Characterization of a polypeptide present in herpes simplex virus type 2-transformed and -infected hamster embryo cells

Transformation of hamster embryo cells by herpes simplex virus stimulated the production of a 35-kilodalton (35K) protein that was specifically immunoprecipitated, along with other polypeptides, by rabbit hyperimmune serum. This 35K polypeptide was further analyzed by partial digestion with Staphylococcus aureus V8 protease in parallel with a 35K polypeptide from herpes simplex virus type 2-infected cells. These polypeptides had almost identical partial-proteolytic cleavage maps, indicating that they are probably the same or that they are very similar polypeptides.     

Structure-function analysis of plasma cholesteryl ester transfer protein by protease digestion and expression of cDNA fragments in Escherichia coli

In an attempt to define an active domain of the protein, fragments of cholesteryl ester transfer protein (CETP) were obtained by limited digestion of the native, plasma-derived protein with trypsin, chymotrypsin, or Staphylococcus aureus V8 protease or by expression of CETP cDNA restriction fragments in Escherichia coli. Although digestion of native CETP with these proteases resulted in extensive fragmentation of the protein and loss of the intact 74-kDa molecule as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, CE transfer activity was unaffected (trypsin or chymotrypsin treatment) or only partially lost (V8 protease treatment). Analysis by molecular sieve chromatography showed that the CE transfer-active product of this proteolysis consisted of polypeptide fragments which remained associated, retaining the native molecular weight of CETP. These proteolyzed complexes were resistant to dissociation by dithiothreitol, 8 M urea, or delipidating agents. As shown by CE transfer activity, native CETP was found to possess a stable conformation which remained unchanged in buffers containing up to 4.5 M urea, or following exposure to even higher (8 M) urea concentrations. CETP polypeptides from bacterially expressed cDNA fragments were found to be catalytically inactive although they contained the epitope for an inhibitory anti-CETP monoclonal antibody and had emulsion binding properties similar to native CETP. Selected synthetic CETP peptides (including the peptide containing the inhibitory monoclonal antibody epitope) were also devoid of CE transfer activity. Thus, no evidence was found for an independently active subunit of the CETP. Together, the results indicate that the CETP possesses a distinct and highly stable tertiary structure which is required for CE transfer catalytic activity.     

The native metastability and misfolding of serine protease inhibitors

The native metastability of serine protease inhibitors (serpins) is believed to facilitate the conformational change required for biological function. However, energetically unfavorable structural features that contribute to metastability of the native serpin conformation, such as buried polar groups, cavities, and over-packing of side-chains, also appear to hinder proper folding. Hence, folding of serpin polypeptides appears prone to error; in particular, the folding polypeptides are readily diverted toward a non-productive folding pathway culminating in a more stable but inactive conformation. In a survey of deficient serpin mutants, various folding defects, such as retarded protein folding, destabilized native conformation, and spontaneous conversion into more stable, inactive conformations such as the latent form and loop-sheet polymers, have been discovered.     

Analysis of the peanut agglutinin molten globule-like intermediate by limited proteolysis

These studies attempt to characterize the molten globule-like intermediate in the unfolding pathway of peanut agglutinin (PNA). PNA is the only known example of a homotetrameric protein that lacks the 2,2,2 or the fourfold symmetry. Previous studies have shown that PNA describes a non two-state unfolding process populated with a clearly defined intermediate. The intermediate is monomeric and has lost most of its tertiary structure and has a substantial amount of secondary structure still intact, thus described as a molten-globule (MG)-like intermediate. It was also shown by isothermal titration calorimetry to bind to lactose and some other ligands with an affinity similar to that of the native protein. This paper describes limited protease cleavage experiments on the intermediate using trypsin and protease V8 for its structural characterization. There are two hydrophobic cores in the PNA subunit. These experiments suggest that in the MG-like intermediate, the second hydrophobic core, near the sugar-binding loop of the protein loosens up. This effect is significantly reduced by the presence of 90% saturating lactose, as deduced by a reduction in cleavage propensity. This is also supported by the gain in the tertiary structure as observed by near-UV CD.     

Biogenesis of a photosystem I light-harvesting complex. Evidence for a membrane intermediate.

