JOURNAL OF BACTERIOLOGY, Mar. 1975, p. 1102-1110 Copyright 0 1975 American Society for Microbiology

Vol. 121, No. 3 Printed in U.S.A.

Solubilization of Escherichia coli Nitrate Reductase by Membrane-Bound Protease

a

C. H. MACGREGOR Department of Microbiology, University of Virginia Medical School, Charlottesville, Virginia 22901 Received for publication 25 November 1974

Nitrate reductase extracted from the membrane of Escherichia coli by alkaline heat treatment was purified to homogeneity and used to prepare specific antibody. Nitrate reductase, precipitated by this antibody from Triton extracts of the membrane, contained a third subunit not present in the purified enzyme used to prepare the antibody. Nitrate reductase precipitated by antibody from alkaline heat extracts was composed of peptide fragments of various sizes. These fragments were produced by a membrane-bound protease which was activated by alkaline pH and heat. It is the action of this protease that releases the enzyme from the membrane, as shown by the observations that protease inhibitors decreased the amount of solubilization of the enzyme, and the enzyme remaining in the membrane after heating showed much less proteolytic cleavage than that which was released. The group of proteins referred to as mem- characterized in regard to shape, size, subunit brane bound includes a wide variety of proteins composition, and metal content (9). Such a well with various means of attachment to or inser- studied molecule should provide a good model tion into a membrane. At one end of the for investigating the question of the means of spectrum are proteins like cytochrome c, which membrane attachment. The means of attachare attached primarily by electrostatic linkages ment of this enzyme, in turn, could provide and can be removed from the membrane quite information concerning its operation during easily by washing with high salt concentrations electron transport. To study this problem, anti(3). A different means of attachment is used for body has been prepared to the purified enzyme Streptococcus adenosine 5'-triphosphatase. and the technique of immune precipitation has This system uses a separate protein, nectin, to been used to elucidate the differences between anchor it to the membrane (1). Cytochrome b5 hydrophobic (Triton solubilized) and hydrohas a region of hydrophobic amino acids which philic (heat solubilized) forms of the enzyme. is inserted into the membrane (18, 19). Finally, MATERIALS AND METHODS at the other end of the spectrum are some extremely hydrophobic proteins, such as polyiGrowth medium. E. coli strain RK7 (9) was grown soprenol phosphokinase, which are tightly on the basal medium of Lester and DeMoss (4) bound to lipid and appear to be an integral part supplemented with 1 MM Na2MoO4, 1 MM NaSeOs, 7.4 MM thiamine, and 0.5% glucose. The medium was of the membrane themselves (13). made anaerobic by continuous bubbling with a mixNitrate reductase from Escherichia coli is a ture nitrogen and 5% carbon dioxide. To label membrane-bound enzyme. It is induced by cells,of0.495% MCi of [3 H ]leucine or 0.04 gCi of ["4C ]leucine growth in the presence of nitrate and functions was added per ml of the final medium and 20 ,ug of as a terminal electron acceptor during anaero- cold leucine per ml was added as carrier. In experibic growth (17). This enzyme is tightly bound to ments involving labeling with 32P, N-2-hydroxyethylthe cytoplasmic membrane in the sense that it piperazine-N'-2-ethanesulfonic acid buffer was used cannot be removed by washing the membrane in place of phosphate buffer and 3.5 uCi of [32P ]orthounder various conditions. Two means of solubi- phosphate per ml was added to the medium, along lizing the enzyme have been demonstrated. One with 1 mM sodium phosphate as a carrier. Isolation of membrane. Cells were broken in a method involves the use of the nonionic detergent Triton X-100 (7). The other method, which French pressure cell in 50 mM phosphate buffer, pH A trace of deoxyribonuclease and ribonuclease was not previously understood, involves heating 7.2. and 10 mM MgCl2 were added, and whole cells were a dilute membrane preparation to 60 C under removed by centrifugation for 5 min at 3000 x g. The alkaline conditions (9, 20). This heat solubilized supernatant was centrifuged for 45 min at 200,000 x form of the enzyme has been purified and g. The pellet was resuspended in the same buffer and 1102

