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Biomedical subjects

J S Lazo

Publications and source records attributed to J S Lazo.

At least 127 records · Page 7Linked to original sources
PubMed1990

Disposition of radioiodinated benzylguanidines in perfused rabbit lung: pharmacokinetics and effect of an organo-gold complexed antineoplastic agent.

SK&F 104524 (bis-[1,2-bis(diphenylphosphino)-ethane]-gold(I)-lactate) ([Au(dppe)2]+) is a lipophilic phosphine-coordinated gold complex that has significant antineoplastic activity. However, this agent has significant hepato- and cardiotoxicity and preclinical preliminary experiments indicate potential pneumotoxic effects. Accordingly, we sought to evaluate the acute effects of [Au(dppe)2]+ on pulmonary removal of two radioiodinated benzylguanidines [metaiodobenzylguanidine (MIBG) and 3-iodo-4-amino-benzylguanidine (AIBG)]. Pulmonary removal of MIBG or AIBG was measured by application of indicator dilution techniques in rabbit lungs, perfused with Kreb's bicarbonate (3% albumin; 50 ml/min). A bolus containing either [125I]MIBG or--AIBG and [99mTc]sulfur colloid was injected into the pulmonary artery and effluent was diverted to an in-line nuclear detection system. In initial experiments we characterized some pharmacokinetic aspects of the disposition of these amines. Removal of MIBG [R(MIBG)] and R(AIBG) were 59 +/- 3% (n = 14) and 24 +/- 2% (n = 7), respectively. Addition of unlabeled norepinephrine, MIBG or AIBG (3-100 nmol) to the injection caused similar dose-dependent reductions in removal of either amine. Lowering the temperature (13 degrees C) or reducing the sodium concentration caused significant reductions in removal. After 15 min of perfusion with [Au(dppe)2]+ (50 micrograms/ml), perfusion pressure doubled and R(MIBG) and R(AIBG) were each decreased significantly. At 30 min, perfusion pressure returned to control values whereas there was still a 36 and 61% reduction in R(MIBG) and R(AIBG), respectively. These data confirm that removal of benzylguanidines involves a saturable, sodium- and temperature-dependent process similar to that responsible for the removal of norepinephrine.(ABSTRACT TRUNCATED AT 250 WORDS)

3-Iodobenzylguanidine↗
PubMed1989

Bleomycin hydrolase: molecular cloning, sequencing, and biochemical studies reveal membership in the cysteine proteinase family.

Bleomycin (BLM) hydrolase catalyzes the inactivation of the antitumor drug BLM and is believed to protect normal and malignant cells from BLM toxicity. The normal physiological function of BLM hydrolase is not known. We now provide evidence for its membership in the cysteine proteinase family. BLM hydrolase was purified to homogeneity from rabbit lungs, and a partial amino acid sequence was determined from a tryptic digest peptide. On the basis of this sequence a 36-mer oligonucleotide was synthesized. The 36-mer oligonucleotide probe hybridized to a single mRNA species of 2.5 kb from several species and was used to isolate an 832-bp cDNA insert from a lambda gt11 rabbit liver cDNA library. This insert encoded the tryptic digest peptide previously identified in rabbit lung BLM hydrolase by amino acid sequencing. Analysis of the predicted amino acid sequence coded by the 832-bp BLM hydrolase cDNA fragment indicated no significant homology with any currently known proteins except for a 15 amino acid portion, which displayed remarkable homology with the active site of cysteine proteinases. Within this active-site region, 10 of the amino acid residues of papain and 9 of aleurain, cathepsin H, and cathepsin L were identical with those of rabbit liver BLM hydrolase. The catalytic cysteine of thiol proteinases was also conserved in BLM hydrolase, and cysteine proteinase specific inhibitors, such as E-64, were found to be potent inhibitors of BLM hydrolase activity. Furthermore, bleomycin hydrolase exhibited cathepsin H like enzymatic activity. Bleomycin hydrolase had, however, no significant cathepsin B or L activities.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1989

Initial single-strand DNA damage and cellular pharmacokinetics of bleomycin A2.