CAB-7p is a chlorophyll a/b binding protein of photosystem I (PSI). It is found in light-harvesting complex I 680 (LHCI-680), one of the chlorophyll complexes produced by detergent solubilization of PSI. Two types of evidence are presented to indicate that assembly of CAB-7p into PSI proceeds through a membrane intermediate. First, when CAB-7p is briefly imported into chloroplasts or isolated thylakoids, we initially observe a fast-migrating membrane form of CAB-7p that is subsequently converted into PSI. The conversion of the fast-migrating form into PSI does not require stroma or ATP. Second, trypsin treatment of thylakoids containing radiolabeled CAB-7p indicates that there are at least two membrane forms of the mature 23-kD protein. The predominant form is completely resistant to proteolysis; a second form of the protein is cleaved by trypsin into 12- and 7-kD polypeptides. We interpret this to mean that the intermediate is a cleavable form that becomes protease resistant during assembly. This notion is supported by the observation that CAB-7p in LHCI-680 is largely cleaved by trypsin into 12- and 7-kD polypeptides, whereas CAB-7p in isolated PSI particles is trypsin resistant. In vitro, we generated a mutant form of CAB-7p, CAB-7/BgI2p, that was able to integrate into thylakoid membranes but was unable to assemble into PSI. The membrane form of CAB-7/BgI2p, like LHCI-680, was predominantly cleaved by trypsin into 12- and 7-kD fragments. We suggest that the mutant protein is arrested at an intermediate stage in the assembly pathway of PSI. Based on its mobility in nondenaturing gels and its susceptibility to protease cleavage, we suggest that the intermediate form is LHCI-680. We propose the following distinct stages in the biogenesis of LHCI: (a) apoprotein is integrated into the thylakoid, (b) chlorophyll is rapidly bound to apoprotein forming LHCI-680, and (c) LHCI-680 assembles into the native PSI complex.     

Polypeptide components of the apamin receptor associated with a calcium activated potassium channel

Photoaffinity labeling of rat brain membranes with [125I]ANPAA-apamin incorporated radioactivity into polypeptides of 86 and 59 kDa and occasionally a more weakly labeled component of 45 kDa. These polypeptides were immunoprecipitated with anti-apamin antibodies and treated with glycosidases. Neither the 86 nor the 59 kDa polypeptide appeared to be N-glycosylated. Partial proteolytic mapping of affinity labeled polypeptides with chymotrypsin or V8 protease generated an identical pattern. These results suggest that the 59 and 45 kDa components are not additional subunits of an oligomeric protein but result from cleavage of the 86 kDa polypeptide.     

Secretion of cytoplasmic and nuclear proteins from animal cells using novel secretion modules

Production of recombinant proteins that are not secreted outside the producing cells usually requires purification steps that can result in significant yield reductions and loss of biological activity. Using insect cells as a model system to devise the means for secreting recombinant proteins that are not normally destined for secretion outside the producing cells, we initially examined the ability of an insect-specific signal peptide sequence to direct secretion of two intracellular proteins (the cytoplasmic enzyme chloramphenicol acetyl transferase [CAT] and the nuclear protein Bombyx mori chorion factor 1 [BmCF1]) expressed in transfected silkmoth cells. Although this signal sequence functioned efficiently as a chimera with normally secreted proteins, it failed to secrete CAT and BmCF1, suggesting that additional signals are required for passage of these polypeptides through the secretion pathway. For this reason, we also generated a secretion module consisting of the secreted protein juvenile hormone esterase (JHE), a spacer region containing a histidine tag and an endopeptidase cleavage site, to which coding sequences of choice can be cloned as C-terminal extensions. In C-terminal fusions with the CAT and BmCF1 open reading frames, the N-terminal JHE moiety was able to provide all the signals necessary for secretion of CAT and BmCF1 into the extracellular environment. The histidine tag present in the spacer region allowed purification of fusion proteins by metal affinity chromatography under nondenaturing conditions, and the enteropeptidase cleavage site was recognized and cleaved by the cognate protease causing the release of the intracellular proteins from the secretion module. We also show that another secreted protein, human granulocyte-macrophage colony stimulating factor (GM-CSF) can substitute for JHE in the secretion module and that these secretion modules can function in mammalian cells.     

Bacterioopsin, haloopsin, and sensory opsin I of the halobacterial isolate Halobacterium sp. strain SG1: three new members of a growing family.

