Newcastle disease virus therapy of human tumor xenografts: antitumor effects of local or systemic administration.

Previously we showed that a single local injection of the avian paramyxovirus Newcastle disease virus (NDV) strain 73-T caused long-lasting, complete tumor regression of human neuroblastoma and fibrosarcoma xenografts in athymic mice. Here we report the antitumor effects of NDV administered by either the intratumoral (IT) route to treat a variety of human carcinoma xenografts or by the systemic (intraperitoneal, IP) route to treat neuroblastoma xenografts (6.5-12 mm in diameter). For IT treatments, mice were randomized into treatment groups and given a single IT injection of NDV 73-T, vehicle (phosphate buffered saline, PBS), or UV-inactivated NDV. For systemic therapy, mice (n=18) with subcutaneous IMR-32 human neuroblastoma xenografts received IP injections of NDV (5 x 10(9) PFU). Significant tumor growth inhibition (77-96%) was seen for epidermoid (KB8-5-11), colon (SW620 and HT29), large cell lung (NCIH460), breast (SKBR3), prostate (PC3), and low passage colon (MM17387) carcinoma xenografts treated IT with NDV. In all cases, tumors treated IT with PBS or replication-incompetent, UV-inactivated NDV displayed rapid tumor growth. After a single IP injection of NDV, complete regression of IMR-32 neuroblastomas was observed in 9 of 12 mice without recurrence for the 3-9 month follow-up period. Six mice with recurrent neuroblastomas after one IP injection received one to three additional IP treatments with NDV. Three of these six mice showed complete regression without recurrence. These data show that: (1) NDV administered either IT or IP is an effective antitumor therapy in this system, (2) replication competency is necessary for maximal effect, and (3) multiple NDV doses can be more effective than a single dose. These studies provide further rationale for the preclinical study of NDV as an oncolytic agent.

Complete regression of human fibrosarcoma xenografts after local Newcastle disease virus therapy.

We have recently demonstrated that a single local injection of the avian pathogen Newcastle disease virus (NDV; strain 73-T) causes complete regression of human neuroblastoma xenografts in athymic mice (R. M. Lorence, K. W. Reichard, B. B. Katubig, H. M. Reyes, A. Phuangsab, B. R. Mitchell, C. J. Cascino, R. J. Walter, and M. E. Peeples. J. Natl. Cancer Inst., 86: 1228-1233, 1994). In this report, we tried to determine if this in vivo antineoplastic effect of NDV extends to human sarcomas. Athymic mice with s.c. HT1080 fibrosarcoma xenografts (7-14 mm) were randomly divided into two groups and treated i.t. with a single injection of either 10(7) plaque-forming units of NDV or phosphate-buffered saline. Complete tumor regression occurred in 8 of 10 mice treated with NDV while unabated tumor growth occurred in all 9 mice treated with phosphate-buffered saline (P < 0.001). To determine if complete tumor regression was long lasting, the 8 mice were monitored for 1 year, during which time no tumor recurred. To test the antitumor effects of NDV on tumors derived from a fresh human sarcoma, a similar experiment was performed in athymic mice using TH15145 synovial sarcoma xenografts at their first and second passages. Of 9 mice with TH15145 xenografts, a single i.t. injection of NDV (10(7) plaque-forming units) caused complete regression of 3 tumors and > 80% regression in 3 more tumors. In contrast, tumors in all 5 mice treated with phosphate-buffered saline exhibited unabated growth (P < 0.03 for > 80% tumor regression). Since HT1080 fibrosarcoma cells express the N-ras oncogene, we explored the effects that transfection of this oncogene has on the sensitivity to NDV. Cultured human fibroblasts that were made tumorigenic following N-ras-transfection were found to be 1000-fold more sensitive to NDV than normal fibroblasts in a cytotoxicity assay. Oncogene expression by the HT1080 fibrosarcoma may therefore contribute to the long-lasting complete regression of this sarcoma following a single local injection of NDV.