The cellular association and fate of high specific activity [3H]bleomycin A2 (BLM A2) were examined in three previously untreated cultured cell lines. Human head and neck A-253 carcinoma cells were 10-fold more sensitive to a 1-hr exposure to BLM A2 than either murine leukemic L1210 or human ovarian SK-OV cells. Both murine and human cells displayed rapid drug association with steady-state drug levels being reached within 15-30 min. At steady state, the T1/2 of drug dissociation was slow (between 65 and 155 min), unaltered by 100-fold excess of unlabeled BLM A2, and unrelated to cellular sensitivity to BLM. Approximately 15% of the total cellular drug was found in the nuclei at steady state. In intact cells, BLM hydrolase activity appeared latent; significant BLM hydrolase activity was detected using broken cell homogenates with all cell types, but no extensive drug metabolism was evident in intact cells. Murine L1210 cells differed from both human cell lines in that they had only 50% of the steady-state drug levels, had lower nuclear drug content, and had markedly less initial single-strand DNA damage. Human SK-OV cells had 2.4-fold greater initial single-strand DNA damage despite similar nuclear content and a much lower rate of DNA repair. Thus, cellular or nuclear factors, in addition to BLM A2 content, affect initial single-strand DNA damage. Collectively, our data support the proposition that lesions other than single-strand DNA breaks contribute to the cytotoxicity of BLM.

Animals↗
PubMed1989

Substrate specificity of bleomycin hydrolase.

Bleomycin (BLM) hydrolase is believed to protect both malignant and normal tissue from the toxicity of the antitumor drug BLM. Little is known about the substrate specificity of BLM hydrolase. Thus, we developed ion-paired reverse phase high speed liquid chromatography systems to assay for the metabolism of several BLM analogs. We found that BLM A2, BLM B2, tallysomycin S10b (TLM S10b), peplomycin (PEP), butylamino-3-propylamino-3-propylamine bleomycin (BAPP), deglyco bleomycin A2 (dgBLM A2) and bleomycinic acid were each metabolized by rabbit lung BLM hydrolase to a single metabolite. When compared to their corresponding parent compounds, these metabolites were 6- to 35-fold less potent in their ability to inhibit the proliferation of A-253 human head and neck squamous carcinoma cells in culture. Furthermore, we found that substitutions in various regions of the BLM molecule greatly affected the kinetic parameters of BLM hydrolase. For example, the Km with BLM B2 (0.056 +/- 0.005 mM) was 15-fold lower than that seen with BLM A2 (0.83 +/- 0.11 mM). In contrast, the Vmax was not affected markedly by these terminal amine substitutions but was influenced greatly by deletion of the carbohydrate groups of BLM. For example, a 4-fold higher Vmax was observed with dgBLM A2 compared to BLM A2. Thus, these results demonstrate that BLM hydrolase can recognize and metabolize a broad spectrum of BLM analogs regardless of their structural features. This enzymatic conversion resulted in the inactivation of the BLMs as demonstrated by a substantial decrease in their cytotoxicity. Furthermore, the terminal amine and carbohydrate regions, respectively, dictate the apparent affinity and the rate of metabolism of BLM hydrolase substrates.

Animals↗
PubMed1989

Characteristics of bleomycin-resistant phenotypes of human cell sublines and circumvention of bleomycin resistance by liblomycin.