The genes coding for bacterioopsin, haloopsin, and sensory opsin I of a halobacterial isolate from the Red Sea called Halobacterium sp. strain SG1 have been cloned and sequenced. The deduced protein sequences were aligned to the previously known halobacterial retinal proteins. The addition of these new sequences lowered the number of conserved residues to only 23 amino acids, or 8% of the alignment. Data base searches with two highly conserved peptides as well as with an alignment profile yielded no significant similarity to any other protein, so the halobacterial retinal proteins should be regarded as a distinct protein family. The protein alignment was used to make predictions about the structure of the retinal proteins as well as about the amino acids in contact with retinal proteins. These results were in excellent agreement with the structural model of bacteriorhodopsin of Halobacterium halobium as well as with mutant studies, indicating that (i) structure predictions based on the sequences of a membrane protein family can be quite accurate; (ii) halorhodopsin and sensory rhodopsin I have tertiary structures similar to that of bacteriorhodopsin; (iii) conserved amino acids do not take part in reactions specific for one group of proteins, e.g., proton translocation for bacteriorhodopsins, but have a crucial role in determining the conformation and reactions of the chromophore; and (iv) the general mode of action (light-induced chromophore and protein movements) is the same for all halobacterial retinal proteins, ion pumps as well as sensors.     

Limited Digestion of Guinea Pig Myelin Basic Protein and Its Carboxy-Terminal Fragment (Residues 89–169) with Staphylococcus aureus V8 Protease

Staphylococcus aureus V8 protease has been reported to have a strict specificity for cleavage of the Glu-X bond in ammonium bicarbonate (pH 7.9). With myelin basic protein and one of its major peptic fragments (residues 89-169) as substrates, selective cleavage of Asp(32)-Thr(33), Asp(37)-Ser(38), and Glu(118-Gly(119) bonds was observed, as well as the unusual cleavage of the Gly(127)-Gly(128) bond. The Asp-Glu and Glu-Asn bonds in the sequence of Gln-Asp-Glu-Asn-Pro(81-84) were resistant to V8 protease attack. The following peptides were identified as products of limited cleavage of basic protein by V8 protease: (1-32), (1-37), (33-169), (38-169), (33-118), (38-118), (33-127), (38-127), (119-169), and (128-169). Cleavage of the peptic peptide (89-169) yielded fragments (89-118), (89-127), (119-169), and (128-169). All peptides were identified by amino acid analysis, as well as NH2- and COOH-terminal analyses. Time course studies with basic protein showed that V8 protease initially attacked the bonds between Asp(32) and Thr(33) and Asp(37) and Ser(38). With peptide (89-169) the initial cleavage was between Glu(118) and Gly(119). Peptides (89-118) and (89-127) were encephalitogenic in the Lewis rat. The activity of these peptides in the rat confirms the presence of a minor encephalitogenic site in guinea pig basic protein. Peptide (89-127) was encephalitogenic in the guinea pig, as expected, because it contains the intact encephalitogenic site. V8 protease digestion of basic protein yields some interesting new fragments, not previously available for biologic studies.     

Proteases and proteolytic cleavage of storage proteins in developing and germinating dicotyledonous seeds

Proteolytic cleavage plays an important role in storage proteindeposition and reactivation in seeds. Precursor polypeptidesare processed by limited proteolysis to mature subunits of reserveproteins in storage tissue cells of developing seeds. Stepsof proteolytic processing are closely related to steps in intracellularprotein transfer through the endomembrane system and to thedeposition in the storage vacuole. In germinating seeds specialendopeptidases trigger storage protein breakdown by limitedproteolysis. The induced conformation changes of storage proteinsopen them to attack by additional endo- and exopeptidases whichdegrade the protein reserves completely. Proteases that catalyselimited cleavage or complete degradation are synthesized asprecursors which also undergo stepwise limited proteolysis whenthey are formed in cotyledons of developing or germinating seeds.In general, this processing transforms enzymatically inactiveproenzymes into active proteases. Different compartments participatein the processing steps. Many of the proteases are encoded bysmall multigene families. Different members of the correspondingprotease families seem to act during seed development and germination.Proteolytic processes that contribute to the molecular maturationand to the reactivation of storage proteins in dicotyledonousseeds seem to be controlled by (1) differential expression ofmembers of the protease-encoding gene families; (2) stepwiseprocessing and activation of protease precursor polypeptides;(3) transient differential compartmentation of precursors andmature polypeptides of proteases and storage proteins, respectively;and (4) interacting changes in storage protein structure andprotease action. The present knowledge on these processes isreviewed. Key words: Dicotyledons, seeds, storage proteins, proteolytic cleavage, proteases     

