Properties of spores of Bacillus subtilis with or without a transposon that decreases spore germination and increases spore wet heat resistance.

AIMS: This work aimed to determine how genes on transposon Tn1546 slow Bacillus subtilis spore germination and increase wet heat resistance, and to clarify the transposon's 3 gene spoVA operon's role in spore properties, since the seven wild-type SpoVA proteins form a channel transporting Ca2+ -dipicolinic acid (DPA) in spore formation and germination. METHODS AND RESULTS: Deletion of the wild-type spoVA operon from a strain with Tn1546 gave spores with slightly reduced wet heat resistance but some large decreases in germination rate. Spore water content and CaDPA analyses found no significant differences in contents of either component in spores with different Tn1546 components or lacking the wild-type spoVA operon. CONCLUSIONS: This work indicates that the SpoVA channel encoded by Tn1546 functions like the wild-type SpoVA channel in CaDPA uptake into developing spores, but not as well in germination. The essentially identical CaDPA and water contents of spores with and without Tn1546 indicate that low core water content does not cause elevated wet heat resistance of spores with Tn1546. SIGNIFICANCE AND IMPACT OF THE STUDY: Since wet heat resistance of spores of Bacillus species poses problems in the food industry, understanding mechanisms of spores' wet heat resistance is of significant applied interest.

Dodecylamine rapidly kills of spores of multiple Firmicute species: properties of the killed spores and the mechanism of the killing.

AIMS: Previous work showed that Bacillus subtilis dormant spore killing and germination by dodecylamine take place by different mechanisms. This new work aimed to optimize killing of B. subtilis and other Firmicutes spores and to determine the mechanism of the killing. METHODS AND RESULTS: Spores of seven Firmicute species were killed rapidly by dodecylamine under optimal conditions and more slowly by decylamine or tetradecylamine. The killed spores were not recovered by additions to recovery media, and some of the killed spores subsequently germinated, all indicating that dodecylamine-killed spores truly are dead. Spores of two species treated with dodecylamine were more sensitive to killing by a subsequent heat treatment, and spore killing of at least one species was faster with chemically decoated spores. The cores of dodecylamine-killed spores were stained by the nucleic acid stain propidium iodide, and dodecylamine-killed wild-type and germination-deficient spores released their stores of phosphate-containing small molecules. CONCLUSIONS: This work indicates that dodecylamine is likely a universal sporicide for Firmicute species, and it kills spores by damaging their inner membrane, with attendant loss of this membrane as a permeability barrier. SIGNIFICANCE AND IMPACT OF THE STUDY: There is a significant need for agents that can effectively kill spores of a number of Firmicute species, especially in wide area decontamination. Dodecylamine appears to be a universal sporicide with a novel mechanism of action, and this or some comparable molecule could be useful in wide area spore decontamination.

Killing of bacterial spores by dodecylamine and its effects on spore inner membrane properties.

AIMS: The objective of this study was to determine the effects of Ca-dipicolinic acid (CaDPA), cortex-lytic enzymes (CLEs), the inner membrane (IM) CaDPA channel and coat on spore killing by dodecylamine. METHODS AND RESULTS: Bacillus subtilis spores, wild-type, CaDPA-less due to the absence of DPA synthase or the IM CaDPA channel, or lacking CLEs, were dodecylamine-treated and spore viability and vital staining were all determined. Dodecylamine killed intact wild-type and CaDPA-less B. subtilis spores similarly, and also killed intact Clostridiodes difficile spores ± CaDPA, with up to 99% killing with 1 mol l-1 dodecylamine in 4 h at 45°C with spores at ~108  ml-1 . Dodecylamine killing of decoated wild type and CLE-less B. subtilis spores was similar, but ~twofold faster than for intact spores, and much faster for decoated CaDPA-less spores, with ≥99% killing in 5 min. Propidium iodide stained intact spores ± CaDPA minimally, decoated CaDPA-replete spores or dodecylamine-killed CLE-less spores peripherally, and cores of decoated CaDPA-less spores and dodecylamine-killed intact spores with CLEs. The IM of some decoated CaDPA-less spores was greatly reorganized. CONCLUSIONS: Dodecylamine spore killing does not require CaDPA channels, CaDPA or CLEs. The lack of CaDPA in decoated spores allowed strong PI staining of the spore core, indicating loss of these spores IM permeability barrier. SIGNIFICANCE AND IMPACT OF THE STUDY: This work gives new information on killing bacterial spores by dodecylamine, and how spore IM's relative impermeability is maintained.