Three bleomycin (BLM)-resistant sublines were isolated from a human head and neck squamous cell carcinoma cell line (A-253); these sublines (C-10, D-10, and G-11) were 4-, 9-, and 21-fold resistant to BLM A2, respectively. These sublines were selectively resistant to other members of the BLM class, namely BLM B2, peplomycin, talisomycin S10b, and bleomycinic acid; none of the sublines displayed cross-resistance to vincristine, doxorubicin, cis-diamminedichloroplatinum or melphalan; only one subline (G-11) was cross-resistant to X-irradiation. None of the BLM-resistant cell lines demonstrated resistance to the novel BLM analogue liblomycin, which contains a lipophilic terminal amine. The cell cycle distributions of the clonally derived BLM-resistant cell populations were similar to the distribution of the parental cell population. In vitro BLM hydrolase activity in homogenates of D-10 and G-11 BLM-resistant cell lines was two- to threefold higher than that in homogenates of A-253 or C-10 cells. Nonetheless, no deamido BLM A2 was found associated with any cell type or in the culture medium and more than 80% of the radioactivity in all cells appeared as unmetabolized BLM A2 by high pressure liquid chromatography. Thus, the appearance of large quantities of the deamido BLM metabolite was not a prominent feature of acquired resistance to BLM in these human tumor cells. The cellular accumulation of radiolabeled BLM A2 by C-10 and G-11 cells during a 1-h incubation with [3H]BLM A2 was 1/2 that seen with A-253 and D-10 cells. C-10 cells maintained a lower nuclear content of radioactivity than A-253, G-11, or D-10 cells. Initial single strand DNA damage, based upon alkaline elution analysis, also was lower in C-10 cells compared to A-253 cells. D-10 cells, in contrast, exhibited high initial genomic DNA damage but demonstrated a greater repair rate than either A-253 or C-10 cells. Thus, multiple BLM-resistant phenotypes can be obtained from a population of human squamous carcinoma cells, and modification of the terminal amine in the BLM molecule can produce compounds capable of circumventing all of these BLM-resistant phenotypes. Liblomycin, which appears to be a nonclassical BLM, may be a useful therapeutic agent with a spectrum of activity distinct from other members of the BLM class.

Antineoplastic Agents↗
PubMed1989

Phase I trial of combined therapy with bleomycin and the calmodulin antagonist, trifluoperazine.

Calmodulin antagonists, such as trifluoperazine, can enhance the cytotoxic effects of bleomycin both in tissue culture and in vivo. Therefore, we evaluated the effects of combination treatment with these drugs in a phase I clinical trial. Patients with objectively measurable or evaluable cancer refractory to conventional treatment who had an acceptable performance status (ECOG 0-2) and acceptable laboratory studies were eligible. All patients gave written informed consent. A cycle of therapy consisted of three weekly treatments with trifluoperazine (days 1-4) and 30 IU bleomycin (day 3). After three patients completed a cycle of therapy without experiencing dose-limiting toxicity, new patients were entered in the study and received a higher dose of trifluoperazine. The dose of bleomycin remained constant. Evaluable patients received at least 2 weeks of treatment and survived for 6 weeks; of 19 patients, 2 were unevaluable. The major toxicities were neurological and pulmonary and included one case of fatal pneumonia with interstitial pulmonary fibrosis. There was no hematologic toxicity. Two patients underwent partial responses (PRs) and two had complete responses (CRs). We conclude that trifluoperazine can safely be given with bleomycin and that further study of the potential efficacy of this treatment is indicated.

Adult↗
PubMed1989

Pharmacological modulators of DNA-interactive antitumor drugs.

The poor therapeutic index and limited efficacy of current cancer chemotherapeutic agents represent an important pharmacological problem. Although there has been a significant increase in our understanding of the mechanisms by which anticancer drugs kill mammalian cells, identification of new, effective anticancer agents during the last decade has been exceeding slow. Thus, attention has focused on understanding the causes of drug resistance and on either sensitizing tumor cells to existing anticancer agents using what could be called 'chemoenhancers', or protecting non-malignant tissues against serious untoward effects using 'chemoprotectors'. John Lazo and Robert Bahnson review recent strategies attempting to modulate the activity of antineoplastic drugs.

Antineoplastic Agents↗
PubMed1989

Immobilized adriamycin: toxic potential in vivo and in vitro.