A new strategy for glycoprotein synthesis: ligation of synthetic glycopeptides with truncated proteins expressed in E. coli as TEV protease cleavable fusion protein

We report here the use of TEV protease cleavable fusion proteins to produce glycosylated bioactive peptides and proteins. Bacterial expression was utilized to produce two fusion proteins, GPRT-C37-H6 and His-tagged interleukin-2 (amino acids 6-133), which when cleaved by the tobacco etch virus NIa protease (TEV protease) to generate HIV entry inhibitor peptide C37-H6 and a truncated version of the cytokine interleukin-2, both containing N-terminal cysteines. The N-terminal cysteine containing C37-H6 and truncated interleukin-2 were then joined to a synthetic glycopeptide thioester utilizing native chemical ligation under nondenaturing and denaturing conditions, respectively. The ligations of the glycopeptide to the C37-H6 peptide and the truncated interleukin-2 protein both proceeded in high yield, though the size, and physical properties of the two polypeptides differ greatly.     

Eukaryotic proteins expressed in Escherichia coli: an improved thrombin cleavage and purification procedure of fusion proteins with glutathione S-transferase

Several systems have been developed to allow for rapid and efficient purification of recombinant proteins expressed in bacteria. The expression of polypeptides in frame with glutathione S-transferase (GST) allows for purification of the fusion proteins from crude bacterial extracts under nondenaturing conditions by affinity chromatography on glutathione agarose (D. B. Smith and K. S. Johnson, 1988, Gene 67, 31-40). This vector expression system has also incorporated specific protease cleavage sites to facilitate proteolysis of the bacterial fusion proteins. In our hands, the cleavage of these fusion proteins at a thrombin cleavage site proceeded slowly. To facilitate the cleavage of fusion proteins, we have introduced a glycine-rich linker (glycine kinker) containing the sequence P.G.I.S.G.G.G.G.G located immediately following the thrombin cleavage site. This glycine kinker greatly increases the thrombin cleavage efficiency of several fusion proteins. The introduction of the glycine kinker into fusion proteins allows for the cleavage of the fusion proteins while they are attached to the affinity resin resulting in a single step purification of the recombinant protein. More than 2 mg of the highly purified protein was obtained from the equivalent of 100 ml of bacterial culture within a few hours when a protein tyrosine phosphatase was employed as a test protein. The vector, pGEX-KG, has also been modified to facilitate cloning of a variety of cDNAs in all reading frames and has been successfully used to express several eukaryotic proteins.     

Expression of functional hypodermin A,a serine protease from Hypoderma lineatum (Diptera,Oestridae), in Schneider 2 cells

Hypodermin A (HA) is a serine protease secreted by first-instar Hypoderma lineatum larvae (Oestridae, Diptera). It plays a crucial role in induced immunosuppression during hypodermosis. This report describes the production of recombinant HA in Drosophila melanogaster Schneider 2 cells, its purification and its characterization, and compares it with the protease extracted form parasite larvae. The recombinant protein and the native HA have similar biochemical and biological features. Activity of the recombinant protease on the lymphocyte proliferation inhibition and on membrane antigen cleavage was tested and shown to be similar to the native one. Tunicamycin treatment of the recombinant HA shows that the two putative glycosylation sites carry glycan residues. Unglycosylated recombinant HA has the same enzymatic activity as the fully glycosylated protein, indicating that glycosylation is not important for the protease activity of HA.     

Regulatory role of C-terminal residues of SulA in its degradation by Lon protease in Escherichia coli

The SulA protein is a cell division inhibitor in Escherichia coli, and is specifically degraded by Lon protease. To study the recognition site of SulA for Lon, we prepared a mutant SulA protein lacking the C-terminal 8 amino acid residues (SA8). This deletion protein was accumulated and stabilized more than native SulA in lon(+) cells in vivo. Moreover, the deletion SulA fused to maltose binding protein was not degraded by Lon protease, and did not stimulate the ATPase or peptidase activity of Lon in vitro, probably due to the much reduced interaction with Lon. A BIAcore study showed that SA8 directly interacts with Lon. These results suggest that SA8 of SulA was recognized by Lon protease. The SA8 peptide, KIHSNLYH, specifically inhibited the degradation of native SulA by Lon protease in vitro, but not that of casein. A mutant SA8, KAHSNLYH, KIASNLYH, or KIHSNAYH, also inhibited the degradation of SulA, while such peptides as KIHSNLYA did not. These results show that SulA has the specified rows of C-terminal 8 residues recognized by Lon, leading to facilitated binding and subsequent cleavage by Lon protease both in vivo and in vitro.     