Lack of efficient killing of purified dormant spores of Bacillales and Clostridiales species by glycerol monolaurate in a non-aqueous gel.

Inactivation of Bacillales and Clostridiales spores is of interest, since some cause food spoilage and human diseases. A recent publication (mSphere 3: e00597-1, 2018) reported that glycerol monolaurate (GML) in a non-aqueous gel (GMLg) effectively killed spores of Bacillus subtilis, Bacillus cereus and Clostridioides difficile, and Bacillus anthracis spores to a lesser extent. We now show that (i) the B. subtilis spores prepared as in the prior work were impure; (ii) if spore viability was measured by diluting spores 1/10 in GMLg, serially diluting incubations 10-fold and spotting aliquots on recovery plates, there was no colony formation from the 1/10 to 1/1000 dilutions due to GMLg carryover, although thorough ethanol washes of incubated spores eliminated this problem and (iii) GMLg did not kill highly purified spores of B. subtilis, B. cereus, Bacillus megaterium and C. difficile in 3-20 h in the conditions used in the recent publication. GMLg also gave no killing of crude B. subtilis spores prepared as in the recent publication in 5 h but gave ~1·5 log killing at 24 h. Thus, GMLg does not appear to be an effective sporicide, although the gel likely inhibits spore germination and could kill spores somewhat upon long incubations. SIGNIFICANCE AND IMPACT OF THE STUDY: Given potential deleterious effects of spores of Bacillales and Clostridiales, there is an ongoing interest in new ways of spore killing. A recent paper (mSphere 3: e00597-1, 2018) reported that glycerol monolaurate (GML) in a non-aqueous gel (GMLg) effectively killed spores of many species. We now find that (i) the Bacillus subtilis spores prepared as in the previous report were impure and (ii) GMLg gave no killing of purified spores of Bacillales and Clostridiales species in ≤5 h under the published conditions. Thus, GMLg is not an effective sporicide, though may prevent spore germination or kill germinated spores.

Mechanisms of killing of Bacillus thuringiensis Al Hakam spores in a blast environment with and without iodic acid.

AIMS: To determine the mechanism of killing of spores of Bacillus thuringiensis Al Hakam, a Bacillus anthracis spore surrogate, in a blast environment with or without HIO3 and whether the spores are truly dead. METHODS AND RESULTS: Spores exposed to an aluminium-based blast environment with or without HIO3 with dynamic peak gas phase temperatures near 1000°C persisting for 10's of ms, were killed 97 and 99·99% without and with HIO3 respectively and the spores were truly dead. The survivors of the detonations did not acquire mutations, did not become wet heat sensitive, became sensitive to elevated NaCl but not lack of glucose in recovery media, and many dead spores remained phase bright and retained their Ca-dipicolinic acid. A large fraction of the dead spores could germinate, but most of these germinated spores were dead. CONCLUSIONS: Most spores exposed to a blast environment are truly dead, and HIO3 increases spore death. The likely mechanism of spore killing in these blast environments is damage to some essential spore protein, although spore inner membrane damage could contribute. SIGNIFICANCE AND IMPACT OF THE STUDY: This work shows that spores of a surrogate for B. anthracis spores are killed in a blast environment without or with HIO3 present, this approach could inactivate up to 99·99% of dry B. anthracis spores, and the spores are likely killed by damage to some essential spore protein.

Effects of the microbicide ceragenin CSA-13 on and properties of Bacillus subtilis spores prepared on two very different media.