We report experiments which test the toxicity of a new potential therapeutic agent, agarose-bound adriamycin (ImA). In C57Bl/6N mice this preparation is almost completely devoid of untoward effects when administered intraperitoneally; ImA lacks all the usual toxic repercussions of free adriamycin including abdominal adhesions, inflammatory peritonitis, weight loss and cardiotoxicity. The immobilized adriamycin is also inactive in a fetal mouse heart model of cardiac toxicity. This lack of toxicity is not due to an intrinsic inactivity of the drug, however, since previous studies have shown that polymer-bound adriamycin can kill actively dividing cells. We also show here that the immobilized drug can undergo redox reactions and interact with enzymes from isolated respiratory chain preparations, so the lack of cardiac toxicity in vivo is most likely due to inaccessibility of the target. These results suggest that polymer immobilized adriamycin lacks the toxicity of the parent compound and may present a useful approach to regional chemotherapy.

Animals↗
PubMed1989

Translocation of protein kinase C is associated with inhibition of 5-HT uptake by cultured endothelial cells.

Acute exposure (30 min-1 h) of bovine pulmonary artery endothelial cells in culture (BPAEC) to phorbol myristate acetate (PMA 10(-10)-10(-6) M) resulted in concentration-dependent decrement in serotonin (5-HT) uptake. Neither cell viability (trypan blue exclusion or release of deoxyglucose) nor activity of another plasma membrane function, angiotensin-converting enzyme activity, were affected. A decrease in 5-HT uptake was also noted with phorbol 12,13 dibutyrate and mezerein, but not with 4-alpha-phorbol 12,13 didecanoate, which does not stimulate protein kinase C (PKC). Inhibition of 5-HT uptake by PMA (160 nM) was reversed in a concentration-dependent manner by pretreatment of cells with the PKC inhibitor staurosporine (3-100 nM). BPAEC were treated with PMA (160 nM) for 1 h, and activities of PKC in cytosolic and membrane compartments were determined. PMA did not significantly affect total cellular PKC activity but resulted in a translocation of activity from cytosol to membrane (control membrane activity 67 +/- 4%; PMA-treated membrane activity 97 +/- 1% of total cellular PKC). Thus we propose that translocation of PKC from cytosol to membrane results in inhibition of 5-HT uptake by BPAEC.

Animals↗
PubMed1989

Acute pulmonary toxicity of bleomycin: DNA scission and matrix protein mRNA levels in bleomycin-sensitive and -resistant strains of mice.

The severity of bleomycin (BLM)-induced pulmonary fibrosis in mice varies markedly among several different murine strains. We have examined the DNA from lungs of sensitive (i.e., C57BL/6N) and resistant (i.e., BALB/c) strains of mice using a nucleoid sedimentation technique to detect early in vivo changes in the integrity of DNA after intravenous BLM. Mice received intravenous injections of BLM (80 mg/kg) or vehicle; lung nucleoids were prepared 15 min to 6 hr later. BLM produced striking decreases in nucleoid sedimentation distance versus paired controls in both strains within 15 min after injection, indicating extensive DNA scission. Repair of DNA strand breaks was complete in the resistant (BALB/c) mice by 5 hr; in contrast, only partial repair occurred in the sensitive (C57BL/6N) strain during that time. We then examined lungs for subsequent changes in steady state poly-(A)+ RNA levels and mRNA levels for lung matrix proteins (type I procollagen, type III procollagen, and fibronectin). Steady state levels of poly-(A)+ RNA were depressed to 50% of control 1 through 6 days after BLM injection in the lungs of sensitive mice. Resistant mice had pulmonary poly-(A)+ RNA levels similar to those of C57BL/6N mice, except for a 2-fold elevation 1 day after BLM injection. BLM injection affected the steady state levels of mRNA encoding lung matrix proteins differently than total poly-(A)+ RNA. Fibronectin mRNA/poly(A)+ RNA was elevated 2-fold 1 day after BLM treatment only in the sensitive strain and remained elevated at 3 and 6 days. In contrast, alpha 2I procollagen mRNA increased in both murine strains and alpha 1III procollagen mRNA decreased in both strains. Thus, a 7-fold or greater increase in the type I: type III procollagen mRNA ratio was seen in both strains 3 to 6 days after BLM injection. These data demonstrate that BLM treatment rapidly produces extensive pulmonary DNA damage in vivo, that persistence of DNA damage rather than the initial level of strand scission is associated with sensitivity to BLM lung disease in these mice, and that changes in the levels of mRNA encoding pulmonary matrix proteins occur in vivo within 1 to 3 days after intravenous BLM treatment.