Complete regression of human neuroblastoma xenografts in athymic mice after local Newcastle disease virus therapy.

BACKGROUND: Neuroblastoma is the most common pediatric extra-cranial solid cancer. Using conventional therapies, children older than 1 year of age with advanced neuroblastoma have a poor prognosis. The development of new approaches for treating such children with neuroblastoma continues to be one of the most important goals today in pediatric oncology. Despite numerous anecdotal reports of human tumor regression during viral infections, the use of viruses to directly lyse neuroblastoma cells has never been reported as a potential therapy. Newcastle disease virus (NDV) has been shown to replicate in and kill cultured human and rat neuroblastoma cells but not normal human fibroblasts. PURPOSE: Our purpose was to determine if this selective killing of human neuroblastoma (IMR-32) cells is maintained during the in vivo treatment of established tumors. METHODS: Two experiments were performed using NDV strain 73-T. Athymic mice with subcutaneous IMR-32 human neuroblastoma xenografts (6-12 mm) were treated intralesionally with live NDV, UV-inactivated NDV, or phosphate-buffered saline (PBS). To study virus replication in situ, mice were given intratumoral or intramuscular injections of NDV. These mice were then killed at various times, and the amount of infectious virus present in tumor or muscle was determined. RESULTS: After one injection of live NDV, 17 of 18 tumors regressed completely, whereas rapid tumor growth occurred in all 18 mice treated with PBS and in all nine mice treated with UV-inactivated NDV (P < .0001). The one tumor that showed only a partial response to a single injection regressed completely after a second NDV treatment. Six months following virus-induced regression, only one tumor had recurred. No significant acute or chronic side effects of live NDV were noted in athymic mice given doses up to 500 times that used in this study. Virus levels increased more than 80-fold between 5 and 24 hours in virus-injected tumors (P < .04), while no infectious virus was produced in NDV-injected muscle tissue. CONCLUSIONS: NDV 73-T appears to replicate selectively in human IMR-32 neuroblastoma xenografts, leading directly to a potent antitumor effect as demonstrated by long-lasting, complete tumor regression occurring after a single local injection of virus. IMPLICATION: These experiments may provide an important step in the development of new therapeutic approaches to challenging cancers such as neuroblastoma.

Retinoic acid enhances killing of neuroblastoma cells by Newcastle disease virus.

Newcastle disease virus (NDV), an avian pathogen, selectively replicates in and kills neuroblastoma (NB) cells, but not normal fibroblasts in vitro and in vivo in nude mice. NDV cytotoxicity towards NB cells is enhanced by N-myc oncogene amplification. To further define the antineoplastic effects of NDV, we examined NDV's interaction with NB cells following short-term exposure to the differentiating agent, all-trans retinoic acid (RA), and to neuraminidase. The human NB cell line IMR-32, after treatment with 50 mumol/L RA, became eight times more sensitive to NDV in a cytotoxicity assay. A time course study to determine the optimal incubation period of IMR-32 cells with RA indicated that a fourfold increase in sensitivity towards NDV killing occurred after only 8 hours of RA incubation prior to addition of virus. Maximal sensitivity was achieved at 24 hours of RA incubation and remained constant for longer incubation periods (up to 72 hours). The sensitization of IMR-32 NB cells to NDV was constant for RA doses between 3 mumol/L and 50 mumol/L. Plaque formation, which indicates replication, virus spread and cytotoxicity by a single infectious virus particle, was also enhanced by RA. This effect does not appear to require N-myc amplification in the target NB cells since RA had similar effects upon the high N-myc (IMR-32) and the low N-myc expressing cells (SK-N-SH). Enhanced sialylation has been shown by others to mediate the growth inhibitory effects of RA on a variety of tumor lines. Removal of sialic acid from the IMR-32 NB cell surface using Clostridium neuraminidase (2.7 mg/mL) inhibited 75% of NDV plaque formation. These results demonstrate that NDV killing of two NB cell lines is enhanced using clinically achievable levels of RA and that sialylation of the NB cell surface is important for virus binding and cytotoxicity.