AIMS: To determine how the microbicide ceragenin-13 (CSA-13) kills Bacillus subtilis spores prepared on growth or sporulation media, and these spores' properties. METHODS AND RESULTS: Spores made on Luria broth (LB) growth or double-strength Schaeffer's-glucose (2xSG) sporulation plates found that spores made on LB plates have coat defects as evidenced by their lower hypochlorite resistance, faster germination with dodecylamine and slower germination with Ca2+ -dipicolinic acid (CaDPA) than 2xSG plate spores. CSA-13 triggered CaDPA release from spores, an early step in germination, but only well at 70°C and better with spores made on LB than on 2xSG plates. Approximately 90% of spores with elevated levels of SpoVA proteins that form a CaDPA release channel, released CaDPA with CSA-13 at 70°C, and faster with spores made on LB than 2xSG plates. Levels of CSA-13 killing of spores made on LB and 2xSG plates were similar to levels of CaDPA release triggered by this agent. CONCLUSIONS: CSA-13 kills bacterial spores, but only at high concentrations and temperatures, and is preceded by CaDPA release. SIGNIFICANCE AND IMPACT OF THE STUDY: CSA-13 is not a direct sporicide as reported previously, but most likely germinates spores via activation of spores' CaDPA channel, albeit inefficiently, and then killing the germinated spores.

Killing of spores of Bacillus species by cetyltrimethylammonium bromide.

AIMS: To investigate effects of the cationic surfactant cetyltrimethylammonium bromide (CTAB), a disinfectant, on spores of Bacillus species. METHODS AND RESULTS: The ability of CTAB to trigger release of Bacillus spores' large depot of dipicolinic acid (DPA) in a 1 : 1 chelate with Ca2+ (CaDPA), and to kill spores was investigated. CTAB-triggered CaDPA release from spores of Bacillus subtilis, Bacillus cereus and Bacillus megaterium, but was not followed by completion of germination. CaDPA release triggered by CTAB increased at higher temperatures, and was optimal for B. subtilis spores at pH 9·4 and 30 µg ml-1 CTAB. CTAB also killed Bacillus spores as shown by plate counts and vital staining of treated dormant spores, and after their germination. However, B. cereus and B. megaterium spores were more CTAB-sensitive than were B. subtilis spores. CaDPA release from and killing of CTAB-treated spores of isogenic B. subtilis mutants lacking germination proteins was also examined, and compared with effects of the well-known germinant dodecylamine on spores, to determine how CTAB exerts its effects on spores. CONCLUSIONS: The results of this investigation showed that CTAB kills spores of three Bacillus species, perhaps by damaging the spore inner membrane, although it is also possible that some killing by this agent follows its triggering of spore germination. SIGNIFICANCE AND IMPACT OF THE STUDY: The results of this work indicate that CTAB is also a disinfectant, but also a sporicide, and may be a useful adjunct in spore decontamination, especially at higher temperatures.

Killing the spores of Bacillus species by molecular iodine.

AIMS: To determine the responses of spores of Bacillus subtilis and Bacillus anthracis surrogate Bacillus thuringiensis Al Hakam to I2 treatment. METHODS AND RESULTS: Spores of B. subtilis and B. thuringiensis killed by aqueous 30°C-I2 could germinate, and their inner membrane (IM) was intact. Spore coats were important in I2 resistance, DNA-protective proteins were not important, and survivors of I2 treatment were not mutagenized. Viabilities of I2 -treated, 90-98% killed spores were much lower on high-salinity media, and the treated spores were more heat sensitive than the untreated spores. Germinated I2 -killed spores were dead as determined by staining with nucleic acid dyes, and many appeared to have been lysed. CONCLUSIONS: Aqueous I2 appeared to kill B. subtilis and B. thuringiensis spores such that spores lyse soon after they germinate, and not by causing DNA damage or rupture of spores' IM. I2 treatment also generated many damaged spores that could only be recovered under nonstressful conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: This work shows that spores of the model organism B. subtilis, and B. thuringiensis, a surrogate for B. anthracis spores, exhibit similar mechanisms of resistance to and killing by I2 . Generation by I2 treatment of conditionally dead spores indicates that appropriate media are essential to efficiently enumerate viable I2 -treated spores.

Effects of steam autoclave treatment on Geobacillus stearothermophilus spores.