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PROTEOLYSIS OF NITRATE REDUCTASE

centrifuged again as above. This second pellet is referred to as the membrane fraction. Solubilization of the membrane. Triton solubilization was done by suspending the membrane fraction in 50 mM phosphate buffer, pH 7.2, containing 2% Triton X-100 and by incubating 15 min at 23 C. Insoluble material was removed by centrifugation at 200,000 x g for 45 min. This procedure has previously been shown to solubilize only the inner (cytoplasmic) membrane (15). For heat solubilization, the membrane fraction was suspended at a protein concentration of 0.4 mg/ml in 5 or 10 mM phosphate buffer, pH 8.3, and heated at 60 C for 8 to 10 min. Insoluble material was removed by centrifugation at 200,000 x g for 45 min. Preparation of antibody. Three milligrams of the pure enzyme was added to 1.5 ml of Freund adjuvant and injected subcutaneously at six sites on the rabbit's back. After 5 weeks, 3 mg of enzyme was injected intravenously. The rabbit was then bled on day 5, 6, and 7 after the last injection.

Precipitation of nitrate reductase with antibody. Triton- or heat-solubilized extracts were diluted to a concentration of approximately 1,000 mU of nitrate reductase/ml. Twenty microliters of anti-nitrate reductase or preimmune serum was added to 0.2 ml of the soluble extract. This was incubated at 37 C for one h. If immune co-precipitation was done, goat antirabbit gamma globulin was added in an amount sufficient to precipitate all the rabbit gamma globulin, and the mixture was again incubated at 37 C for 1 h. A final incubation was carried out at 4 C overnight. The precipitate was then removed by centrifugation and washed once in 0.15 M NaCl containing 1% Triton. The supernatants from immune precipitation were assayed for nitrate reductase and in all cases less than 1% remained. As noted below, antibody did not inhibit enzyme activity. Gel electrophoresis. The antibody precipitates were dissolved in a solution containing 3% sodium dodecyl sulfate (SDS), 7.5 mM ethylenediaminetetraacetate and 0.15% 2-mercaptoethanol in 0.1 M phosphate buffer, pH 7.2. The amount of solution was added such that there were at least 4 mg of SDS per mg of protein in the precipitate. Urea was added to a final concentration of 8 M and the sample was boiled for 5 min. Gels were prepared as previously described (14). If possible, 5,000 to 8,000 counts/min of 3H were applied to a gel and the gels were run for 7 h at 5 mA per gel. Gels were sliced and counted as previously described (14). Protease inhibitors. The inhibitors e-amino-ncaproic acid, p-aminobenzamidine hydrochloride (PAB), and N-a-p-tosyl-L-lysine chloromethylketone hydrochloride were prepared as concentrated solutions in water and 25 ul of each was added per ml of membrane suspension to be heated. L-1-Tosylamide2-phenyl-ethylchloromethyl ketone was dissolved in dimethyl formamide and 8 Ml of this was used per ml. Diphenylcarbamyl chloride (DPCC) was dissolved in isopropanol and 50 ul of this was used per ml. Controls showed that isopropanol alone did not inhibit heat solubilization of nitrate reductase but slightly enhanced its solubilization. Assays. Protein was measured by the method of

1 103

Lowry et al. (5). Nitrate reductase activity was measured colorimetrically using methyl viologen as the electron donor. This assay and the enzyme purification have been described elsewhere (10). One unit of activity represents the formation on 1 lmol of nitrite per min at 23 C. RESULTS Nitrate reductase can be solubilized from the cytoplasmic membrane of E. coli by heating a washed membrane preparation at 60 C at alkaline pH. This soluble form of the enzyme has

been purified and found to be a spherical molecule with a molecular weight of 774,000. It is composed of subunits with molecular weights of 142,000 and 58,000, and the complete enzyme is an octamer containing four of each of these subunits. This octamer also contains approximately 4 mol of molybdenum as well as nonheme iron (10). Protein profiles on SDS gels of different purified preparations of this enzyme showed a variability in size and amount of the smaller subunit. An example is shown in Fig. 1. Often more than one small subunit was found. The molar ratio of the large subunit (A) to the sum of the small (B) subunits was approximately 1:1. The variability in size and amount of the smaller subunit led to the hypothesis that proteolysis occurred during the preparation of the enzyme and the smaller subunit was partially degraded. It was assumed that these various partially degraded small subunits were functionally equivalent in the intact enzyme (10). To test this hypothesis, specific antibody was prepared to the heat-solubilized nitrate reductase which had been purified to homogeneity (10). The resulting antibody did not alter the activity of either crude or purified enzyme. However, it did prevent reconstitution of active enzyme in mixtures of extracts of chlorateA

JB FIG. 1. Protein profile on an SDS gel of nitrate reductase purified to homogeneity from heat solubilized membrane protein (10). The gel was stained with Coomassie blue and scanned (20).