In vivo administration of recombinant growth hormone or gamma interferon activities macrophages: enhanced resistance to experimental Salmonella typhimurium infection is correlated with generation of reactive oxygen intermediates.

Purified and recombinant forms of growth hormone (GH) as well as of recombinant rat gamma interferon (IFN-gamma) enhance the survival of rats deprived of endogenous pituitary GH secretion by hypophysectomy (HX rats) and infected with virulent Salmonella typhimurium. Macrophages obtained from rats with intact pituitaries (pituitary-intact rats) or HX rats that were treated in vivo with either GH or the closely related hormone prolactin released elevated (P less than 0.05) levels of superoxide anion (O2-) after in vitro opsonized-zymosan stimulation compared with those from placebo-treated animals. These levels of O2- release were similar in magnitude to those of macrophages from rats treated in vivo with IFN-gamma. In time course in vivo macrophage activation studies, both IFN-gamma and GH significantly increased O2- secretion within 24 h, with maximal secretion occurring at day 3. Macrophages obtained from pituitary-intact and HX rats injected in vivo with GH also released elevated (P less than 0.05) levels of hydrogen peroxide (H2O2) and displayed enhanced (P less than 0.01) phagocytic activity toward opsonized Listeria monocytogenes in vitro. The mechanism of action of GH in vivo is likely to be a direct one because resident peritoneal macrophages from rats could be primed in vitro for enhanced secretion of O2- following triggering of these cells with opsonized zymosan. These data show that in vivo administration of two closely related pituitary hormones, GH and prolactin, can effectively prime macrophages, which is consistent with the hypothesis that GH mediates resistance to S. typhimurium by a direct stimulatory action on macrophages.

Newcastle disease virus selectively kills human tumor cells.

Newcastle disease virus (NDV), strain 73-T, has previously been shown to be cytolytic to mouse tumor cells. In this study, we have evaluated the ability of NDV to replicate in and kill human tumor cells in culture and in athymic mice. Plaque assays were used to determine the cytolytic activity of NDV on six human tumor cell lines, fibrosarcoma (HT1080), osteosarcoma (KHOS), cervical carcinoma (KB8-5-11), bladder carcinoma (HCV29T), neuroblastoma (IMR32), and Wilm's tumor (G104), and on nine different normal human fibroblast lines. NDV formed plaques on all tumor cells tested as well as on chick embryo cells (CEC), the native host for NDV. Plaques did not form on any of the normal fibroblast lines. To detect NDV replication, virus yield assays were performed which measured virus particles in infected cell culture supernatants. Virus yield increased 10,000-fold within 24 hr in tumor and CEC supernatants. Titers remained near zero in normal fibroblast supernatants. In vivo tumoricidal activity was evaluated in athymic nude Balb-c mice by subcutaneous injection of 9 x 10(6) tumor cells followed by intralesional injection of either live or heat-killed NDV (1.0 x 10(6) plaque forming units [PFU]), or medium. After live NDV treatment, tumor regression occurred in 10 out of 11 mice bearing KB8-5-11 tumors, 8 out of 8 with HT-1080 tumors, and 6 out of 7 with IMR-32 tumors. After treatment with heat-killed NDV no regression occurred (P less than 0.01, Fisher's exact test). Nontumor-bearing mice injected with 1.0 x 10(8) PFU of NDV remained healthy. These results indicate that NDV efficiently and selectively replicates in and kills tumor cells, but not normal cells, and that intralesional NDV causes complete tumor regression in athymic mice with a high therapeutic index.

The pituitary gland is required for protection against lethal effects of Salmonella typhimurium.

