Sodium Dodecyl Sulfate-urea Polyacrylamide Gel Research Articles

Mechanisms of ertapenem resistance in Enterobacteriaceae isolates in a tertiary university hospital

The aim of this study was to investigate the molecular mechanisms of ertapenem resistance among Enterobacteriaceae isolates in a clinical microbiology laboratory at a tertiary university hospital. A total of...

Detection of plasmid-mediated IMP-1 metallo-β-lactamase and quinolone resistance determinants in an ertapenem-resistant Enterobacter cloacae isolate

To investigate the mechanism of carbapenem resistance and the occurrence of plasmid-mediated quinolone resistance determinants qnr and aac(6')-Ib-cr in a clinical isolate of Enterobacter cloacae. An ertapenem-resistant E. cloacae ZY106, which was isolated from liquor puris of a female gastric cancer patient in a Chinese hospital, was investigated. Antibiotic susceptibilities were determined by agar dilution method. Conjugation experiments, isoelectric focusing, polymerase chain reaction (PCR), and DNA sequence analyses of plasmid-mediated carbapenemases and quinolone resistance determinants were preformed to confirm the genotype. Outer membrane proteins (OMPs) were examined by urea-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Urea-SDS-PAGE). Minimum inhibitory concentrations (MICs) of imipenem, meropenem, and ertapenem for ZY106 were 2, 4, and 16 microg/ml, respectively. Conjugation studies with Escherichia coli resulted in the transfer of significantly reduced carbapenem susceptibility. ZY106 produced IMP-1 metallo-beta-lactamase and CTX-M-3 extended-spectrum beta-lactamase, and E. coli transconjugant produced IMP-1. Plasmid-mediated quinolone resistance determinant qnrS1 was detected in ZY106. Transfer of the qnrS1-encoding-plasmid into E. coli by conjugation resulted in intermediate resistance to ciprofloxacin in E. coli transconjugant. Urea-SDS-PAGE analysis of OMPs showed that ZY106 lacked an OMP of approximately 38 kDa. It is the first IMP-1-producing Enterobacteriaceae in China and the first report of a clinical isolate that harbors both blaIMP and qnrS genes as well. The blaIMP-1, blaCTX-M-3, and qnrS1 are encoded at three different plasmids. IMP-1 combined with the loss of an OMP possibly resulted in ertapenem resistance and reduced imipenem and meropenem susceptibility in E. cloacae.

A unique glycosyltransferase involved in the initial assembly of Moraxella catarrhalis lipooligosaccharides

Moraxella catarrhalis express three predominant forms of lipooligosaccharide (LOS) molecules on the bacterial surface. These major glycolipids contain specific carbohydrate epitopes that distinguish each glycoform into serotype A, B, or C LOS. All three serotypes, however, share a common glucose containing inner-core structure, consisting of an alpha-glucose attached to 2-keto-3-deoxyoctulosonic acid (KDO), which is unique among Gram-negative bacteria. Many of the LOS glycosyltransferase genes (lgt) responsible for assembly of the extended M. catarrhalis LOS structure have been identified. In this report, we now describe the identification and characterization of Lgt6, a unique glycosyltransferase that is responsible for the addition of the first glucose to the inner core thus initiating the assembly of full length LOS. Isogenic mutants defective in the expression of lgt6 were constructed in all three M. catarrhalis LOS serotypes and the resulting LOS glycoforms consisted of KDO(2)-lipid A-OH as analyzed by urea sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometry. In addition, the expression of lgt6 in trans in a heptose-deficient Neisseria meningitidis NMB gmhX mutant resulted in the addition of a hexose to the LOS of this strain. These studies demonstrate that Lgt6 functions as an alpha-(1-5)-glucosyltransferase in M. catarrhalis adding the primary glucose to the KDO(2)-lipid A-OH in LOS biosynthesis. The function of Lgt6 is required for the completion of both the major and minor oligosaccharide chains in M. catarrhalis.