In vitro synthesis of lactose permease to probe the mechanism of membrane insertion and folding

Insertion and folding of polytopic membrane proteins is an important unsolved biological problem. To study this issue, lactose permease, a membrane transport protein from Escherichia coli, is transcribed, translated, and inserted into inside-out membrane vesicles in vitro. The protein is in a native conformation as judged by sensitivity to protease, binding of a monoclonal antibody directed against a conformational epitope, and importantly, by functional assays. By exploiting this system it is possible to express the N-terminal six helices of the permease (N(6)) and probe changes in conformation during insertion into the membrane. Specifically, when N(6) remains attached to the ribosome it is readily extracted from the membrane with urea, whereas after release from the ribosome or translation of additional helices, those polypeptides are not urea extractable. Furthermore, the accessibility of an engineered Factor Xa site to Xa protease is reduced significantly when N(6) is released from the ribosome or more helices are translated. Finally, spontaneous disulfide formation between Cys residues at positions 126 (Helix IV) and 144 (Helix V) is observed when N(6) is released from the ribosome and inserted into the membrane. Moreover, in contrast to full-length permease, N(6) is degraded by FtsH protease in vivo, and N(6) with a single Cys residue at position 148 does not react with N-ethylmaleimide. Taken together, the findings indicate that N(6) remains in a hydrophilic environment until it is released from the ribosome or additional helices are translated and continues to fold into a quasi-native conformation after insertion into the bilayer. Furthermore, there is synergism between N(6) and the C-terminal half of permease during assembly, as opposed to assembly of the two halves as independent domains.     

Amino acid sequence of the L-arabinose-binding protein from Escherichia coli B/r.

The amino acid sequence of the L-arabinose-binding protein of Escherichia coli B/r was determined by sequenator analyses of reduced and S-pyridylethylated L-arabinose-binding protein and fragments derived by chemical and enzymatic cleavage of the native protein. The fragments were the products of cleavage by cyanogen bromide. BNPS-skatole, hydroxylamine, mild acid hydrolysis, limited trypsin digestion, chymotrypsin subdigestion, and subdigestion with Staphylococcus aureus protease V8. The COOH-terminal sequence was determined using bovine carboxypeptidases A and B and amino acid analyses. The L-arabinose-binding protein was determined to contain 306 amino acid residues, the sequence of which is presented below.     

The amino acid sequence specificity of a protease from spores of Bacillus megaterium

Previous work has shown that the degradation of 20% of total protein which occurs early in germination of Bacillus megaterium spores is initiated by an endoprotease. This enzyme is found only in the spore and is active only on the spore proteins degraded during germination. Action of the spore protease in vitro on the three major proteins (Proteins A, B, and C) which are degraded in vivo during germination results in cleavage of one (A and C protein) or two (B protein) peptide bonds. The sequences surrounding the cleavage sites are -Tyr-Glu- Ile-Ala-Ser-Glu-Phe- in the A protein, -Phe-Glu- Ile-Ala-Ser-Glu-Phe- in the C protein, and -Thr-Glu- Phe-Gly-Ser-Glu-Thr-, and -Thr-Glu- Phe-Ala-Ser-Glu-Thr- in the B protein, with cleavage taking place at the glutamyl bond noted by the arrow. The similarity of these four sequences suggests the possibility that the specificity of the spore protease may be due to its requirement for a specific pentapeptide sequence of the type -R-Glu-(Phe or Ile)-(Gly or Ala)-Ser-Glu-R- for recognition and cleavage. However, it is also possible that it is the conformation of the A, B, and C proteins which determines their site of cleavage by the spore protease.     

