Limited proteolysis of the nitrate reductase from spinach leaves

The functional structure of assimilatory NADH-nitrate reductase from spinach leaves was studied by limited proteolysis experiments. After incubation of purified nitrate reductase with trypsin, two stable products of 59 and 45 kDa were observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The fragment of 45 kDa was purified by Blue Sepharose chromatography. NADH-ferricyanide reductase and NADH-cytochrome c reductase activities were associated with this 45-kDa fragment which contains FAD, heme, and NADH binding fragment. After incubation of purified nitrate reductase with Staphylococcus aureus V8 protease, two major peaks were observed by high performance liquid chromatography size exclusion gel filtration. FMNH2-nitrate reductase and reduced methyl viologen-nitrate reductase activities were associated with the first peak of 170 kDa which consists of two noncovalently associated (75-90-kDa) fragments. NADH-ferricyanide reductase activity, however, was associated with the second peak which consisted of FAD and NADH binding sites. Incubation of the 45-kDa fragment with S. aureus V8 protease produced two major fragments of 28 and 14 kDa which contained FAD and heme, respectively. These results indicate that the molybdenum, heme, and FAD components of spinach nitrate reductase are contained in distinct domains which are covalently linked by exposed hinge regions. The molybdenum domain appears to be important in the maintenance of subunit interactions in the enzyme complex.     

Hog thyroid peroxidase: physical, chemical, and catalytic properties of the highly purified enzyme.

Studies are reported on the purity and on the physical, chemical, and catalytic properties of a highly purified, stable, thyroid peroxidase (TPO). The enzyme was solubilized by treatment with deoxycholate and trypsin, and it was purified by a series of column treatments, including ion-exchange chromatography on DEAE-cellulose, gel filtration through Bio-Gel P-100, and hydroxylapatite chromatography. The final product, designated TPO VII, had a value for A₄₁₀/A₂₈₀ of 0.54, and its specific activity based on the guaiacol assay (794 μmol of guaiacol oxidized/min/mg) was considerably greater than that of any previously described TPO. Specific activity values based on other peroxidase-catalyzed reactions were also higher for TPO VII than for previous TPO preparations. Purity estimates for TPO VII, based on polyacrylamide disc gel electrophoresis and on isoelectric focusing in polyacrylamide gels, ranged from 80 to 95%. The molecular weight, determined by sedimentation equilibrium, was 93,000. Results of sodium dodecyl sulfate-gel electrophoresis also indicated a molecular weight of approximately 90,000. Sodium dodecyl sulfate-gel electrophoresis under reducing conditions indicated that TPO VII is composed of two peptide chains of unequal size, with the larger about 2.5-fold the size of the smaller. Carbohydrate analysis revealed that TPO is a glycoprotein containing about 10% by weight of carbohydrate. The predominant sugars were mannose and N-acetyl glucosamine. A significant amount of glucose was also found, along with small amounts of galactose, fucose, and xylose. The amino acid composition of TPO VII showed a high proline content, a predominance of arginine over lysine, and a ratio of [Asp] plus [Glu] to [Lys] plus [Arg] of over 2. Isoelectric focusing in polyacrylamide gels indicated an isoelectric pH of 5.75. In agreement with observations made on earlier preparations of TPO, heme spectral data showed significant differences between the pyridine hemochromogens of TPO VII and horseradish peroxidase, suggesting that the heme in TPO is not ferriprotoporphyrin IX. Circular dichroism measurements indicated that approximately 40% of TPO VII involves α helix or β structure.     

Identification of a spectrally stable proteolytic fragment of human neutrophil flavocytochrome b composed of the NH2-terminal regions of gp91(phox) and p22(phox)

A heme-bearing polypeptide core of human neutrophil flavocytochrome b(558) was isolated by applying high performance, size exclusion, liquid chromatography to partially purified Triton X-100-solubilized flavocytochrome b that had been exposed to endoproteinase Glu-C for 1 h. The fragment was composed of two polypeptides of 60-66 and 17 kDa by SDS-polyacrylamide gel electrophoresis and retained a native heme absorbance spectrum that was stable for several days when stored at 4 degrees C in detergent-containing buffer. These properties suggested that the majority of the flavocytochrome b heme environment remained intact. Continued digestion up to 4.5 h yielded several heme-associated fragments that were variable in composition between experiments. Digestion beyond 4.5 h resulted in a gradual loss of recoverable heme. N-Linked deglycosylation and reduction and alkylation of the 1-h digestion fragment did not affect the electrophoretic mobility of the 17-kDa fragment but reduced the 60-66-kDa fragment to 39 kDa. Sequence and immunoblot analyses identified the fragments as the NH(2)-terminal 320-363 amino acid residues of gp91(phox) and the NH(2)-terminal 169-171 amino acid residues of p22(phox). These findings provide direct evidence that the primarily hydrophobic NH(2)-terminal regions of flavocytochrome b are responsible for heme ligation.     