Interaction Between the C-Terminal Peptides of Tubulin and Tubulin S Detected with the Fluorescent Probe 4′,6-Diamidino-2-phenylindole

The digestion of tubulin with subtilisin and the reassociation of the digestion products was followed by means of the fluorescent probe 4′,6-diamidino-2-phenylindole (DAPI). The fluorescence spectra of DAPI bound to chicken brain tubulin and to the main products of tubulin digested with subtilisin-agarose (tubulin S and C-terminal peptides) were analyzed. The corrected emission spectrum of DAPI in the presence of tubulin showed an enhancement of fluorescence intensity with a maximum at 452 nm. The digestion reaction was followed by the diminution of the area of DAPI-tubulin emission spectra, which showed biphasic pseudo-first-order kinetics. The values for the rate constants were 1.2 × 10 −2 min −1 and 3.5 × 10 −2 min −1 for the α and β subunits, respectively, and were similar to those determined from the undigested subunits using polyacrylamide gel electrophoresis. Tubulin S and the C-terminal peptides were purified by means of a Bio-Gel P-60 column. The C-terminal peptides obtained from this column were analyzed by urea-sodium dodecyl sulfate polyacrylamide gel electrophoresis, and an apparent molecular weight around 3000 was determined. The corrected emission spectrum of DAPI in the presence of tubulin S showed a maximum shifted to 460 nm and a lower enhancement of fluorescence than the emission spectrum of the DAPI-tubulin complex. Titration of purified tubulin S with the C-terminal peptides of tubulin showed, after the addition of DAPI, an increase in the fluorescence intensity at 460 nm with a saturation function dependent on the concentration of peptides added. On the other hand, the emission spectrum of DAPI in the presence of the C-terminal peptides was unchanged from that of free DAPI in the solution. From these results we propose that the DAPI binding site is located on tubulin S and that the C-terminal peptides interact with tubulin S after digestion with subtilisin.

Outer membrane protein profiles and multilocus enzyme electrophoresis analysis for differentiation of clinical isolates of Proteus mirabilis and Proteus vulgaris.

Outer membrane protein (MP) profiles and multilocus enzyme electrophoresis (MEE) analysis were used as tools for differentiating clinical isolates of Proteus spp. Fourteen distinct MP profiles were established by sodium dodecyl sulfate-urea polyacrylamide gel electrophoresis in 54 clinical isolates of Proteus spp. (44 strains identified as P. mirabilis and 10 strains identified as P. vulgaris). Forty-one isolates of P. mirabilis and eight isolates of P. vulgaris were grouped within six and three MP profiles, respectively. The remaining P. mirabilis and P. vulgaris isolates had unique profiles. MEE analysis was used to further discriminate among the strains belonging to the same MP groups. Thirty-five distinct electrophoretic types (ETs) were identified among P. mirabilis isolates. The isolates of P. mirabilis from the four most common MP groups were subgrouped into 30 ETs. All of the P. vulgaris strains had unique ETs. The results suggest that upon biochemical classification of Proteus isolates as P. mirabilis or P. vulgaris, further differentiation among strains of the same species can be obtained by the initial determination of MP profiles followed by MEE analysis of strains with identical MPs.

The Tumor Suppressor p53 Is Bound to RNA by a Stable Covalent Linkage

We have previously shown that the carboxyl-terminal tryptic peptide of the tumor suppressor p53 coeluted from reverse-phase high-performance liquid chromatography (HPLC) with ribonucleotides, suggesting the possible linkage of RNA to p53. In this report, we establish that p53 is covalently linked to RNA, using biochemical criteria at the levels of both tryptic peptide and intact protein: the electrophoretic properties of a tryptic peptide containing phosphorylated Ser-389 and the HPLC chromatographic properties of p53 depend on the linked RNA, p53, purified through urea-sodium dodecyl sulfate-polyacrylamide gel electrophoresis and HPLC, copurifies with RNA, and Ser-389 liberates ribonucleotides upon RNase or alkali treatment. Wild-type and mutant p53s from both simian virus 40 (SV40)-transformed and SV40-nontransformed cells are RNA linked, indicating that RNA linkage may be a general property of p53. The RNA is labeled in vivo with 3H-uridine and in vitro by RNA ligase, suggesting that the RNA is bound by a 5' linkage. The RNA is a long-lived, integral component of p53 rather than a transient reaction intermediate. RNA linkage occurs at an evolutionarily conserved site on p53. We propose that RNA-linked p53 is a major biologically active form of p53 and that its interaction with RNA-linked SV40 T antigen reflects a role in RNA metabolism.