One-half of pituitary-intact or sham-operated rats survive infection with 10(9) colony-forming units of Salmonella typhimurium, whereas rats without a pituitary gland all die within a few days. When the dose of S. typhimurium is reduced 600-fold, 15-25% of the hypophysectomized rats survive, and the survival rate is significantly enhanced by administration of tetracycline, recombinant interferon gamma (IFN-gamma), or recombinant growth hormone (GH). The protective effect of GH is abolished by heat inactivation or with an antibody to GH. Spleens from normal and hypophysectomized rats treated with tetracycline, IFN-gamma, or GH have 59-99% fewer bacteria 5 days after infection as compared to control rats. Peritoneal macrophages from hypophysectomized rats that are infected in vitro with S. typhimurium kill half as many extracellular bacteria as compared to pituitary-intact rats, and this bactericidal capacity is significantly augmented 75-95% by either GH or IFN-gamma. These data establish that the pituitary gland is essential for homeostasis during an infectious episode and that GH plays an important role in host resistance by augmenting the ability of macrophages to kill S. typhimurium.

Hypophysectomy inhibits the synthesis of tumor necrosis factor alpha by rat macrophages: partial restoration by exogenous growth hormone or interferon gamma.

We recently demonstrated that GH and interferon-gamma (IFN gamma) act in a similar manner to prime macrophages in vitro and in vivo for enhanced superoxide anion release. In this report we investigated the physiological role of the pituitary gland and GH in in vivo priming of resident peritoneal macrophages for the synthesis of tumor necrosis factor-alpha (TNF alpha) in vitro. Compared to normal rats, hypophysectomized animals had an 83% reduction in macrophage production of TNF alpha after in vitro stimulation with lipopolysaccharide. Sham operation had no significant effect on the ability of macrophages to secrete TNF alpha in response to lipopolysaccharide. Both native pituitary-derived porcine GH (48 micrograms/rat.9 days) and native pituitary-derived rat GH (96 micrograms/rat.9 days) more than tripled the in vitro production of TNF alpha by macrophages from hypophysectomized rats (342 and 358 vs. 112 U/mg protein for placebo-treated rats, respectively). Each of these preparations of GH also increased growth more than 6-fold in hypophysectomized rats (32 and 30 g vs. 5 g in placebo controls). Heat inactivation of native pituitary-derived porcine GH significantly reduced its in vivo ability to augment both TNF alpha synthesis by macrophages and body growth. Recombinant rat IFN gamma (2000 U/rat.9 days) more than tripled the production of TNF alpha by macrophages from hypophysectomized rats (343 vs. 112 U/mg protein). In contrast to its in vivo effects, addition of GH in vitro to macrophages from hypophysectomized rats did not prime these cells for the synthesis of TNF alpha, indicating an indirect mechanism of action for GH. To further test the biological relevancy of GH with respect to synthesis of TNF alpha, hemorrhagic necrosis of TNF alpha-sensitive murine methyl-cholanthrene-induced tumors was assessed in pituitary-intact mice. Native porcine GH (133 micrograms/mouse.7 days) significantly augmented both the necrosis to tumor ratio and the hemorrhage to tumor ratio. These findings establish the physiological relevance of the pituitary gland and GH in the priming of macrophages for TNF alpha synthesis.

In vivo effects of recombinant interferon-gamma: augmentation of endotoxin-induced necrosis of tumors and priming of macrophages for tumor necrosis factor-alpha production.

Recombinant interferon-gamma (rIFN-gamma) is currently undergoing clinical trials in cancer patients. In this paper, we assessed the in vivo antitumor effects of this lymphokine in rodents. Recombinant murine IFN-gamma or control medium was injected intraperitoneally for 5 days into mice with subcutaneous Meth A tumors. An injection of a suboptimal dose of endotoxin (2 micrograms) on the fifth treatment day caused significant necrosis of tumors in the IFN-gamma-treated group while causing essentially no necrosis of tumors in the control group. Next, we examined macrophages isolated from rats treated for 9 days with either IFN-gamma or saline. Endotoxin stimulated release of significantly higher amounts of TNF-alpha from macrophages from the IFN-gamma-treated group compared to macrophages from the control group. A polyclonal antiserum against recombinant murine TNF-alpha abrogated all of the TNF cytotoxic activity from these rat macrophage supernatants, while control rabbit serum had no effect. These results provide strong evidence that rIFN-gamma can prime macrophages in vivo for TNF-alpha synthesis.