Porcine thyroid peroxidase: relationship between the native enzyme and an active, highly purified tryptic fragment

We previously described the preparation of highly purified porcine thyroid peroxidase by a procedure that involved initial solubilization of the enzyme with trypsin plus detergent. Recently, the complete amino acid sequence of porcine thyroid peroxidase (TPO) was determined by cDNA cloning, and it became of interest to compare the structure of the purified trypsin-solubilized enzyme with that of the native enzyme. For this purpose we employed antibodies to the purified enzyme and to two synthetic peptides representing defined regions of the protein. We also obtained N-terminal amino acid sequence data on TPO fragments separated by gel electrophoresis. Trypsin cleavage sites in the purified enzyme were observed after arg residues 109 and 561, and also at two undetermined sites close to the putative membrane spanning region at the carboxyl end. Major fragments of approximately 60, 32, and 29 kilodaltons were observed when the purified enzyme was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. This observation is explained by assuming that the cleavage site after arg residue 561 occurred within a disulfide loop. The Mr of the trypsin-solubilized enzyme is approximately 88,000 compared to approximately 106,000 for the native enzyme. The difference can be accounted for by the loss of approximately 90 residues from the amino terminus and of at least 80 residues from the carboxyl end. Despite the loss of these fragments totaling approximately 18 kilodaltons and cleavage of the peptide bond after arg residue 561, the purified trypsin-solubilized TPO appears to retain full enzyme activity.     

Binding of a monoclonal antibody E12 to Gc globulin (vitamin D-binding protein) is inhibited by actin.

A monoclonal antibody, E12, to human Gc globulin was raised in murine somatic cell using purified Gc. The antibody was subtyped IgG2b kappa and had a kd of 3.0 x 10(-8) M for antigen Gc. Monospecificity for Gc was demonstrated by Western blotting of normal human serum using nondenaturing polyacrylamide gel electrophoresis. As judged by ELISA, actin inhibited binding of E12 to Gc in dose-dependent fashion. Affinity chromatography studies further showed that ternary complexes of actin-Gc-E12 were not formed, and actin displaced Gc from Gc-E12 complexes. Proteolytic digestion of Gc with trypsin showed that the monoclonal antibody E12 reacted with the major 30-kDa tryptic fragment containing the amino terminal fragment of Gc, but actin did not react with this fragment. These results indicate that interaction of actin with Gc causes conformational changes which inhibit binding of E12.     

Structural analysis of NADPH-cytochrome P-450 reductase from porcine hepatic microsomes. Sequences of proteolytic fragments, cysteine-containing peptides, and a NADPH-protected cysteine peptide

Detergent-solubilized NADPH-cytochrome P-450 reductase was purified from porcine hepatic microsomes and compared to the rabbit enzyme isolated under identical conditions. The porcine enzyme had an equivalent specific activity toward cytochrome c compared to the rabbit enzyme. When analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the porcine enzyme exhibited a major band at Mr = 80,000 and two additional bands at Mr = 20,000 and 60,000. The 20-kDa fragment was shown to be the COOH-terminal portion of the protein which contains a hydrophobic sequence of 28 residues homologous to the pyrophosphate-binding portion of the FAD-binding protein p-hydroxybenzoate hydroxylase. The 60-kDa fragment corresponded to the NH2-terminal portion of the protein since this peptide and the intact protein have blocked NH2 terminal. The trypsin-solubilized porcine enzyme has an NH2-terminal sequence which is homologous to the equivalent trypsin-solubilized enzymes from rat and rabbit (80% sequence homology). Eight cysteine-containing peptides were isolated from a tryptic digest of the S-carboxymethylated pig enzyme. Significant sequence homology was not found between these peptides and other flavoproteins, except for one peptide (Glu-Val-Gly-Glu-Thr-Leu-Leu-Tyr-Tyr-Gly-Cys-Arg) which exhibited partial homology with the known NADPH-binding site of glutathione reductase. When the NADPH-protected enzyme was first S-alkylated with unlabeled iodoacetate, NADPH depleted, and further alkylated with 14C-labeled iodoacetate, the above radiolabeled peptide was isolated from a tryptic digest. The equivalent peptide was also isolated by a similar procedure from rabbit liver cytochrome P-450 reductase.     