The tumor suppressor p53 is bound to RNA by a stable covalent linkage.

We have previously shown that the carboxyl-terminal tryptic peptide of the tumor suppressor p53 coeluted from reverse-phase high-performance liquid chromatography (HPLC) with ribonucleotides, suggesting the possible linkage of RNA to p53. In this report, we establish that p53 is covalently linked to RNA, using biochemical criteria at the levels of both tryptic peptide and intact protein: the electrophoretic properties of a tryptic peptide containing phosphorylated Ser-389 and the HPLC chromatographic properties of p53 depend on the linked RNA, p53, purified through urea-sodium dodecyl sulfate-polyacrylamide gel electrophoresis and HPLC, copurifies with RNA, and Ser-389 liberates ribonucleotides upon RNase or alkali treatment. Wild-type and mutant p53s from both simian virus 40 (SV40)-transformed and SV40-nontransformed cells are RNA linked, indicating that RNA linkage may be a general property of p53. The RNA is labeled in vivo with 3H-uridine and in vitro by RNA ligase, suggesting that the RNA is bound by a 5' linkage. The RNA is a long-lived, integral component of p53 rather than a transient reaction intermediate. RNA linkage occurs at an evolutionarily conserved site on p53. We propose that RNA-linked p53 is a major biologically active form of p53 and that its interaction with RNA-linked SV40 T antigen reflects a role in RNA metabolism.

Site-directed mutagenesis of the m2 muscarinic acetylcholine receptor. Analysis of the role of N-glycosylation in receptor expression and function.

The cardiac m2 muscarinic acetylcholine receptor (mAChR) is a sialoglycosylated transmembrane protein which has three potential sites for N-glycosylation (namely, Asn2, Asn3, and Asn6). To investigate the role of N-linked oligosaccharide(s) in the expression and function of the receptor, we constructed glycosylation-defective mutant receptor genes in which the three asparagine codons were substituted by codons for either aspartate (Asp2,3,6), lysine (Lys2,3,6), or glutamine (Gln2,3,6). The glycosylation-defective and wild-type receptor genes were stably expressed in Chinese hamster ovary cells. Binding experiments with the membrane-permeable radioligand [3H]quinuclidinyl-benzilate and the membrane-impermeable radioligand [3H]N-methylscopolamine revealed that the Asp2,3,6, Gln2,3,6, and wild-type receptors were located exclusively on the cell surface and expressed in similar numbers. The Lys2,3,6 mutant receptor was expressed at a relatively low level and was therefore not included in subsequent experiments. Wheat germ agglutinin-Sepharose chromatography and sodium dodecyl sulfate-urea polyacrylamide gel electrophoresis demonstrated that the wild-type receptor, but not the Asp2,3,6 and Gln2,3,6 mutant receptors were N-glycosylated. The Asp2,3,6 and Gln2,3,6 mutant receptors had the same affinities for mAChR ligands as wild-type receptors. The time courses for degradation of the Asp2,3,6, Gln2,3,6, and wild-type receptors were also similar. In vivo functional analysis of the ability of the glycosylation mutant receptors to inhibit forskolin-stimulated cAMP accumulation revealed that maximal inhibition of adenylate cyclase activity was similar in the mutant and wild-type receptors. The Asp2,3,6 mutant receptor had an unaltered IC50 value for carbachol while the IC50 value of the Gln2,3,6 mutant receptor was 2-fold higher than that of the wild-type receptor. These results indicate that N-glycosylation of the m2 mAChR is not required for cell surface localization or ligand binding and does not confer increased stability against receptor degradation. Furthermore, N-glycosylation of the m2 mAChR is not required for functional coupling of the m2 mAChR to inhibition of adenylate cyclase.