Animals↗
PubMed1989

Early increases in pulmonary mRNA encoding procollagens and transforming growth factor-beta in mice sensitive to cyclophosphamide-induced pulmonary fibrosis.

Pulmonary fibrosis was induced 7 weeks after a single i.p. injection of cyclophosphamide (200 mg/kg b.wt.) in BALB/c mice; C57Bl/6 mice were unaffected. There was a corresponding strain variation in the effects of cyclophosphamide on levels of pulmonary mRNA encoding alpha 2I and alpha 1III procollagen, and transforming growth factor-beta. In BALB/c mice, the ratios of alpha 2I and alpha 1III procollagen mRNA to polyadenylated RNA were increased 1 week after cyclophosphamide injection. No increases in levels of either procollagen mRNA occurred in C57Bl/6 mice. The ratio of fibronectin mRNA to polyadenylated RNA was elevated to a similar extent in both murine strains during the 1st week after cyclophosphamide treatment. The pulmonary content of transforming growth factor-beta mRNA and its ratio to polyadenylated RNA increased 2-fold at 1 and 2 weeks in BALB/c but not C57Bl/6 mice. Thus, collagen accumulation in cyclophosphamide-sensitive mice is preceded by increased pulmonary alpha 2I and alpha 1III procollagen mRNA. The early strain selective elevation of transforming growth factor-beta mRNA in response to cyclophosphamide suggests a role, in vivo, for transforming growth factor-beta in drug-induced pulmonary fibrosis.

Animals↗
PubMed1988

Overexpression of metallothionein confers resistance to anticancer drugs.

Resistance to antineoplastic agents is the major obstacle to curative therapy of cancer. Tumor cell lines with acquired resistance to the antineoplastic agent cis-diamminedichloroplatinum(II) overexpressed metallothionein and demonstrated cross-resistance to alkylating agents such as chlorambucil and melphalan. Human carcinoma cells that maintained high levels of metallothionein because of chronic exposure to heavy metals were resistant to cis-diamminedichloroplatinum(II), melphalan, and chlorambucil. Furthermore, cells transfected with bovine papilloma virus expression vectors containing DNA encoding human metallothionein-IIA were resistant to cis-diamminedichloroplatinum(II), melphalan, and chlorambucil but not to 5-fluorouracil or vincristine. Thus, overexpression of metallothionein represents one mechanism of resistance to a subset of clinically important anticancer drugs.

Animals↗
PubMed1988

Antitumor and toxic effects of combination chemotherapy with bleomycin and a phenothiazine anticalmodulin agent.

Phenothiazines and structurally related calmodulin antagonists acted synergistically with bleomycin in killing malignant cells in culture. For exploration of the potential clinical importance of this observation, the effects of administering the combination of chlorpromazine and bleomycin in vitro and in vivo against the transplantable B16 melanoma were studied. The toxicity of the combination to bone marrow and lungs was also determined. Combined chlorpromazine and bleomycin therapy reduced the concentration of chlorpromazine required to inhibit cellular growth of B16 melanoma cells by greater than half (25 vs. 60 microM). Against the transplanted tumor, after 3 weeks of treatment the tumor sizes (in cubic millimeters) were 540 +/- 100 (vehicle), 520 +/- 120 (chlorpromazine), 170 +/- 10 (bleomycin), and 80 +/- 20 (bleomycin + chlorpromazine, P less than .01 vs. bleomycin alone). The median time required to reach a tumor size of 100 mm3 was 15 days for vehicle and chlorpromazine, 17 days for bleomycin, and 26 days for chlorpromazine and bleomycin together. The doubling time of the tumor was also increased 3.5-fold above control with the two-drug combination. At doses of bleomycin and chlorpromazine that increased antitumor activity, no pulmonary fibrosis, measured histologically and biochemically, was detected. At higher doses of bleomycin, the addition of chlorpromazine diminished lung fibrosis. The combination of drugs was not more toxic to hematopoietic precursors than chlorpromazine alone. These studies suggested that the addition of a phenothiazine to bleomycin can augment the therapeutic efficacy of bleomycin in vivo without substantially increasing its toxicity.