Functional characterization of protease-treated Bacillus anthracis protective antigen.

Characterization of the functional domains of Bacillus anthracis protective antigen (PA, 83-kDa), the common cellular binding molecule for both anthrax edema toxin and anthrax lethal toxin, is important for understanding the mechanism of entry and action of the anthrax toxins. In this study, we generated both biologically active (facilitates killing of J774A.1 cells in combination with lethal factor, LF) and inactive preparations of PA by protease treatment. Limited proteolytic digestion of PA in vitro with trypsin generated a 20-kDa fragment and a biologically active 63-kDa fragment. In contrast, limited digestion of PA with chymotrypsin yielded a preparation containing 37- and 47-kDa fragments defective for biological activity. Treatment with both chymotrypsin and trypsin generated three major fragments, 20, "17," and 47 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This PA preparation was also biologically inactive. To investigate the nature of the defect resulting from chymotrypsin treatment, we assayed PA preparations for the ability to bind to the cellular receptor and to bind and internalize 125I-LF. All radiolabeled PA preparations bound with specificity to J774A.1 cells and exhibited affinities similar to native 83-kDa PA. Once bound to the cell surface receptor, both trypsin-treated PA and chymotrypsin/trypsin-treated PA specifically bound 125I-LF with high affinity. Finally, these PA preparations delivered 125I-LF to a Pronase-resistant cellular compartment in a time- and temperature-dependent fashion. Thus, the biological defect exhibited by chymotrypsin-treated PA is not at the level of cell binding or internalization but at a step later, such as toxin routing or processing by J774A.1 cells. These protease-treated preparations of PA should prove useful in both elucidating the intracellular processing of anthrax lethal toxin and determining the structure-function relationship of PA and LF.     

Isolation of a low molecular weight active fragment of potato proteinase inhibitor IIb.

A low molecular weight active fragment of potato proteinase inhibitor IIPB was obtained by incubating the inhibitor with an equimolar amount of trypsin [EC 3.4.21.4] at pH 8 and 30 degrees for 16 hr, followed by gel filtration through Sephadex G-50, treatment with trichloroacetic acid, and CM-cellulose chromatography. The purified active fragment consisted of a single peptide chain with a molecular weight of 4,300, comprising 39 amino acid residues. It retained very strong inhibitory activity against chymotrypsin [EC 3.4.21.1] and subtilisin [EC 3.4.21.14]. However, the yield of this active fragment was rather low and was variable. On further incubation with trypsin, it was converted into smaller inactive peptides.     

Purification and characterization of human recombinant precursor interleukin 1 beta

We have purified the 31-kDa precursor of human interleukin 1 beta (proIL1 beta) from recombinant Escherichia coli expressing the protein. The recombinant precursor was characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, spectroscopy, Western blot, and for biological and receptor binding activity. The protein migrates at the expected molecular weight on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and analytical gel filtration columns. The specific activity of the recombinant precursor is less than 10(2) units/mg in the EL4 thymoma assay compared with 5 x 10(8) units/mg for the recombinant 17-kDa mature protein. The inactivity of the precursor is attributable to the inability of the protein to bind the IL1 receptor on EL4 cells as shown by receptor competition studies using 125I-labeled 17-kDa IL1 beta. Inactivity of the IL1 beta precursor is not due to degradation of the protein in either the bioactivity or receptor binding assays. The inactive IL1 beta precursor is converted to an active form following proteolysis with chymotrypsin which generates a carboxyl-terminal fragment of 17 kDa that is 6 orders of magnitude more active than the starting IL1 beta precursor. Removal of the first 114 amino acids from proIL1 beta generates a fully active molecule. In contrast, removal of the first 77 amino acids by treatment with trypsin only partially restores activity. The resultant 22-kDa protein exhibits a 600-fold increase in both biological and receptor binding activity, demonstrating a direct correlation between the ability of sequences within the pro-region to inhibit biological activity and inhibit binding to the IL1 receptor. Far-UV circular dichroism spectroscopy indicates that proIL1 beta is similar in secondary structure to mature IL1 beta; both proteins are nonhelical beta sheet proteins.     

