinase activity to become iodine-sensitive.
These data also show that when the ammonium sulfate is removed by dialysis the newly formed iodine-sensitive penicillinase activity spontaneously reverts to the iodine-insensitive form. These results, summarized in Table II, also indicate that the specific activity of the iodine-sensitive and iodine-insensitive forms of penicillinase activity are approximately equal. Hence, the ammonium sulfate treatment appears to produce a quantitative interconversion between the two forms of penicillinase activity. From these data it is concluded that a-and y-penicillinase correspond to the iodine-insensitive and iodine-sensitive states, respectively, and that the two penicillinases are different conformational states of the same enzyme in dynamic equilibrium. This conclusion is supported by the observation that penicillinase can readily undergo reversible denaturation (3) and that attempts to separate the iodine-insensitive and the iodine-sensitive forms of penicillinase by acrylamide gel electrophoresis and phosphocellulose chromatography were unsuccessful. 2 It is interesting to note that the y-penicillinase activit,y associated with the bacterial cell is reported to be attached largely to the cell membrane (9). Since approximately 90% of the penicillinase activity is secreted by the cell (Table I), it is possible that the "membrane-bound" y, which represents approximately 5% of the total penicillinase activity, is newly synthesized penicillinase that is undergoing secretion from the cell by some, as yet, unknown process. If this prediction is correct it implies that the secretion process requires approximately 5% of a cell doubling time, or about 2 min. Although it has been reported that the 'Lmembrane-bound" y does not serve as a direct precursor to extracellular penicillinase, the data from which that claim was derived were obtained with nonexponentially growing cells and hence do not permit the cited conclusion (10).