A cleavage enzyme-cytometric bead array provides biochemical profiling of resistance mutations in HIV-1 Gag and protease

Most protease-substrate assays rely on short, synthetic peptide substrates consisting of native or modified cleavage sequences. These assays are inadequate for interrogating the contribution of native substrate structure distal to a cleavage site that influences enzymatic cleavage or for inhibitor screening of native substrates. Recent evidence from HIV-1 isolates obtained from individuals resistant to protease inhibitors has demonstrated that mutations distal to or surrounding the protease cleavage sites in the Gag substrate contribute to inhibitor resistance. We have developed a protease-substrate cleavage assay, termed the cleavage enzyme- cytometric bead array (CE-CBA), which relies on native domains of the Gag substrate containing embedded cleavage sites. The Gag substrate is expressed as a fluorescent reporter fusion protein, and substrate cleavage can be followed through the loss of fluorescence utilizing flow cytometry. The CE-CBA allows precise determination of alterations in protease catalytic efficiency (k(cat)/K(M)) imparted by protease inhibitor resistance mutations in protease and/or gag in cleavage or noncleavage site locations in the Gag substrate. We show that the CE-CBA platform can identify HIV-1 protease present in cellular extractions and facilitates the identification of small molecule inhibitors of protease or its substrate Gag. Moreover, the CE-CBA can be readily adapted to any enzyme-substrate pair and can be utilized to rapidly provide assessment of catalytic efficiency as well as systematically screen for inhibitors of enzymatic processing of substrate.     

Structural basis for coevolution of a human immunodeficiency virus type 1 nucleocapsid-p1 cleavage site with a V82A drug-resistant mutation in viral protease

Maturation of human immunodeficiency virus (HIV) depends on the processing of Gag and Pol polyproteins by the viral protease, making this enzyme a prime target for anti-HIV therapy. Among the protease substrates, the nucleocapsid-p1 (NC-p1) sequence is the least homologous, and its cleavage is the rate-determining step in viral maturation. In the other substrates of HIV-1 protease, P1 is usually either a hydrophobic or an aromatic residue, and P2 is usually a branched residue. NC-p1, however, contains Asn at P1 and Ala at P2. In response to the V82A drug-resistant protease mutation, the P2 alanine of NC-p1 mutates to valine (AP2V). To provide a structural rationale for HIV-1 protease binding to the NC-p1 cleavage site, we solved the crystal structures of inactive (D25N) WT and V82A HIV-1 proteases in complex with their respective WT and AP2V mutant NC-p1 substrates. Overall, the WT NC-p1 peptide binds HIV-1 protease less optimally than the AP2V mutant, as indicated by the presence of fewer hydrogen bonds and fewer van der Waals contacts. AlaP2 does not fill the P2 pocket completely; PheP1' makes van der Waals interactions with Val82 that are lost with the V82A protease mutation. This loss is compensated by the AP2V mutation, which reorients the peptide to a conformation more similar to that observed in other substrate-protease complexes. Thus, the mutant substrate not only binds the mutant protease more optimally but also reveals the interdependency between the P1' and P2 substrate sites. This structural interdependency results from coevolution of the substrate with the viral protease.     

Isolation and characterization of protease do from Escherichia coli, a large serine protease containing multiple subunits

A new cytoplasmic proteolytic enzyme in Escherichia coli, named protease Do, has been purified to near homogeneity. The enzyme is an endoprotease that degrades casein, denatured bovine serum albumin, and globin but shows little or no hydrolytic activity against insulin, growth hormone, native bovine serum albumin, or a variety of commonly used peptide substrates. The molecular size of the enzyme was large, and it could be isolated in different preparations in either of two forms. One showed a molecular weight of about 500,000 on gel filtration and a sedimentation coefficient of 15.9 S on sucrose gradient centrifugation. The other appeared to be about 300,000 and sedimented at 12.7 S. No interconversion between the two forms and no other difference in the properties was found. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS) shows that both forms contain a major 54,000-dalton band and three additional minor polypeptides with molecular weights of 45,000, 44,000, and 42,000. These minor polypeptides appear to result from autolytic degradation of the major protein as demonstrated by peptide mapping with Staphylococcus aureus V8 protease. Thus, protease Do appears to contain a single subunit of 54,000, and can exist either as a decamer or as a hexamer or pentamer. The enzyme is a serine protease. It is sensitive to diisopropyl fluorophosphate (DFP) but not to metal chelating agents, sulfhydryl blocking groups, certain chloromethyl ketones, or various peptide aldehyde inhibitors. The enzyme covalently binds [3H]DFP, and the labeled subunit was visualized on SDS-polyacrylamide gels by fluorography. When cells growing in rich broth enter stationary phase, the relative concentration of protease Do increases more than twofold.