Purification of the human thyroid peroxidase and its identification as the microsomal antigen involved in autoimmune thyroid diseases

Human thyroid peroxidase (TPO) has been purified from thyroid microsomes by immunoaffinity chromatography using a monoclonal antibody (mAb) to TPO. The eluted material had a specific activity of 381 U/mg and exhibited a peak in the Soret region. The ratio of A411 to A280 ranged from 0.20 to 0.25. Upon SDS-polyacrylamide gel electrophoresis, the purified enzyme gave two contiguous bands in the 100 kDa region. Further, it has been demonstrated that sera with anti-microsomal autoantibodies from patients presenting Graves' or Hashimoto's thyroiditis diseases were able to bind to purified TPO and to inhibit in a dose-dependent manner the mAb binding to purified TPO. This suggests that TPO is the thyroid antigen termed to date the microsomal antigen.     

Controlled tryptic digestion of prostaglandin H synthase. Characterization of protein fragments and enhanced rate of proteolysis of oxidatively inactivated enzyme

Treatment of prostaglandin H (PGH) synthase (70 kDa) with trypsin generates fragments of 33 and 38 kDa. Each of the fragments was purified by reverse-phase high performance liquid chromatography (HPLC) using acetonitrile/water/trifluoroacetic acid gradients. Amino acid sequence analysis indicates that the 33-kDa protein contains the NH2 terminus of PGH synthase. Neither the 33- nor 38-kDa fragment isolated by HPLC exhibits any PGH synthase activity; however, cleavage of intact enzyme to 33- and 38-kDa fragments to the extent of 90% only reduces cyclooxygenase activity by 40%. This implies that the cleaved proteins or a complex formed between them retains the conformation necessary for enzyme activity. Extensive attempts to resolve active fragments from each other or from intact enzyme were unsuccessful; intact enzyme and digestion fragments cochromatograph under all conditions employed. Treatment of PGH synthase with [3H]acetylsalicylic acid followed by trypsin digestion introduces [3H]acetyl moieties into the intact protein and the 38-kDa fragment (0.8-0.9 acetyl group/subunit). Nearly complete conversion of PGH synthase to 33- and 38-kDa fragments by exposure to high concentrations of trypsin prior to [3H]acetylsalicylic acid treatment results in labeling of the 38-kDa fragment, but not the 33-kDa fragment. The present findings are consistent with the presence of a membrane-binding domain (33 kDa) and an active site domain (38 kDa) in the 70-kDa subunit of PGH synthase. They also suggest that, following cleavage, the 38-kDa fragment retains the structural features responsible for the cyclooxygenase activity and selective aspirin labeling of PGH synthase. PGH synthase undergoes self-catalyzed inactivation by oxidants generated during its catalytic turnover. When PGH synthase, inactivated by treatment with arachidonic acid or hydrogen peroxide, was treated with trypsin it was cleaved two to three times faster than unoxidized enzyme. Addition of heme to oxidized PGH synthase did not reconstitute cyclooxygenase activity or resistance to trypsin cleavage. Spectrophotometric studies demonstrated that oxidatively inactivated enzyme did not bind heme. This implies that oxidation of protein residues as well as the heme prosthetic group is an important determinant of proteolytic sensitivity. Oxidative modification may mark PGH synthase for proteolytic cleavage and turnover.     

The steroid binding domain of porcine estrogen receptor

For the purpose of characterizing the estrogen binding domain of porcine estrogen receptor (ER), we have made use of affinity labeling of partially purified ER with [3H]tamoxifen aziridine. The labeling is very efficient and selective particularly after partial purification of ER. A 65,000-dalton (65-kDa) band was detected on the fluorogram of a sodium dodecyl sulfate-polyacrylamide gel, together with a 50-kDa band and a few more smaller bands. The 50-kDa protein appears to be a degradation product of the 65-kDa protein in view of the similar peptide map. ER was affinity labeled before or after controlled limited proteolysis with either trypsin, papain, or alpha-chymotrypsin. The labeling patterns of limited digests indicate that a fragment of about 30 kDa is relatively resistant to proteases and has a full and specific binding activity to estrogen, whereas smaller fragments have lost much of the binding activity. This fragment is very hydrophobic and probably corresponds to the carboxy half of ER.     