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resistant mutants (8). This antibody was used to precipitate nitrate reductase proteins from crude 3H-labeled heat solubilized extracts and crude 3H-labeled Triton solubilized extracts of membrane fractions. These antibody precipitates were run on SDS gels which were subsequently sliced and counted. The resulting protein profiles from nitrate reductase are shown in Fig. 2. All the 3H counts precipitated from the Triton extract appeared in three peaks, whereas that precipitated from the heat extract appeared in numerous peaks, ranging in size from 142,000 to less than 10,000. No 3H counts precipitated when preimmune serum was added to either of the extracts. Figure 3 illustrates the immune co-precipitation of nitrate reductase protein from a Triton extract with rabbit anti-nitrate reductase and goat anti-rabbit gamma globulin. When rabbit anti-nitrate reductase was added to the Triton extract, followed by goat anti-rabbit gamma globulin the polypeptide peaks A, B, and C were obtained (Fig. 2). In the control where preimmune rabbit serum was added in place of rabbit anti-nitrate reductase, only a few minor peaks were observed and these did not correspond to

any of the nitrate reductase polypeptides. These minor peaks are observed only when goat serum is added, and hence must be due to protein which reacts with the goat serum. These minor peaks do not interfere so long as the sample contains a sufficient amount of nitrate reductase, but they do prevent the detection of very small amounts of nitrate reductase in samples. As Fig. 3 illustrates, there are three striking differences between the purified, heat-solubilized enzyme (Fig. 1) and the enzyme obtained from Triton extracts by immune precipitation: (i) subunit B from the Triton extract consisted of a single, sharp peak with a molecular weight of 60,000 rather than a variety of peaks ranging downward from that molecular weight. (ii) In the material precipitated from the Triton extract, the molar ratio of subunit A to subunit B is 1:1, within the limits of experimental error. This has been consistently observed with a number of preparations using both direct immune precipitation with rabbit antiserum and immune co-precipitation using the goat and rabbit antisera. (iii) The material precipitated from the Triton extract contained a third polypeptide (subunit C) which was totally absent from the purified, heat-extracted enzyme. Subunit C has a molecular weight of 19,500 and, as is shown in the next paper in this series (6), is

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FIG. 2. Comparison on SDS gels of heat solubilized (solid line) and Triton solubilized (dotted line) nitrate reductase protein. A 3H-labeled membrane preparation was divided in two parts. One part was solubilized at 23 C with Triton, the other part by heat extraction. Rabbit anti-nitrate reductase, followed by goat antirabbit gamma globulin were added to each of the soluble extracts. The resulting precipitates were solubilized in SDS solution and run on 7.5% gels. The amount of heat-solubilized protein applied to the gel was 2.5 times the amount of Triton solubilized protein. The top of the gel is on the left in this and all the following gel figures. A, B, and C identify the three peptide peaks present in the immune precipitate from the Triton extract.

0

20

40 60 SLICE NUMBER FIG. 3. Comparison on SDS gels of 31H-labeled material precipitated from Triton-solubilized membrane protein by rabbit anti-nitrate reductase (dotted line) and rabbit preimmune serum (solid line). Rabbit anti-nitrate reductase and goat anti-rabbit gamma globulin were added to one sample of 3H-labeled, Triton-solubilized membrane protein. To another sample, preimmune rabbit serum and the same goat serum were added. The resulting precipitates were solubilized with SDS and equal amounts of each precipitate were run on 7.5% gels.

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PROTEOLYSIS OF NITRATE REDUCTASE