Serum protease inhibitor abrogation of Newcastle disease virus enhancement of cytolysis by recombinant tumor necrosis factors alpha and beta.

Newcastle disease virus (NDV) has been used to induce regression of tumors in human cancer patients. We recently demonstrated that human malignant melanoma cells resistant to the lytic effects of tumor necrosis factor-alpha (TNF-alpha) become susceptible after treatment with NDV. We examined the effects of a serine protease inhibitor, N-1-tosylamide-2-phenyl-ethyl-chloromethyl ketone (TPCK), on viral enhancement of TNF cytotoxicity. Virulent NDV (but neither heat- nor UV-inactivated NDV) induced a 100-fold increase in the sensitivity of murine fibroblast L929 cells to recombinant human TNF-alpha (rHuTNF-alpha), rHuTNF-beta, and recombinant murine TNF-alpha (rMuTNF-alpha). TPCK, which is an inhibitor of chymotrypsin-like proteases, blocked between 42% and 93% of the cytolytic activity of rMuTNF-alpha, rHuTNF-alpha, and rHuTNF-beta toward NDV-treated L929 cells. Similarly, TPCK abrogated 62% of the cytotoxicity of rMuTNF-alpha toward dactinomycin-treated L929 cells. In contrast, TPCK had no effect on WEHI 164 clone 13 cells, a murine fibrosarcoma cell line that is much more sensitive to the lytic effects of TNF and does not show enhanced sensitivity to TNF after treatment with either NDV or dactinomycin. These results suggest a role for a cellular protease in the mechanism by which some viruses sensitize tumor cells to the cytolytic activity of TNF.

Spectroscopic and quantitative analysis of the oxygenated and peroxy states of the purified cytochrome d complex of Escherichia coli.

Oxygenated and peroxy states of the cytochrome d complex of Escherichia coli have been proposed as intermediates in the reaction mechanism of this ubiquinol oxidase. In this report, several stable states of the purified enzyme were examined spectroscopically at room temperature. As purified, the cytochrome d complex exists in an oxygenated state characterized by an absorbance band at 650 nm. Removal of oxygen results in loss of absorbance at this wavelength, which is restored upon the return of oxygen. The presence of one oxygen molecule in the oxygenated state was quantified by measuring oxygen released when excess hydrogen peroxide was added to the oxygenated state by passage of argon generates a "partially reduced" state with an absorbance peak at 628 nm, apparently due to reduced cytochrome d. Addition of equimolar hydrogen peroxide to the fully oxidized state produces the peroxy state. This peroxy state is also formed upon addition of excess hydrogen peroxide to the oxygenated state via a stable intermediate termed "peroxy intermediate." It is likely that 1) the oxygenated state consists of one molecule of oxygen bound to reduced heme d, and 2) there are at least two stable states that have bound peroxide at room temperature, the peroxy state and a newly discovered peroxy intermediate.

Newcastle disease virus as an antineoplastic agent: induction of tumor necrosis factor-alpha and augmentation of its cytotoxicity.