Purification and immunochemical properties of tyrosine hydroxylase in human brain

Tyrosine hydroxylase (TH) was isolated from human brain (caudate nucleus + putamen). The major form of the active enzyme in the cytoplasmic fraction was purified to apparent homogeneity. The molecular weight of the purified enzyme was estimated to be 280 kdalton by gel filtration. Sodium dodecyl sulfate gel electrophoresis (SDS-PAGE) of the purified enzyme gave a single subunit with mol. wt 60 kdalton, which is similar to the subunit of human adrenal TH. Using a sandwich enzyme immunoassay (EIA), the presence of inactive form(s) of TH in human brain was demonstrated, and the total content of this immunoinactive form(s) was approx. 8 times higher than that of the active form. By the Western blot technique after two-dimensional (2-D) electrophoresis, TH in the crude fraction of the human brain was found to consist of multiple forms with different pI-values and with the same molecular weight. The pl of the major spots ranged from 5.3 to 5.8, and that of the minor spot was 6.0. Because the pl of the purified enzyme preparation was 6.0, this protein with pI at 6.0 may be the active form of TH.     

Human urinary proteinase inhibitor: inhibitory properties and interaction with bovine trypsin

The major urinary trypsin inhibitor (UTI) was found to inhibit bovine chymotrypsin and human leucocyte elastase strongly, cathepsin G weakly. No inhibition of porcine pancreatic elastase was observed. The stoichiometry of the inhibition of bovine trypsin by UTI was determined spectrophotometrically to be 1:2 (I/E molar ratio). After incubation of UTI with this enzyme in various molar ratios, two complexes (C1 and C2) could be visualized in alkaline polyacrylamide gel electrophoresis. C1 was isolated by affinity chromatography on Con-A Sepharose. In dodecyl sulfate polyacrylamide gel electrophoresis, C1 was dissociated to give an inhibitory band with the same electrophoretic mobility as native UTI. C2 released an active inhibitory fragment with Mr near 20000. A time-course study demonstrated that at a molar ratio I/E of 1.5:1, the C2 complex appears after two hours of incubation.     

Purification to apparent homogeneity of inactive kallikrein from rat urine

Inactive kallikrein was purified from rat urine by a procedure including ammonium sulfate fractionation, DEAE cellulose chromatography, phenyl-Sepharose CL-4B chromatography, and gel filtration on Sephadex G-100 and Sephadex G-75 columns. The resulting preparation was essentially homogeneous, as assessed by polyacrylamide gel electrophoresis. This preparation migrated as a single protein band on a SDS-polyacrylamide gel and the molecular weight was 41000. The purified material underwent marked activation by trypsin, but not by deoxycholate, Triton X-100, SDS or acidification. These results indicate that the purified inactive kallikrein is the precursor rather than a complex with a substance binding to the active form of kallikrein.     

Heterogeneity of human erythrocyte band 3 analyzed by two-dimensional gel electrophoresis

Human erythrocyte membrane and purified band 3 were separated initially by isoelectric focusing and then examined in a second dimension by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Band 3 was segregated into three major bands whether the protein was contained within the membranes or was present in the isolated state. The isoelectric points of these major bands were 5.25, 5.35 and 5.70. Of chymotryptic fragments of band 3, the 60-kDa fragment was also separated into three major bands whose pI values were 4.75, 5.10 and 5.30. The multiplicity of band 3 appears to be due to different charges carried by the peptide(s) and is not ascribed to oxidation of band 3 during its preparation. Isoelectric points of the purified 60-kDa fragment were different from the pI values of the fragment coexisting with the complementary 35-kDa fragment, in which case the pI values were exactly the same as those of intact band 3. This suggests that these fragments interact tightly in situ even after being cleaved by chymotrypsin, and the tight interaction must still be present during electrophoresis in the first dimension.     

Separate domains of the insulin receptor contain sites of autophosphorylation and tyrosine kinase activity