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the apoprotein of cytochrome b,. The molar partially purified enzyme with enzyme precipiratio of subunit C to either subunit A or subunit tated from a Triton extract. It is evident that B is 2:1, within the limits of experimental error. there is much more low-molecular-weight mateIf the enzyme is actually degraded as it is rial in the partially purified, heat-extracted released from the membrane by heating at material than in the Triton solubilized matealkaline pH, one should be able to compare rial, although many of the small fragments seen antibody-precipitable proteins in partially puri- in the unpurified heat extract (Fig. 2) are gone. fied, heat solubilized preparations to antibody- Thus, our previous enzyme purification (9) precipitable proteins in crude heat extracts must have involved the removal of degraded and find fewer fragments in the partially puri- fragments of nitrate reductase protein as well as fied material. When a heat solubilized extract the removal of other protein contaminants. In was carried through only one of the three final the material precipitated by antibody from purification steps, a preparation of approxi- crude, heat-extracted preparations, the ratio of mately half maximal specific activity resulted. subunit A to subunit B was always greater than Nitrate reductase proteins were precipitated 1:1. Therefore, there appears to be a greater from this material with antibody and run on degradation of the B subunit than the A. To see SDS gels. Figure 4 shows a comparison of this if there was also a slight reduction in the molecular weight of the A subunit, the above preparations were compared on 5% SDS gels in which the migration of the A subunit is much A 4 A. 1 greater. No change in molecular weight was detected (Fig. 4). I10 To further substantiate the occurrence of B Bh. proteolysis during heat solubilization of nitrate -5 reductase, various protease inhibitors were added to samples of membrane before heating and the amount of enzyme solubilized after was measured. The conclusion drawn heating ~~~~~~~~B. z from many z Iexperiments with different protease 0 inhibitors is that heating the membrane in the o sO A -5 presence of the proper protease inhibitors re'4 4- sults in a decrease in both nitrate reductase solubilized as well as total protein solubilized. 3~~~~~~~~~Table 1 shows the inhibitors tested and their ability to prevent solubilization of nitrate reductase by heating. Note that nitrate reductase 40 O 20 60~~~~~C assays were performed before heating, in the presence of the inhibitor, and there is no inhibition of enzyme activity by any inhibitor. DPCC nitrate gel of FIG. 4. Comparison on an SDS reductase proteins precipitated from Triton solubi- was the most effective inhibitor of nitrate reduclized membrane (solid line) and partially purified, tase solubilization. Unfortunately, this comheat solubilized nitrate reductase (dotted line). Identi- pound is extremely insoluble in aqueous solucal cultures were labeled with [3Hlleucine or [14C]- tions and, even at 60 C, the majority of the leucine and the membrane fraction was isolated from DPCC added was not dissolved. Therefore, each. The 3H-labeled membrane was heated to 60 C further inhibitor experiments were carried out at pH 8.3, the insoluble material was removed, and with PAB. the soluble proteins were concentrated by adsorption PAB is a competitive inhibitor of trypsin (11). to diethylaminoethyl cellulose. The nitrate reductase activity was eluted with 0.3 M NaCl and this eluent Because of the nature of this inhibition, it was was centrifuged for 3 h at 200,000 x g. The super- necessary to use higher concentrations of this natant was discarded and the pellet, containing the compound than would be needed for the irreverenzyme activity, was suspended in phosphate buffer sible inhibitors. Table 2 shows that solubilizacontaining 0.5% Triton. The enzyme was precipitated tion of nitrate decreases with increasing from this solution with rabbit anti-nitrate reductase. amounts of PAB. A control sample heated in 50 The "4C-labeled membrane was extracted with Triton mM phosphate buffer alone shows that inhibiand nitrate reductase was precipitated from the tion by PAB is not due to the ionic strength of Triton extract as above. The precipitates were solubithis solution lized with SDS and a mixture of 10 ,g of Triton-ex- the PAB in solution. The pH of tracted protein and 21 gg of heat-extracted protein remained stable during heating. Note in Table 2 was run on gels containing 7.5% (A) and 5% (B) acryl- that in the absence of inhibitor there is an increase in enzyme activity after heating. This amide. 2

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TABLE 1. Effect of protease inhibitors on the heat solubilization of nitrate reductasea Nitrate reductase Inhibitor

conc (mMh)r cn(m)Preheat

None EACA TPCK DPCC PAB

20 0.7 2 10

(mU/mi) Post - Soluheat ble

840 1,180 733 820 1,200 826 840 1,180 720 800 1,180 519 860 1,120 559

Soluble

66 69 61 44 50

a A washed membrane preparation was suspended in 10 mM phosphate buffer, pH 8.3. The protein solution was divided into 2-ml samples and the inhibitors were added from concentrated solutions. The samples were preincubated with the inhibitors at 23 C for 20 min and then assayed for nitrate reductase activity. They were then heated to 60 C for 10 min, cooled immediately on ice, and then reassayed. Finally, they were centrifuged at 200,000 x g for 45 min and the supernatants were assayed. Percent solubilized was calculated by dividing the activity in the supernatants by the activity in the heated membrane fraction prior to centrif'ugation. 'EACA, e-amino-n-caproic acid; TPCK, L-1tosylamide-2-phenyl-ethylchloromethyl ketone.