Subunit Composition of Cytochrome c Oxidase in Mitochondria of Zea mays

Cytochrome c oxidase has been purified from Zea mays mitochondria by a solubilization with dodecyl maltoside followed by a simple and rapid two step fast protein liquid chromatographic method involving anion exchange on Mono Q and size exclusion chromatography on Superose 12. The preparation obtained was resolved by urea sodium dodecyl sulfate-polyacrylamide gel electrophoresis and had a subunit composition comprising polypeptides of apparent molecular masses of 48, 31, and 25 kilodaltons at least one at 16 and 11 kilodaltons and three subunits below 10 kilodaltons. Comparison with a purified yeast cytochrome c oxidase revealed that the four largest subunits showed similar electrophoretic mobilities. Subunits I and II cross-reacted with antibodies raised against the yeast homologous polypeptides. Polypeptides of the plant ubiquinone:cytochrome c reductase complex have also been identified by cross-reaction with antibodies raised against yeast cytochrome b and c(1) subunits and by inference from comigration.

Identification and purification of truncated insulin-like growth factor I from porcine uterus. Evidence for high biological potency.

We report the completion of the purification of uterine-derived growth factors (UDGF) described previously by this laboratory [Ikeda, T., & Sirbasku, D.A. (1984) J. Biol. Chem. 259, 4049-4064]. During isolation, the mitogenic activity was monitored by using the human MCF-7 breast cancer cells in serum-free Ham's F12 and Dulbecco's modified Eagle's medium (1:1, v/v) containing 15 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (pH 7.2), 200 micrograms/mL bovine serum albumin, and 10 micrograms/mL human transferrin. This medium sustained growth for several days in response to a single addition of growth factor. The isolation of UDGF began with acetic acid extraction followed by sulfopropyl-Sephadex chromatography, Bio-Gel P-10 molecular sieve fractionation, and a series of reverse-phase high-pressure liquid chromatography separations. Purifications [[(1.0-8.5) X 10(6)]-fold] of three mitogens (5-20 ng each) were achieved. The mitogens were shown by protein microsequencing to be DES 1----3 to DES 1----6 forms of insulin-like growth factor I (truncated IGF-I). An Mr estimated by 125I labeling, urea-sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and autoradiography was consistent with a DES 1----3(4) N alpha truncation. Immunoadsorption and radioimmunoassay confirmed immunological properties equivalent to IGF-I. Radioreceptor assays showed truncated IGF-I was functionally equivalent to recombinant IGF-I. The ED50 values of DES 1----3 IGF-I and recombinant IGF-I for MCF-7 cell growth were 0.8-6.0 and 30-150 pg/mL, respectively. With Balb/c 3T3 mouse fibroblasts, the ED50 of DES 1----3 IGF-I was 100 times lower than that of IGF-I. We conclude that the major acid-stable low-Mr mitogenic activities isolated from uterus are very potent forms of truncated IGF-I capable of stimulating growth of epithelial and mesenchymal cells.

Amyloid angiopathy of Alzheimer's disease: amino acid composition and partial sequence of a 4,200-dalton peptide isolated from cortical microvessels.

The cardinal lesions of Alzheimer's disease are neurofibrillary tangles, senile neuritic plaques, and vascular amyloid, the latter generally involving cortical arteries and small arterioles. All three lesions are composed of amyloid-like, beta-pleated sheet fibrils. Recently, a 4,200-dalton peptide has been isolated from extraparenchymal meningeal vessels, neuritic plaques, and neurofibrillary tangles. The assumption of N-terminal homogeneity in vascular amyloid has been used as an argument for a neuronal (versus blood) origin of the peptide. However, intracortical microvessels from Alzheimer's disease have not been previously isolated. The present studies describe the isolation of a microvessel fraction from Alzheimer's disease and control fresh autopsy human brain. Alzheimer's disease isolated brain microvessels that were extensively laden with amyloid and control microvessels were solubilized in 90% formic acid and analyzed by urea sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The arteriole fraction from the Alzheimer's subject with extensive amyloid angiopathy contained a unique 4,200-dalton peptide, whereas the arterioles or capillaries isolated from two controls and two Alzheimer's disease subjects without angiopathy did not. This peptide was purified by HPLC and amino acid composition analysis showed the peptide is nearly identical to the 4,200-dalton peptide recently isolated from neuritic plaques or from neurofibrillary tangles. Sequence analysis revealed N-terminal heterogeneity. The N-terminal sequence was: Asp-Ala-Glu-Phe-Arg-His-Asp-Ser-Gly-Tyr, which is identical to the N-terminal sequence of the 4,200-dalton peptide isolated previously from extraparenchymal meningeal vessels and neuritic plaques.(ABSTRACT TRUNCATED AT 250 WORDS)