We have studied the structure and function of the solubilized insulin receptor before and after partial proteolytic digestion to define domains in the beta-subunit that undergo autophosphorylation and contain the tyrosine kinase activity. Wheat germ agglutinin purified insulin receptor from Fao cells was digested briefly at 22 degrees C with low concentrations (5-10 micrograms/mL, pH 7.4) of trypsin, staphylococcal V8 protease, or elastase. Autophosphorylation of the beta-subunit was carried out before and after digestion, and the [32P]phosphoproteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, detected by autoradiography, and analyzed by tryptic peptide mapping by use of reverse-phase high-performance liquid chromatography. Mild trypsin digestion reduced the apparent molecular mass of the beta-subunit from 95 to 85 kDa, and then to 70 kDa. The 85-kDa fragment was not immunoprecipitated by an antibody directed against the C-terminal domain of the beta-subunit (alpha Pep-1), indicating that this region of the receptor was lost. The 85-kDa fragment contained about half of the [32P]phosphate originally found in the beta-subunit, and tryptic peptide mapping showed that two major tryptic phosphopeptides (previously called pY2 and pY3) were removed. Three other tryptic phosphopeptides (pY1, pY1a, and pY4) were found in the 85- and 70-kDa fragments. Treatment of the intact receptor with staphylococcal V8 protease also converted the beta-subunit to an 85-kDa fragment that did not bind to alpha Pep-1, contained about 50% of the initial radioactivity, and lacked pY2 and pY3. Elastase rapidly degraded the receptor to inactive fragments between 37 and 50 kDa.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and comparative characterization of cytochrome P-450scc from porcine adrenocortical mitochondria.

Cytochrome P-450scc (cholesterol side-chain cleavage enzyme) was purified from porcine adrenocortical mitochondria. 2. The purified cytochrome P-450scc was found to be homogeneous on SDS-polyacrylamide gel electrophoresis. 3. The heme content of the purified enzyme was 20.6 nmol/mg protein. 4. The enzymatic activity of the reconstituted cytochrome P-450scc-linked monooxygenase system amounted to 7.8 nmol of pregnenolone formed per nmole of P-450 per minute, with cholesterol as a substrate. 5. The amino acid sequence of the amino-terminal region of the cytochrome P-450scc and the amino acid residue at the carboxyl terminal were determined and compared with those of other mammalian cytochromes P-450scc.     

Human neutrophil cytochrome b contains multiple hemes. Evidence for heme associated with both subunits.

Human phagocyte cytochrome b is the terminal component of the microbicidal superoxide generating system. Although the primary structure of this protein has been determined, little is known about the placement of the heme prosthetic groups in this heterodimeric integral membrane protein. Analysis of the cytochrome using lithium dodecyl sulfate-polyacrylamide gel electrophoresis at 0 degree C followed by tetramethylbenzidine heme staining demonstrated the presence of heme in both the 91- and 22-kDa subunits identified by Western blot analysis using peptide specific antisera. Exposure of cytochrome b (purified or in isolated neutrophil plasma membranes) to Staphylococcal protease V8 or trypsin did not affect absorbance spectra. However, such treatment resulted in degradation of both subunits to smaller fragments, including characteristic immunoreactive 20-kDa fragments of both the large and small subunits of the cytochrome that retained one or both of the hemes. The spectral stability to proteolysis and size of the proteolytic heme-containing fragments generated explains previous reports which suggested that the heme resided in the small subunit. Our current results indicate that human neutrophil cytochrome b is a bi-heme or possibly tri-heme molecule with at least one heme residing in the large subunit and one shared between both subunits and that the heme-containing regions of the cytochrome probably lie within the membrane lipid bilayer. Such a multi-heme structure would be consistent with an electron transfer function for this cytochrome by providing an efficient mechanism for transferring electrons across the plasma membrane to the extracellular surface where oxygen could be reduced to create superoxide.     

Interaction of Bordetella pertussis adenylate cyclase with calmodulin. Identification of two separated calmodulin-binding domains

The structural organization of Bordetella pertussis adenylate cyclase was examined by limited proteolysis with trypsin and/or cross-linking with azido-calmodulin a photoactivable derivative of its activator, calmodulin (CaM). Adenylate cyclase (which consists of three structurally related peptides of 50, 45, and 43 kDa as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) formed a 1:1 complex with CaM or azido-CaM. CaM-bound adenylate cyclase was cleaved by trypsin into two separate trypsin-resistant fragments of 25 and 18 kDa which both interacted with CaM as judged by their ability to be cross-linked with azido-CaM. These two fragments remained associated with CaM in a catalytically active conformation resembling that of the undigested complex. When proteolysis was carried out in the absence of CaM, the adenylate cyclase was completely inactivated in less than 3 min. Sodium dodecyl sulfate-polyacrylamide gel revealed a single 24-kDa trypsin-resistant fragment. Since this fragment cannot be cross-linked with azido-CaM we suggest that the CaM-binding site on the 25-kDa moiety of the adenylate cyclase is located on a short segment of 1 kDa.