solution to less than pH 5.5, and it is the low pH which prevents the solubilization of the enzyme. After the pH has been readjusted to 8.3 at 60 C, PMSF becomes completely soluble in aqueous solution (prior to heating at alkaline pH it is quite insoluble) and it remains soluble, even at 4 C. This suggests that PMSF- is unstable at alkaline pH. When the pH was maintained at 8.3, PMSF did not prevent heat solubilization. The same problem was encountered with N-a-p-tosyl-L-lysine chloromethylketone hydrochloride. An alkaline pH could not be maintained in the presence of this compound. The heat solubilization of nitrate reductase is dependent on the pH and, to a lesser extent, the ionic strength of the heat extraction buffer. This is shown in Table 3. The amount of solubilization varies somewhat from one preparation to the next, and this is reflected in the amount of inhibition by PAB (compare Tables 2 and 3). A considerable solubilization of membrane protein occurs when samples are stored overnight in the cold at alkaline pH and low ionic strength. There are several differences between TABILE 3. Effect of buffer molarity and pH on the heat solubilization of nitrate reductasea

TABLE 2. Effect of increasing amounts of PAB on the heat solubilization of nitrate reductasea Addition

Nitrate reductase (mU/ml) Soluble

(MM) (mM) Pre-heat Post-heat

None PAB (5) PAB (10) PAB (20) PAB (30) PAB (40) PO4 (50)

J. BACTERIOL.

MACGREGOR

940 900 900 920 860 900 860

1,520 1,500 1,420 1,360 1,180 1,180 1,420

1,213 1,026 786 613 546 306 959

Heating conditions

%T NR solubilized

A Soluble Tr~~~~~~~

80 68 55 45 46 25 68

a A washed membrane preparation was suspended in 10 mM phosphate buffer, pH 8.3, or where indicated, 50 mM phosphate buffer, pH 8.3. The protein solution was divided into 2-ml samples and increasing amounts of PAB were added. Samples were preincubated 2 h at 23 C and then assayed for nitrate reductase activity. They were then heated and centrifuged as in Table 1. Percent solubilized was calculated as in Table 1.

heat activation always occurs and increases as the time of heating is increased. Increasing amounts of PAB also decreased this heat activation. We had previously presented preliminary evidence that phenylmethyl sulfonyl fluoride (PMSF) also prevented heat solubilization of nitrate reductase (9, 10). Further investigation has shown that PMSF lowers the pH of the

5mMPO4 10 mM PO4 20mMPO4 40 mM PO4 50 mM PO4 5 mM PO4 + 20 mM PAB

75 79 71 63 66 24

pH6.5 pH 6.8 pH7.3 pH 7.9 pH 8.2

3 7 39

B

pH8.5

47 47 42

a A and B were done with different membrane preparations. (A) A washed membrane preparation was suspended at a protein concentration of' 25 mg/ml in 5 mM phophate buffer, pH 8.3, and then diluted to a concentration of 0.4 mg/ml in various molarities of phosphate buffer, all pH 8.3, one of which contained PAB. These samples were heated, assayed, and centrifuged as in Table 1. The supernatants were assayed and % nitrate reductase (NR) solubilized was calculated as in Table 1. (B) A washed membrane preparation was suspended to a protein concentration of' 21 mg/ml in 0.1 M NaCl then diluted to 0.4 mg/ml in 10 mM phosphate buffer solutions of various pH. These samples were then heated and centrifuged as in Table I and '7. NR solubilized was calculated as in Table 1. The pH recorded was that determined after heating.

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PROTEOLYSIS OF NITRATE REDUCTASE