Human tumor cells synthesize and secrete alpha‐2‐macroglobulin in vitro

In previous studies we showed that human sarcoma and melanoma cell lines synthesize and secrete into culture medium a glycoprotein, migrating in urea sodium dodecyl sulfate-polyacrylamide gel electrophoresis at Mr 140,000. It is not detected in cultures of the corresponding normal cells. Conditioned medium of the melanoma cell line HMB-2, producing among the cell lines tested the largest amounts of this glycoprotein, has now been used as a source for purification of the protein. NH2-terminal amino-acid sequence determination of the purified glycoprotein showed that it is identical to human alpha 2-macroglobulin (alpha 2M). Rabbit antibodies raised against the glycoprotein specifically reacted in immunoblotting and immunodiffusion tests with alpha 2M present in human plasma. Likewise, these antibodies immunoprecipitated from the conditioned media of 35S-methionine-labelled melanoma and osteosarcoma cell lines the protein which had a molecular weight corresponding to alpha 2M. alpha 2M was also synthesized and secreted by 2 strains of fetal lung fibroblasts but not by fetal skin fibroblasts or adult skin fibroblasts autologous to the osteosarcoma cell line.

Rabbit placental relaxin: purification and immunohistochemical localization.

Rabbit placentas were extracted with 0.7 N HCl-acetone (3:5, vol/vol) containing protease inhibitors. Gel filtration (Sephadex G-50) followed by ion exchange chromatography (carboxymethyl cellulose) separated a bioactive relaxin-like fraction with a specific activity of 8.5 U/mg protein as determined by the in vitro mouse uterus bioassay. Column chromatography using Sepharose CL-4B in 6 M guanidine HCl was employed to purify the bioactive sample. The yield of the purified relaxin-like protein was 12 micrograms/g placenta and the specific activity was 23 U/mg protein. The bioactive sample was also immunoreactive after being electrophoretically transferred to nitrocellulose paper and stained using rabbit antiporcine relaxin serum and peroxidase-antiperoxidase immunochemistry. Isoelectrofocusing of the purified relaxin-like protein revealed one band with an isoelectric point of approximately 6.5. The apparent molecular weight of the rabbit relaxin was approximately 7200 as determined by sodium dodecyl sulfate-urea slab gel electrophoresis. Upon reduction with 5.0% mercaptoethanol and electrophoresis on sodium dodecyl sulfate-urea polyacrylamide gels, both the 7200 dalton rabbit immunoreactive relaxin-like polypeptide and porcine relaxin migrated as a lower molecular weight protein. These results suggest that rabbit relaxin, like pig, rat, shark, and human relaxin, consists of two chains linked by disulfide bonds. At the light microscopy level, immunohistochemical staining with guinea pig antiporcine relaxin serum indicated that relaxin was located in the syncytiotrophoblast cells of the placental labyrinth of day-23 and day-30 pregnant rabbits. The syncytiotrophoblast cells from day 16 of pregnancy did not stain for relaxin.

Stability of transmembrane regions in bacteriorhodopsin studied by progressive proteolysis.

Proteinase K digestions of bacteriorhodopsin were carried out with the aim of characterizing the membrane-embedded regions of the protein. Products of digestions for two, eight or 24 hours were separated by high-pressure liquid chromotography. A computerized search procedure was used to compare the amino acid analyses of peptide-containing peaks with segments of the bacteriorhodopsin sequence. Molecular weight distributions of the products were determined by sodium dodecylsulfate-urea polyacrylamide gel electrophoresis. The structural integrity of the protein after digestion was monitored through the visible absorption spectrum, by X-ray diffraction of partially dried membranes, and by following release of biosynthetically-incorporated 3H leucine from the digested membranes. During mild proteolysis, bacteriorhodopsin was cleaved near the amino and carboxyl termini and at two internal regions previously identified as being accessible to the aqueous medium. Longer digestion resulted in cleavage at new sites. Under conditions where no fragments of bacteriorhodopsin larger than 9000 mol wt were observed, a significant proportion of the digested membranes retained diffraction patterns similar to those of native purple membranes. The harshest digestion conditions led to complete loss of the X-ray diffraction patterns and optical absorption and to release of half the hydrophobic segments of the protein from the membrane in the form of small soluble peptides. Upon cleavage of aqueous loop regions of the protein, isolated transmembrane segments may experience motion in a direction perpendicular to the plane of the membrane, allowing them access to protease.