The oncolytic strain 73-T of Newcastle disease virus (NDV) has been reported to be beneficial in the treatment of cancer patients, but little is known about its mechanism of action. In this study, NDV strain 73-T and a wild-type isolate of NDV were found to be potent inducers of tumor necrosis factor (TNF) production by both human peripheral blood mononuclear cells (PBMCs) and rat splenocytes. Antibody inhibition experiments identified TNF-alpha as the major species of TNF induced by NDV in PBMCs. The effect of recombinant human TNF-alpha (rHuTNF-alpha) on human cancer cells was then examined. Neither rHuTNF-alpha nor supernatants from NDV-stimulated PBMCs were cytotoxic toward the TNF-resistant human malignant melanoma cell line MEL-14. However, when MEL-14 cells were treated with NDV strain 73-T, both rHuTNF-alpha and supernatants from NDV-stimulated PBMCs killed 48% and 55%, respectively, of these tumor cells. Treatment with NDV also conferred TNF susceptibility to the TNF-resistant human malignant melanoma cell line MEL-21 and the human myelogenous leukemia cell line K562. In contrast to its enhanced cytotoxicity toward NDV-treated cancer cells, rHuTNF-alpha had no effect on NDV-treated normal human PBMCs proliferating in response to concanavalin A. These results suggest two important mechanisms for the antineoplastic activity of NDV: (a) induction of TNF-alpha secretion by human PBMCs and (b) enhancement of the sensitivity of neoplastic cells to the cytolytic effects of TNF-alpha.

Trypsin proteolysis of the cytochrome d complex of Escherichia coli selectively inhibits ubiquinol oxidase activity while not affecting N,N,N',N'-tetramethyl-p-phenylenediamine oxidase activity.

The cytochrome d complex is one of two membrane-bound terminal oxidases of the Escherichia coli aerobic respiratory chain. Previous studies have shown that this enzyme reconstituted into proteoliposomes rapidly oxidizes ubiquinol-8 as well as the soluble homologue, ubiquinol-1, and that quinol oxidase activity is accompanied by the formation of a transmembrane H+ electrochemical gradient. The enzyme also oxidizes the artificial reductant, N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) with the generation of a H+ electrochemical gradient. In this work, it is established that trypsin digestion of the purified cytochrome d complex cleaves subunit I while subunit II is unaffected. Proteolysis of subunit I is correlated with loss of ubiquinol-8 and ubiquinol-1 oxidase activities. Trypsin digestion has no effect on TMPD oxidase activity. The cytochrome d complex is concluded to possess three distinct active sites for 1) ubiquinol oxidation, 2) TMPD oxidation, and 3) oxygen binding and reduction. Data also suggest that both sites of ubiquinol and TMPD oxidations are located on the periplasmic side of the E. coli membrane while the site of oxygen reduction is on the opposite side.

Cytochrome b558 monitors the steady state redox state of the ubiquinone pool in the aerobic respiratory chain of Escherichia coli.

The aerobic respiratory chain of Escherichia coli contains two terminal oxidases, the cytochrome o complex and the cytochrome d complex. These both function as ubiquinol-8 oxidases and reduce molecular oxygen to water. Electron flux is funneled from a variety of dehydrogenases, such as succinate dehydrogenase, through ubiquinone-8, to either of the terminal oxidases. A strain was examined which lacks the intact cytochrome d complex, but which overproduces one of the two subunits of this complex, cytochrome b558. This cytochrome, in the absence of the other subunit of the oxidase complex, does not possess catalytic activity. It is shown that the extent of reduction of cytochrome b558 in the E. coli membrane monitors the extent of reduction of the quinone pool in the membrane. The activity of each purified oxidase was examined in phospholipid vesicles as a function of the amount of ubiquinone-8 incorporated in the bilayer. A ratio of ubiquinol-8:phospholipid as low as 1:200 is sufficient to saturate each oxidase. The maximal turnover of the oxidases in the reconstituted system is considerably faster than observed in E. coli membranes, demonstrating that the rate-limiting step in the E. coli respiratory chain is at the dehydrogenases which feed electrons into the system.

Use of an azido-ubiquinone derivative to identify subunit I as the ubiquinol binding site of the cytochrome d terminal oxidase complex of Escherichia coli.