this solubilization and the solubilization of nitrate reductase which occurs at 60 C. First, there is no activation of nitrate reductase. Second, the amount of nitrate reductase which is solubilized represents only about 20 to 30% of that which is solubilized at 60 C. Third, a great deal of protein which cannot be precipitated with rabbit anti-nitrate reductase is solubilized. Presumably, this represents other membrane proteins which have been degraded-when this solubilized material is examined on SDS gels a variety of low-molecular-weight polypeptides are observed which are not seen in Triton extracts of fresh membrane samples, and the molecular weights of all of the polypeptides appear lower than those seen in Triton extracts. The addition of 10 mM PAB or 40 mM E-amino caproic acid is very effective in inhibiting this cold alkaline solubilization, resulting in each case in a reduction of 86% in the amount of nitrate reductase solubilized. Since these inhibitors are much less effective at 60 C, it is possible either that the inhibitors are somewhat heat labile or that the protease(s) differ at the higher temperature. Clearly, proteolysis occurs in the cold at alkaline pH, but it is neither as rapid nor as effective with respect to the release of nitrate reductase. To show that PAB was preventing both degradation and solubilization of nitrate reductase, a comparison of the nitrate reductase protein remaining in the membrane after heating in the presence or absence of PAB was made. Half of a sample of membrane was heated with PAB, and the remainder was heated without the inhibitor. The material which was insoluble after the heating was isolated by centrifugation and extracted with Triton X-100. The nitrate reductase in these Triton extracts was precipitated with antibody and compared on SDS gels to enzyme precipitated from a Triton extract from an unheated membrane sample. This experiment is shown in Fig. 5. The gel patterns demonstrate the following points. (i) The nitrate reductase remaining in the membrane after heating is essentially undegraded, the only evidence of proteolysis being the partial splitting of the B subunit into B1 and B2. This is true both in the sample heated with the inhibitor (Fig. 5A) and the sample heated without inhibitor (Fig. 5B). The only difference between the samples with and without inhibitor is in the amount of nitrate reductase remaining (note the difference in amount of protein in Figs. 5A and 5B). (ii) Similarly, in the enzyme remaining in the membrane in both cases, the ratio of subunit A to subunit B was 1:1. (iii) The

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FIG. 5. Comparison of SDS gels of nitrate reductase proteins which remain attached to the membrane after heating in the presence (A) and in the absence (B), of PAB and those which are released after heating in the absence of PAB (C). Identical membrane preparations were heated at 60 C with and without 40 mM PAB. After heating, the samples were centrifuged and the pellets were extracted with Triton. (A and B) The material precipitated from these Triton extracts with rabbit anti-nitrate reductase and goat anti-rabbit gamma globulin. The precipitate in (A) contained 2.5 times the nitrate reductase protein as that in (B). The dotted lines represent a "4C marker of Triton extracted membrane which is the same as in Fig. 4. (C) An immune precipitate of the supernatant of the sample heated without PAB. The inset in (C) shows the release of labeled protein and the distribution of nitrate reductase activity in samples heated with and without PAB. These are expressed as percent of the protein or activity present after heating but before centrifugation.

molecular weight of subunit B1 in the sample heated with PAB is significantly larger than that of the subunit B of the marker (Fig. 5A), which consisted of material precipitated from a Triton extract of unheated membrane. This suggests that even in Triton extracts of fresh, unheated membrane samples there has been some proteolysis which is prevented by PAB. (iv) No subunit C was found in the heated samples, regardless of whether PAB was present or not.

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Figure 5C shows an immune precipitate of the material which was solubilized by heating in the absence of PAB. An immune precipitate of the material which was solubilized by heating in the presence of PAB was also examined (data not shown). Although it was much smaller in amount, the material was as extensively fragmented as that shown in Fig. 5C. The inset data in Fig. 5 shows that, at a concentration of 40 mM, PAB reduced, but did not entirely eliminate, the release of the enzyme and also reduced the total amount of protein released. The total recovery is somewhat low, because there was some loss of material from the particulate fractions due to the manipulation of small samples. The apparent discrepancy between the amount of nitrate reductase activity released and the amount of protein released is due to the heat activation in the absence of the inhibitor. The data shown in Fig. 5 indicates that when only a slight degradation of the enzyme occurs, only a small amount is solubilized. Consequently, the solubilization must be due to limited proteolysis of the enzyme. Since nitrate reductase can only be removed from the membrane by disrupting the membrane with detergent or by the action of a protease, it might be possible that the undegraded enzyme is held to the membrane by covalently bound phospholipid. To look for such a phospholipid, strain RK7 was grown in high levels of [32P]orthophosphate along with [3H]leucine. The membrane was isolated and extracted with Triton, and nitrate reductase was precipitated from the extract with antibody. In the total Triton extract, the 32P/3H ratio was 23/1, whereas in the antibody precipitate the ratio was 2/1. When the precipitate was run on SDS gels, none of the 32p was associated with subunit A, B, or C and all the counts were at the bottom of the gel. To insure that no hydrolysis occurred during boiling of the samples in SDS, an unboiled antibody precipitate was run on gels. The result (Fig. 6) was identical to the boiled sample. Therefore, the enzyme complex is not held to the membrane by covalently bound phospholipid. This does not rule out binding by other kinds of lipids. DISCUSSION Two lines of evidence point to the fact that nitrate reductase undergoes proteolysis during its release from the membrane by heat: (i) the enzyme precipitated by antibody from crude heat extracts shows a fragmented peptide pattern on SDS gels as compared to enzyme

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