Identification and characterization of Vibrio cholerae surface proteins by radioiodination.

Whole cells and isolated outer membrane from Vibrio cholerae (Classical, Inaba) were radiolabeled with Iodogen or Iodo-beads as catalyst. Radiolabeling of whole cells was shown to be surface specific by sodium dodecyl sulfate-urea polyacrylamide gel electrophoresis of whole cells and cell fractions. Surface-labeled whole cells regularly showed 16 distinguishable protein species, of which nine were found in radiolabeled outer membrane preparations obtained by a lithium chloride-lithium acetate procedure. Eight of these proteins were found in outer membranes prepared by sucrose density gradient centrifugation and Triton X-100 extraction of radiolabeled whole cells. The mobility of several proteins was shown to be affected by temperature, and the major protein species exposed on the cell surface was shown to consist of at least two different peptides.

Identification and occurrence of Vibrio cholerae flagellar core proteins in isolated outer membrane.

Sodium dodecyl sulfate-urea polyacrylamide gel electrophoresis of outer membranes from a flagellated and an isogenic nonflagellated strain of Vibrio cholerae (classical, Inaba) suggested that two proteins were absent from the nonflagellated strain. Immunoblot examination of such preparations demonstrated that two proteins, present only in outer membrane from the flagellated strain, were associated with flagella. Analysis of purified flagellar cores from strains CA401 and N16961 (El Tor, Inaba) by electron microscopy, sodium dodecyl sulfate-urea polyacrylamide gel electrophoresis, and immunoblotting showed that these two proteins, with apparent molecular weights of 47,000 and 49,000, composed the flagellar core. Antiserum specific for flagellar core proteins did not agglutinate or inhibit the motility of intact V. cholerae. These latter findings suggested that, for intact cells, the flagellar core proteins are not accessible to antibody.

Ion-exchange high-performance liquid chromatographic purification of bovine cardiac and rabbit skeletal muscle troponin subunits

Bovine cardiac and rabbit skeletal troponin complexes were separated into their respective subunits employing high-performance liquid chromatographic (HPLC) techniques on CM-300 and Q-300 ion-exchangers. Bovine cardiac and rabbit skeletal subunits were separated on the strong anion-exchanger, Q-300, in 8 M urea, 50 m M Tris, 2 m M EGTA, 0.5 m M dithiothreitol, pH 7.5, employing a linear salt gradient and on the weak cation-exchanger, CM-300, in 8 M urea, 50 m M potassium dihydrogen phosphate, 2 m M EGTA, 0.5 m M dithiothreitol, pH 6.5, using a linear salt gradient. To obtain complete purification of all components of troponin both ion-exchangers were required. The initial separation of troponin was carried out on the strong anion-exchanger followed by weak cation-exchange chromatography of the troponin I collected from the strong anion-exchange column. The troponin T subunits obtained from Q-300 chromatography demonstrated heterogeneity (three components: T1, T2 and T3) while the troponin I collected from both sources on the Q-300 column were both resolved into major doublets (I1 and I2) when rechromatographed on the CM-300 column. The three troponin T fractions and two troponin I fractions isolated from ion-exchange HPLC were examined by sodium dodecyl sulfate—urea polyacrylamide gel electrophoresis and two-dimensional gel electrophoresis to confirm that the heterogeneity was due to differences in charge and not molecular weight. These results were in agreement with the charge differences observed from retention times on ion-exchange HPLC. When comparing the same troponin subunit from different muscle sources, considerable differences in the content of charged amino acid residues were also observed.