Animals↗
PubMed1988

Alterations in pulmonary protective enzymes following systemic bleomycin treatment in mice.

Repeated bleomycin administration in animals and humans produces significant lung fibrosis. The pathogenesis of this toxicity may be multifactorial, but it appears to be initiated through the production of radical oxygen species by an activated bleomycin-iron-oxygen ternary complex. Protection of lung tissue from bleomycin-induced toxicity may occur through both specific metabolic inactivation of bleomycin by the enzyme bleomycin hydrolase, as well as by such non-specific antioxidants as catalase and the glutathione system. The effect of chronic, systemic administration of bleomycin on the activities and levels of these enzymes and proteins in pulmonary tissue is unknown. C57BL/6 mice were injected subcutaneously with saline, non-fibrogenic (2 mg/kg) and fibrogenic (10 mg/kg) doses of bleomycin twice-weekly for 6 weeks. Animals were killed at 0, 1.5, 3, and 6 weeks after initiation of bleomycin treatment. Catalase activity was increased more than 50% at 3 weeks in the low-dose animals, and was decreased over 40% at 6 weeks in the high-dose animals. Total lung glutathione levels were unaffected in both groups, although glutathione reductase activity was increased significantly (over 2-fold) at 1.5 and 3 weeks in the high-dose animals. At 6 weeks glutathione reductase was increased 7- and 12-fold in low and high-dose animals respectively. Glutathione peroxidase activity also was elevated more than 2-fold above control values at 6 weeks in both sets of animals. There was no evidence of induction of bleomycin hydrolase activity at any time point. Rather, bleomycin hydrolase activity was decreased significantly to 30 and 40% of control values at 3 and 6 weeks, respectively, in mice receiving the fibrogenic doses of bleomycin. These results demonstrate that chronic, systemic administration of non-fibrogenic and fibrogenic doses of bleomycin produces changes in activity of lung antioxidant defense mechanisms. The early loss of lung bleomycin hydrolase activity may contribute to the pathogenesis of bleomycin-induced pulmonary toxicity following repeated drug exposure.

Animals↗
PubMed1988

Alterations in pulmonary mRNA encoding procollagens, fibronectin and transforming growth factor-beta precede bleomycin-induced pulmonary fibrosis in mice.

Female C57Bl/6 mice treated by constant s.c. infusion for 1 week with 100 mg of bleomycin per kg of body weight develop a more pronounced pulmonary fibrosis than BALB/c mice. Within 4 weeks after bleomycin treatment, the pulmonary content of mRNAs encoding fibronectin, alpha 2I procollagen and alpha 1III procollagen was increased. The increases were greater and occurred earlier in C57Bl/6 mice compared to BALB/c mice. Fibronectin mRNA increased 12-fold in C57Bl/6 mice and only 3-fold in BALB/c mice, whereas alpha 1III procollagen mRNA increased 4-fold in C57Bl/6 mice and 2-fold in BALB/c mice. alpha 2I procollagen mRNA was increased only in C57Bl/6 mice (2-fold). The increases were sequential in C57Bl/6 mice: fibronectin mRNA was elevated first, followed by alpha 2I procollagen, then alpha 1III procollagen mRNA. The temporal relationship between these mRNA elevations and extracellular matrix accumulation, and the exaggerated responses in C57Bl/6 mice, suggest that matrix accumulation is a function of the mRNA levels. Transforming growth factor-beta mRNA relative to total polyadenylated RNA was elevated 5-fold in C57Bl/6 mice and depressed 80% in BALB/c mice 1 week after treatment. Early alterations in transforming growth factor-beta mRNA may contribute to murine strain variation in bleomycin-induced pulmonary fibrosis and suggest the involvement of transforming growth factor-beta in this disease.