AIMS: To determine the mechanism of autoclave killing of Geobacillus stearothermophilus spores used in biological indicators (BIs) for steam autoclave sterilization, and rates of loss of spore viability and a spore enzyme used in BIs. METHODS AND RESULTS: Spore viability, dipicolinic acid (DPA) release, nucleic acid staining, α-glucosidase activity, protein structure and mutagenesis were measured during autoclaving of G. stearothermophilus spores. Loss of DPA and increases in spore core nucleic acid staining were slower than loss of spore viability. Spore core α-glucosidase was also lost more slowly than spore viability, although soluble α-glucosidase in spore preparations was lost more rapidly. However, spores exposed to an effective autoclave sterilization lost all viability and α-glucosidase activity. Apparently killed autoclaved spores were not recovered by artificial germination in supportive media, much spore protein was denatured during autoclaving, and partially killed autoclave-treated spore preparations did not acquire mutations. CONCLUSIONS: These results indicate that autoclave-killed spores cannot be revived, spore killing by autoclaving is likely by protein damage, and spore core α-glucosidase activity is lost more slowly than spore viability. SIGNIFICANCE AND IMPACT OF THE STUDY: This work provides insight into the mechanism of autoclave killing of spores of an organism used in BIs, and that a spore enzyme in a BI is more stable to autoclaving than spore viability.

Analysis of α-glucosidase enzyme activity used in a rapid test for steam sterilization assurance.

AIMS: This study was to determine the sources, location and identity of α-glucosidases in dormant/germinating/outgrowing spores and growing cells of Geobacillus stearothermophilus ATCC 7953, an enzymatic activity in spores used in rapid tests of steam sterilization. METHODS AND RESULTS: α-Glucosidase activity in spores and cells was determined measuring methylumbelliferyl-α-d-glucoside (α-MUG) or α-MUG-6-phosphate hydrolysis fluorometrically. While α-MUG-6-phosphate was not hydrolysed by cell or spore extracts, assays with α-MUG showed that: (1) the α-glucosidase activity was inside and outside spores, and the activity outside spores was largely removed by buffer washes or heat activation, whereas α-glucosidase activity was only inside vegetative cells; (2) most α-glucosidase activity in cells and spores was soluble; (3) Western blots and enzyme inhibition using an anti-α-glucosidase antiserum identified ≥2 α-glucosidases in spores and growing cells; (4) α-glucosidase-specific activities were similar in dormant, germinated and outgrowing spore and growing cell extracts; and (5) significant α-glucosidase was synthesized during spore germination and outgrowth and cell growth, this synthesis was not repressed by glucose nor induced by α-MUG, but glucose inhibited α-MUG uptake. CONCLUSIONS: α-MUG hydrolysis by G. stearothermophilus is by α-MUG uptake and hydrolysis by ≥2 α-glucosidases associated with dormant spores and synthesized by germinating and outgrowing spores. The enzyme activity observed by sterilization assurance assays appears likely to come from heat-stable enzyme in the spore core and enzyme(s) synthesized in spore outgrowth. SIGNIFICANCE AND IMPACT OF THE STUDY: The results of this work provide new insight into the science behind a rapid test for steam sterilization assurance.

Mechanism of Bacillus subtilis spore inactivation by and resistance to supercritical CO2 plus peracetic acid.

AIMS: Determine how supercritical CO2 (scCO2 ) plus peracetic acid (PAA) inactivates Bacillus subtilis spores, factors important in spore resistance to scCO2 -PAA, and if spores inactivated by scCO2 -PAA are truly dead. METHODS AND RESULTS: Spores of wild-type B. subtilis and isogenic mutants lacking spore protective proteins were treated with scCO2 -PAA in liquid or dry at 35°C. Wild-type wet spores (aqueous suspension) were more susceptible than dry spores. Treated spores were examined for viability (and were truly dead), dipicolinic acid (DPA), mutations, permeability to nucleic acid stains, germination under different conditions, energy metabolism and outgrowth. ScCO2 -PAA-inactivated spores retained DPA, and survivors had no notable DNA damage. However, DPA was released from inactivated spores at a normally innocuous temperature (85°C), and colony formation from treated spores was salt sensitive. The inactivated spores germinated but did not outgrow, and these germinated spores had altered plasma membrane permeability and defective energy metabolism. Wet or dry coat-defective spores had increased scCO2 -PAA sensitivity, and dry spores but not wet spores lacking DNA protective proteins were more scCO2 -PAA sensitive. CONCLUSIONS: These findings suggest that scCO2 -PAA inactivates spores by damaging spores' inner membrane. The spore coat provided scCO2 -PAA resistance for both wet and dry spores. DNA protective proteins provided scCO2 -PAA resistance only for dry spores. SIGNIFICANCE AND IMPACT OF THE STUDY: These results provide information on mechanisms of spore inactivation of and resistance to scCO2 -PAA, an agent with increasing use in sterilization applications.