Monoclonal antibody to rat uterine peroxidase and its use in identification of the peroxidase as being of eosinophil origin

Peroxidase was purified from uteri of estrogen-treated rats by calcium chloride extraction, affinity chromatography on concanavalin A-Sepharose and hydrophobic interaction chromatography on phenyl-Sepharose. An overall purification of greater than 1700-fold was achieved with a final recovery of 27%. Monoclonal antibodies to peroxidase were subsequently prepared by immunization of male C57BL/10J mice with the highly purified peroxidase from rat uterus. Spleen and lymph node cells from the mice were fused with Sp2/0-Ag 14 mouse myeloma cells. The resultant hybrid cells were screened for production of antibody using a solid-phase, double antibody radioimmunoassay. The mature rat spleen, shown previously to be abundant in eosinophils, contains high peroxidase activity. Spleen peroxidase purified by the same procedure as the uterine enzyme cross-reacted with a monoclonal antibody, designated IgG-107B, used in all subsequent studies. Peroxidase extracted from isolated rat eosinophils also cross-reacted with the antibody and yielded identical titers as the spleen and uterine peroxidases. Spleen, uterine and horse eosinophil peroxidase had the same apparent molecular weight, 57 000, as determined by sodium dodecyl sulfate-urea polyacrylamide gel electrophoresis. Following electrophoretic transfer to nitrocellulose, spleen, uterine and eosinophil peroxidase reacted with monoclonal antibody, using an immunoblotting technique. These results provide biochemical and immunological evidence that the majority of the calcium chloride-extractable peroxidase activity from the uteri of estrogen-treated rats is derived from infiltrating eosinophils.

Cytochrome b558 from (bovine) granulocytes. Partial purification from Triton X-114 extracts and properties of the isolated cytochrome.

A membrane-associated b-type cytochrome (a proposed component in the neutrophil microbicidal superoxide generating system) has been partially purified from nonactivated beef granulocytes to a specific heme content of 20 nmol of heme/mg of protein, a value about 10-fold higher than those previously reported. The hemoprotein was solubilized at low temperature (4 degrees C) from mixed granule (30,000 X g) cell fractions using Triton X-114 detergent. Warming the extract to 25 degrees C allowed separation into detergent and aqueous phases; cytochrome b558 partitioned exclusively into the detergent phase, allowing separation from other visible-absorbing species (e.g. myeloperoxidase) and indicated an intrinsic membrane localization (Bordier, C. (1981) J. Biol. Chem. 256, 1604-1607). The partitioned cytochrome was chromatographed on hydroxylapatite and a hydrophobic affinity matrix, allowing a 185-fold (heme content) purification from the granule extract. The cytochrome preparation revealed three equal-staining protein bands by sodium dodecyl sulfate-urea polyacrylamide gel electrophoresis; apparent molecular weights were 14,000, 12,000, and 11,000. The question of heterogeneity of the preparation versus subunit structure is not resolved at present. The hemoprotein binds carbon monoxide, consistent with a proposed role as a terminal oxidase, and has an unusually negative oxidation-reduction potential (-225 mV) similar to that observed in granulocyte membranes. The preparation is devoid of NAD(P)H-diaphorase and cytochrome c reductase activities.

Preliminary characterization of inhibitors of Neisseria gonorrhoeae produced in vitro by Eubacterium limosum.

Among anaerobic bacteria normally found in the urogenital flora, Eubacterium limosum was found to inhibit the in vitro growth of Neisseria gonorrhoeae. The antigonococcal activity produced by E. limosum was soluble in methanol and in a chloroform--methanol mixture (30:70). The fraction soluble in chloroform--methanol (30:70) yielded eight absorbance peaks when chromatographed on Bio-Gel P-2 and the inhibitory activity was found in the first two peaks. This activity was not absorbed on DEAE Sephacel and was eluted with distilled water in a peak considered as peak 1, on which preliminary characterization was done. The inhibitory activity of peak 1 was found to be heat and pH resistant and not susceptible to proteases, lipase, or amylases. When peak 1 was chromatographed on cellulose paper using a butanol--acetic acid (4:1) solvent system, eight different spots were detected upon spraying the paper with ninhydrin. No spot was detected with anthrone, bromothymol, nor Sudan black reagents used for the detection of carbohydrates and lipids. Based on sodium dodecyl sulfate-urea polyacrylamide gel electrophoresis and gel chromatography on Sephadex G-25, peak 1 appeared either as a diffuse band and as a single peak, respectively. The molecular weight of the inhibitory complex was estimated to be 2400. All these results suggest that the antigonococcal activity produced by E. limosum is composed of more than one low molecular weight amino compound.