Animals↗
PubMed1988

Plasma and pulmonary pharmacokinetics of bleomycin in murine strains that are sensitive and resistant to bleomycin-induced pulmonary fibrosis.

Previous studies have shown that C57Bl/6N mice are sensitive and BALB/c mice are resistant to the pulmonary fibrotic effects of bleomycin (BLM). We assessed the plasma elimination and pulmonary content of BLM in C57Bl/6N and BALB/c mice treated with a single dose of [3H]BLM (80 mg/kg i.v.) to determine whether these murine strains show corresponding differences in BLM pharmacokinetics and pulmonary disposition after systemic administration of the drug. Serial blood samples were obtained from each animal and lungs were collected after pulmonary lavage or vascular perfusion with saline. Administration of BLM (80 mg/kg i.v.) produced significant elevations in lung hydroxyproline (35%) in C57Bl/6N but not in BALB/c mice. In contrast, BALB/c mice were more sensitive to pulmonary fibrosis induced with cyclophosphamide (200 mg/kg i.p.) compared to C57Bl/6N mice, indicating that strain sensitivity to pulmonary fibrosis is drug specific in these mice. BLM showed first order plasma elimination kinetics over 30 min in both strains with a shorter half-life in the sensitive strain (9.6 +/- 0.3 min in C57Bl/6N vs. 12.7 +/- 1.9 min in BALB/c). Plasma elimination deviated from first order kinetics after 30 min in both strains and plasma levels of BLM were up to 2-fold higher in the resistant strain over a 3-hr time course. Radioactivity in saline-perfused lungs was also significantly higher (1.5-2-fold) in BALB/c mice for least 1 hr after BLM injection. A similar fraction of the total lung radioactivity (approximately 80%) was recovered from both strains by pulmonary lavage, suggesting that BLM enters the alveolar spaces relatively freely in each strain.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

Purification, characterization, and amino acid composition of rabbit pulmonary bleomycin hydrolase.

Bleomycin (BLM) hydrolase, a protective enzyme that inactivates the antitumor antibiotic BLM, was purified (6000-fold) to homogeneity from rabbit lungs by DEAE-Sephacel, phenyl-Sepharose chromatography, BLM-Sepharose affinity chromatography, and Mono Q fast protein liquid chromatography. The enzyme had a molecular mass of 250,000 daltons as demonstrated by Superose gel permeation chromatography and polyacrylamide gel electrophoresis (PAGE) under native conditions. Sodium dodecyl sulfate-PAGE revealed a single band of 50,000 daltons, suggesting a pentameric structure. The Km and Vmax for BLM A2 were 1.3 mM and 5.9 mumol mg-1 h-1, respectively. BLM hydrolase activity was labile, had a half-life of 25 min at 56 degrees C, 10 h at 37 degrees C, and 5 days at 4 degrees C, and was stabilized by 2 mM dithiothreitol. The enzyme had a pH optimum of 7.0-7.5 and was inhibited by N-ethylmaleimide, leupeptin, puromycin, and divalent cations such as Cu2+, Cd2+, Zn2+, and Co2+ but was unaffected by chelating agents. On the basis of Mono P chromatofocusing chromatography, three isoforms of BLM hydrolase (apparent pI's of 5.3, 4.5, and 4.3) were present in rabbit pulmonary cytosol. The elution profiles of BLM hydrolase from phenyl-Sepharose and Mono P chromatofocusing indicated that this enzyme is hydrophobic and acidic. This was confirmed by amino acid composition analysis, which demonstrated that 48% of the total amino acids of bleomycin hydrolase were hydrophobic and 37% were acidic.

Amino Acids↗