Induction of stearoyl CoA desaturase is associated with high-level induction of emerin RNA.

The genes encoding enzymes involved in fatty acid metabolism are regulated by sterols. Stearoyl CoA desaturase, a key enzyme in the synthesis of unsaturated fatty acyl-CoAs is transcriptionally regulated by fat-free diet and sterols. To identify other genes that are induced in rat liver by fat-free diet we performed an in vivo gene expression profile analysis using DNA microarrays. Here we report that among the genes highly expressed is emerin, an integral protein of the inner nuclear membrane. Mutated or nonexpressed emerin occurs in patients with muscular dystrophy. Sterol regulatory element binding proteins activate the transcription of several sterol regulated genes. To investigate whether sterol regulatory element binding proteins or indirectly cholesterol activates the transcription of stearoyl CoA desaturase and emerin, we cultured Chinese hamster ovary (CHO), either in cholesterol-rich or cholesterol-depleted mediums. We also transiently transfected the cell culture with plasmid encoding sterol regulatory element binding proteins in cholesterol-rich medium. Our data show that cholesterol-supplemented media as well as the transient transfection induced the expression of stearoyl CoA desaturase RNA 3.5- and 7-fold respectively. However, the RNA level of emerin was not altered under these conditions, implying that the parallel induction of emerin is independent of the sterol regulatory element binding regulation pathway.

The N terminus of microsomal delta 9 stearoyl-CoA desaturase contains the sequence determinant for its rapid degradation.

Stearoyl-CoA desaturase (SCD) is a key regulator of membrane fluidity, turns over rapidly, and represents a model for selective degradation of short-lived proteins of the endoplasmic reticulum (ER). The mechanism whereby specific ER proteins are targeted for degradation in the midst of stable proteins coexisting in the same membrane is unknown. To investigate the intracellular fate of SCD and to identify the determinants involved in the rapid turnover of SCD, we created chimeras of SCD tagged at the C terminus with the green fluorescent protein (GFP). The fusion proteins were expressed in Chinese hamster ovary cells and exhibited an ER localization. Unlike native GFP, the SCD-GFP construct was unstable and had a half life of a few hours. Truncated fusion proteins consisting of residues 27-358 and 45-358 of SCD linked to the N terminus of GFP were stable. To investigate the general applicability of the N terminus of SCD in the destabilization of proteins, we fused residues 1-33 of SCD to the N terminus of GFP. The resulting chimera was extremely short lived. To investigate the effect of membrane sidedness on the fusion protein degradation, we attached a lumenal targeting signal to the N terminus of SCD 1-33-GFP. The construct was localized to the lumen of ER and was metabolically stable, indicating that SCD degradation signal functions on the cytosolic rather than the lumenal side of the ER. These results demonstrate that the N-terminal segment of some 30 residues of SCD constitutes a motif responsible for the rapid degradation of SCD.

Targeting proteins to the lumen of endoplasmic reticulum using N-terminal domains of 11beta-hydroxysteroid dehydrogenase and the 50-kDa esterase.

Previous studies identified two intrinsic endoplasmic reticulum (ER) proteins, 11beta-hydroxysteroid dehydrogenase, isozyme 1 (11beta-HSD) and the 50-kDa esterase (E3), sharing some amino acid sequence motifs in their N-terminal transmembrane (TM) domains. Both are type II membrane proteins with the C terminus projecting into the lumen of the ER. This finding implied that the N-terminal TM domains of 11beta-HSD and E3 may constitute a lumenal targeting signal (LTS). To investigate this hypothesis we created chimeric fusions using the putative targeting sequences and the reporter gene, Aequorea victoria green fluorescent protein. Transfected COS cells expressing LTS-green fluorescent protein chimeras were examined by fluorescent microscopy and electron microscopic immunogold labeling. The orientation of expressed chimeras was established by immunocytofluorescent staining of selectively permeabilized COS cells. In addition, protease protection assays of membranes in the presence and absence of detergents was used to confirm lumenal or the cytosolic orientation of the constructed chimeras. To investigate the general applicability of the proposed LTS, we fused the N terminus of E3 to the N terminus of the NADH-cytochrome b5 reductase lacking the myristoyl group and N-terminal 30-residue membrane anchor. The orientation of the cytochrome b5 reductase was reversed, from cytosolic to lumenal projection of the active domain. These observations establish that an amino acid sequence consisting of short basic or neutral residues at the N terminus, followed by a specific array of hydrophobic residues terminating with acidic residues, is sufficient for lumenal targeting of single-pass proteins that are structurally and functionally unrelated.