The radiolabeled, photoreactive azido-ubiquinone derivative (azido-Q), 3-azido-2-methyl-5-methoxy-6-(3,7-dimethyl-[3H]octyl)- 1,4-benzoquinone, was used to investigate the active site of ubiquinol oxidase activity of the cytochrome d complex, a two-subunit terminal oxidase of Escherichia coli. The azido-Q, when reduced by dithioerythritol, was shown to support enzymatic oxygen consumption by the cytochrome d complex that was 8% of the rate observed with ubiquinol-1. This observation provided the rationale behind further studies of the possible photoinactivation and labeling of the active site by this azido-Q. Ten min of photolysis of the purified cytochrome d complex in the presence of the azido-Q resulted in a 60% loss of the ubiquinol-1 oxidase activity. Uptake of the radiolabeled azido-Q by the cytochrome d complex was correlated to the photoinactivation of the ubiquinol-1 oxidase activity. Both increased linearly during the first 4 min of photolysis and reached 90% of the maximum within 10 min. Photolysis times longer than 10 min resulted in no increase in the maximum of 2 mol of azido-Q incorporated per mol of enzyme. The rate of azido-Q uptake by subunit I, but not subunit II, correlated well with the rate of loss of ubiquinol oxidase activity. Use of ubiquinol-0, which is not oxidized by the enzyme, to competitively inhibit radiolabeling of nonspecific binding sites, resulted in a significant decrease (42%) of azido-Q labeling of subunit II while it did not affect the labeling of subunit I. After photolysis for 4 min, the ratio of radiolabeled azido-Q in subunits I to II of the complex was 4.3 to 1.0. These observations support the conclusion that the ubiquinol substrate binding site is located on subunit I of the cytochrome d complex.

Specific overproduction and purification of the cytochrome b558 component of the cytochrome d complex from Escherichia coli.

In Escherichia coli strain GR84N[pNG10], the cloned gene for subunit I of the membrane-bound cytochrome d complex resulted in the overproduction of cytochrome b558 and facilitated purification of this cytochrome. Extracting membranes with 1% Triton X-100 followed by two chromatographic steps yielded a single band on sodium dodecyl sulfate-polyacrylamide gels corresponding to subunit I (Mr 57 000). Purified cytochrome b558 was in its native state as determined by difference absorption spectroscopy and by potentiometric analysis. Both the membranes of strain GR84N[pNG10] and the purified subunit I lacked the other two spectroscopically defined cytochromes, b595 (previously "a1") and d, of the cytochrome d complex. Reconstitution of cytochrome b558 in phospholipid vesicles demonstrated that cytochrome b558 can be reduced by ubiquinol but that it does not reduce molecular oxygen. Heme extraction of cytochrome b558 yielded an extinction coefficient of 22 000 M-1 cm-1 for the wavelength pair of 560 and 580 nm in the reduced-minus-oxidized spectrum. The mutation on pNG10 that eliminates subunit II was mapped to a 250 base pair DNA fragment.

Coulometric and spectroscopic analysis of the purified cytochrome d complex of Escherichia coli: evidence for the identification of "cytochrome a1" as cytochrome b595.

Coulometric and spectroscopic analyses were performed on the three cytochrome components (cytochrome d, cytochrome b558, and the cytochrome previously described as cytochrome a1) of the purified cytochrome d complex, a terminal oxidase of the Escherichia coli aerobic respiratory chain. On the basis of heme extraction, spectroscopic, and coulometric data, the "cytochrome a1" component was identified as a b-type cytochrome: cytochrome b595. The pyridine hemochromogen technique revealed the presence of two molecules of protoheme IX per cytochrome d complex. This quantity of protoheme IX fully accounted for the sum of the cytochrome b558 and cytochrome b595 components as determined coulometrically. The renaming of cytochrome a1 as cytochrome b595 was further indicated by the lack of any heme a in the complex and by its resolved reduced-minus-oxidized spectrum. The latter was found to be similar to that of cytochrome c peroxidase, which contains protoheme IX. Coulometric titrations and carbon monoxide binding titrations revealed that there are two molecules of cytochrome d per complex. A convenient measurement of the amount of cytochrome b558 was found to be the beta-band at 531 nm since cytochrome b558 was observed to be the only component of the cytochrome d complex with a peak at this wavelength. By use of this method and the extinction coefficient for the purified cytochrome b558, it was estimated that there is one molecule of cytochrome b595 and one of cytochrome b558 per cytochrome complex.