Interactions of serine proteinases with pNiXa, a serpin of Xenopus oocytes and embryos.

In a previous study, kinetic assays showed that pNiXa, an Ni(II)-binding serpin of Xenopus oocytes and embryos, strongly inhibits bovine chymotrypsin, weakly inhibits porcine elastase, and does not inhibit bovine trypsin. In this study, analyses by SDS-PAGE and gelatin zymography showed that an SDS-resistant complex is formed upon the interaction of pNiXa with bovine chymotrypsin. No such pNiXa-enzyme complex was detected after pNiXa interactions with porcine elastase, bovine trypsin, or human cathepsin G. The major products of pNiXa cleavage by the four proteinases were partially sequenced by Edman degradation. The cleavage products were also tested by immunoblotting with an antibody to the His-cluster of pNiXa, and by radio-blotting with 63Ni(II). These assays showed that chymotrypsin and elastase cleave pNiXa at the P1-P1 (Thr-Lys) peptide bond near the C-terminus, while trypsin and cathepsin G cleave pNiXa at specific peptide bonds near the N-terminus, within an interesting 26-residue segment, rich in Lys and Gln, that separates the His-cluster of pNiXa from the rest of the molecule. The segment lacks homology to other serpins, but resembles a domain of Xenopus POU3 transcription factor. This study identifies the specific sites for interactions of four serine proteinases with pNiXa, indicates that pNiXa inhibition of chymotrypsin involves a serpin-like mechanism, and shows that 63Ni(II)-binds to the His-cluster of pNiXa.

Xenopus lipovitellin 1 is a Zn(2+)- and Cd(2+)-binding protein.

This report discusses the identification of a Zn(2+)- and Cd(2+)-binding protein of Xenopus laevis that is abundant in vitellogenic oocytes and in embryos from fertilization to stage 46. Oocyte or embryo homogenates were fractionated by SDS-PAGE, blotted onto nitrocellulose, and probed with 65Zn2+ or 109Cd2+. The resulting autoradiograms showed binding of both radionuclides to a protein, designated pCdZn. Freon extraction of oocyte and embryo homogenates showed pCdZn to be a yolk protein. When pCdZn was isolated from oocyte homogenates by ammonium sulfate precipitation, delipidation, and chromatography, it co-purified with lipovitellin 1. The amino acid composition of pCdZn closely resembled the reported composition of lipovitellin 1 and the molecular weight of purified pCdZn (approximately 115 kD) corresponded to reported values for lipovitellin 1 (111-121 kD). Amino acid sequence analyses of five peptides derived from pCdZn yielded 94% identity to the reported sequence of lipovitellin 1, deduced from the DNA sequence of the Xenopus vitellogenin A2 precursor gene. Based on these findings, pCdZn was identified as lipovitellin 1. This study suggests that lipovitellin 1 is the major storage protein for zinc in mature oocytes and developing embryos of Xenopus laevis.

Human oligosaccharyltransferase: isolation, characterization, and the complete amino acid sequence of 50-kDa subunit.

Oligosaccharyltransferase (OT) catalyzes the glycosylation of asparagine residues in nascent polypeptides in the endoplasmic reticulum. In a previous communication we reported the purification and characterization of this enzyme from chicken oviduct. Here we describe the purification and sequence analysis of OT from human liver microsomes. Oligosaccharyltransferase copurified with three proteins designated 50-kDa, 65-I and 65-II based on their molecular weights by gel electrophoresis. The N-terminal sequence of the 50-kDa component was homologous to the 50-kDa subunit of avian OT. The N-terminal sequences of 65-I and 65-II were identical to the primary structures of human ribophorins I and II, respectively, predicted by cDNA sequencing. The complete amino acid sequence of the 50-kDa subunit of human OT was determined by chemical sequencing of peptides isolated from chemical and enzymatic digests. The 50-kDa subunit of human OT is 98% identical to its canine homolog, 93% identical to its avian homolog, and 25% identical to the beta subunit of yeast OT. These data indicate that structural features of oligosaccharyltransferase are conserved in all eukaryotes.