Isolation and characterization of an Escherichia coli mutant lacking the cytochrome o terminal oxidase.

A respiration-deficient mutant of Escherichia coli has been isolated which is unable to grow aerobically on nonfermentable substrates such as succinate and lactate. Spectroscopic and immunological studies showed that this mutant lacks the cytochrome o terminal oxidase of the high aeration branch of the aerobic electron transport chain. This strain carries a mutation in a gene designated cyo which is cotransducible with the acrA locus. Mutations in cyo were obtained by mutagenizing a strain that was cyd and, thus, was lacking the cytochrome d terminal oxidase. Strain RG99, which carries both the cyd- and cyo- alleles, grows normally under anaerobic conditions in the presence of nitrate. Introduction of the cyd+ allele into the strain restores the respiration function of the strain, indicating that the cytochrome o branch of the respiratory chain is dispensable under normal laboratory growth conditions.

Effects of pH and detergent on the kinetic and electrochemical properties of the purified cytochrome d terminal oxidase complex of Escherichia coli.

The cytochrome d terminal oxidase complex is one of the two terminal oxidases in the aerobic respiratory chain of Escherichia coli. In this paper, effects of pH and detergent on the electrochemical and kinetic properties of the enzyme are investigated. There are two significant conclusions. (1) The oxidation-reduction midpoint potential of the cytochrome b-558 component is sensitive to the detergent used to solubilize the complex. In particular, it is shown that octylglucoside and cholate cause a large decrease in the midpoint potential of cytochrome b-558, while they also result in the reversible inactivation of the oxidase. (2) The midpoint potentials of the cytochrome b-558, a1 and d components are sensitive to pH. More acidic solutions result in stabilizing the reduced forms of the redox-active groups, i.e., raising their midpoint potentials. This may be significant in view of the fact that it has been demonstrated that this enzyme catalyzes an electrogenic reaction and appears to function as a proton pump.

Identification of subunit I as the cytochrome b558 component of the cytochrome d terminal oxidase complex of Escherichia coli.

The cytochrome d terminal oxidase complex was recently purified from Escherichia coli membranes (Miller, M. J., and Gennis , R. B. (1983) J. Biol. Chem. 258, 9159-1965). The complex contains two polypeptides, subunits I and II, as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and three spectroscopically defined cytochromes, b558 , a1, and d. A mutant that failed to oxidize N,N,N',N'-tetramethyl-p-phenylenediamine was obtained which was lacking this terminal oxidase complex and was shown to map at a locus called cyd on the E. coli genome. In this paper, localized mutagenesis was used to generate a series of mutants in the cytochrome d terminal oxidase. These mutants were isolated by a newly developed selection procedure based on their sensitivity to azide. Two classes of mutants which map to the cyd locus were obtained, cydA and cydB . The cydA phenotype included the lack of all three spectroscopically detectable cytochromes as well as the absence of both polypeptides, determined by immunological criteria. Strains manifesting the cydB phenotype lacked cytochromes a1 and d, but had a normal amount of cytochrome b558 . Immunological analysis showed that subunit I (57,000 daltons) was present in the membranes, but that subunit II (43,000 daltons) was missing. These data justify the conclusion that subunit I of this two-subunit complex can be identified as the cytochrome b558 component of the cytochrome d terminal oxidase complex.