The 40 kDa 63Ni(2+)-binding protein (pNiXc) on western blots of Xenopus laevis oocytes and embryos is the monomer of fructose-1,6-bisphosphate aldolase A.

A Ni(2+)-binding protein (pNiXc, 40 kDa), present in Xenopus laevis oocytes and embryos, was isolated from mature oocytes by chromatography on DEAE-cellulose and cellulose phosphate, followed by FPLC on Ni-iminodiacetate-Agarose, or reverse-phase HPLC on a C-4 column. Size-exclusion HPLC showed that intact pNiXc is approximately 155 kDa, consistent with tetrameric structure. After cleavage with Lys-C proteinase or cyanogen bromide, six peptides were separated by HPLC and sequenced by Edman degradation, providing sequence data for 83 residues. Data-base search showed similarity of pNiXc to eukaryotic aldolases, with 96% identity to human aldolase A. pNiXc demonstrated aldolase activity with fructose 1,6-bisphosphate as substrate (Km, 30 microM Vmax 26 mumol min-1 mg-1); the aldolase activity was inhibited non-competitively by Cu2+, Cd2+, Co2+, or Ni2+. Equilibrium dialysis showed high affinity binding (Kd, 7 microM) of 1 mole of Ni per mole of 40 kDa subunit. Based on metal-blot competition assays, the abilities of metals to compete with 63Ni2+ for binding to pNiXc were ranked: Cu2+ >> Zn2+ > Cd2+ > Co2+. This study identifies pNiXc as the monomer of fructose-1,6-bisphosphate aldolase A, and raises the possibility that aldolase A is a target enzyme for metal toxicity.

Lipovitellin 2 beta is the 31 kD Ni(2+)-binding protein (pNiXb) in Xenopus oocytes and embryos.

An Ni(2+)-binding protein (pNiXb, 31 kD) present in mature Xenopus laevis oocytes and in embryos from fertilization in N/F stage 42, was isolated and characterized. After oocytes or embryos were fractionated by PAGE, electroblotted onto nitrocellulose, and probed with 63Ni2+, pNiXb was detected by autoradiography. pNiXb, a yolk protein located in the embryonic gut, was purified from yolk platelets by ammonium sulfate precipitation, delipidation, gel filtration chromatography, and HPLC analysis. During these steps, pNiXb copurified with lipovitellin 2. The N-terminal sequence of purified pNiXb exactly matched that of Xenopus lipovitellin 2 beta, deduced from the DNA sequence of the Xenopus vitellogenin A2 precursor gene. Since pNiXb and lipovitellin 2 beta agree in N-terminal sequence, amino acid composition, and apparent molecular weight, they appear to be identical. Based on a metal-blot competition assay, the abilities of metal ions to compete with 63Ni2+ for binding to pNiXb were ranked: Zn2+ approximately Cu2+ approximately Co2+ > Cd2+ approximately Mn2+ > Sn2+. This study shows that Xenopus lipovitellin 2 beta is a metal-binding protein in vitro, and raises the possibility that it may function similarly in vivo.

pNiXa, a Ni(2+)-binding protein in Xenopus oocytes and embryos, shows identity to Ep45, an estrogen-regulated hepatic serpin.

A Ni(2+)-binding protein (pNiXa, 45 kD, pI 8.5) discovered in Xenopus embryos, was isolated from oocytes. Based on amino acid sequences, pNiXa belongs to the serpin superfamily and shows identity to the cDNA sequence of Ep45, an estrogen-regulated hepatic serpin that contains an (HX)n-motif found in eukaryotic transcription factors. Nondenatured pNiXa, purified by Ni-affinity chromatography, inhibited bovine alpha-chymotrypsin. The presence of pNiXa in embryos when they are susceptible to Ni2+, the high avidity of pNiXa for Ni2+, and the (HX)n-motif point to pNiXa as a molecular target of Ni(2+)-teratogenesis.