Engineering topics

Stan-Lotter, Helga

Publications and source records attributed to Stan-Lotter, Helga.

At least 19 records

NASA NTRS1997

The Evolution of Energy-Transducing Systems. Studies with an Extremely Halophilic Archaebacterium

The F-type ATPases are found in remarkably similar versions in the energy-transducing membranes of bacteria, chloroplasts and mitochondria (1). Thus, it is likely that they have originated early in the evolution of life, which is consistent with their function as key enzymes of cellular metabolism. The archaea (formerly called archaebacteria) are a group of microorganisms which, as shown by molecular sequencing and biochemical data, have diverged early from the main line of prokaryotic evolution (2). From studies of members of all three major groups of archaea, the halophiles, methanogens and thermoacidophiles, it emerged that they possess a membrane ATPase, which differs from the F-ATPases. The goal of this project was a comparison of the ATPase from the halophilic archaebacterium Halobacterium saccharovorum with the well-characterized F-type ATPases on the molecular level. The results were expected to allow a decision about the nature of archaebacterial ATPases, their classification as one of the known or, alternatively, novel enzyme complex, and possibly a deduction of events during the early evolution of energy-transducing systems.

Stan-Lotter, Helga↗

NASA NTRS1996

The Evolution of Energy-Transducing Systems. Studies with Archaebacteria

The dicyclohexyl carbodiimide (DCCD)- binding site of the membrane ATPase from Halobacterium saccharovorum was investigated during earlier periods of this Cooperative Agreement and was localized to a cyanogen bromide fragment of subunit 2 from amino acids 379 (Glu) to 442 (Met). Although the exact position of the reactive amino acid (probably a glutamic acid) has not yet been determined, the data, together with recently obtained immuno reactions and sequences of Cyanogen Bromide (CNBr) fragments from E.coli F-ATPase, suggested subunit interactions in the halobacterial ATPase which had not been recognized before. They also provided evidence for the presence of a gamma subunit in the halobacterial ATPase, and for a stretch of a amino acids similar to the 'catch' between beta and gamma in bovine F-ATPase. The evolutionary implications of these findings are twofold: first, halobacterial (or archaebacterial) ATPases appear as complex as those from higher organisms - no simpler versions of these membrane enzymes are known to date; second, a monophyletic origin of the energy-transducing ATPases is becoming more apparent, and - together with other data - the split into V- and F-ATPases may have occurred much later than had been previously thought (i.e., after the split into Archaea and Bacteria). Other work included the characterization of an extremely halophilic isolate (Halococcus salifodinae ) from Permian salt sediments. This organism appeared to be an autotrophic halobacterium; its incorporation of C02 was investigated.

Stan-Lotter, Helga↗

NASA NTRS1996

Western Blot of Stained Proteins from Dried Polyacrylamide Gels

Western blotting of proteins is customarily performed following their separation on polyacrylamide gels, either prior to staining (1) or, as recently reported, following staining (2). We describe here Western blotting with stained gels, which had been dried and some of which had been stored for years. This procedure permits immunological analysis of proteins, to which antisera may have become available only later, or where the application of newly developed sensitive detection methods is desired. Once rehydration of the gels is achieved, proteins can be-transferred to blotting membranes by any appropriate protocol. Proteins stained with Coomassie Blue have to be detected with a non-chromogenic method, such as the film-based enhanced chemiluminescence (ECL)2) procedure (3). Silver stained proteins, which transfer in the colorless form, may be visualized by any detection method, although, because of the usually very low amounts of proteins, detection by ECL is preferable. Blotting of stained proteins from rehydrated gels is as rapid and as quantitative as from freshly prepared gels, in contrast to blotting from wet stained gels, which requires extensive washing and results in low transfer efficiency (2). Together with a photographic record of the gel pattern, unambiguous identification of immunoreactive proteins from complex mixtures is possible. Some further applications of this work are discussed.

Gruber, Claudia↗

NASA NTRS1995

Inhibition of the ATPase from Halobacterium Saccharovorum by Thiol Inhibitors: Evidence for the Presence of More Than One Essential Cysteinyl Residue

The vacuolar-like ATPase from Halobacterium saccha vorum is inhibited by N-ethylmaleimide and p-chloromercudphenylsulfonate. The failure of adenine nucleotides to protect against p-chloromercuriphenyisulfonate inhibition, of p-chloromercuriphenylsulfonate to protect against N-ethylmaleimide inhibition, and the difference in the temperature dependence of inactivation infers that the enzyme contains at least two thiols that are essential for enzyme activity. CNBr cleavage of C-14-N-ethylmaleimide labeled subunit results in two radioactive peptides that locates the N-ethylmaleimide-reactive cysteinyl residue as cysteine-262 in the H. salinarium sequence.

Hochstein, Lawrence I.↗

NASA NTRS1994

Halococcus salifodinae sp. nov., an Archaeal Isolate from an Austrian Salt Mine

A novel extremely halophilic archaeon (archaebacterium) was isolated from rock salt obtained from an Austrian salt mine. The deposition of the salt is thought to have occurred during the Permian period (225 x 106 to 280 x 10(exp 6) years ago). This organism grew over a pH range of 6.8 to 9.5. Electron microscopy revealed cocci in tetrads or larger clusters. The partial 16S rRNA sequences, polar lipid composition, and menaquinone content suggested that this organism was related to members of the genus Halococcus, while the whole-cell protein patterns, the presence of several unknown lipids, and the presence of pink pigmentation indicated that it was different from previously described coccoid halophiles. We propose that this isolate should be recognized as a new species and should be named Halococcus salifodinae. The type strain is Bl(sub p) (= ATCC 51437 = DSM 8989). A chemotaxonomically similar microorganism was isolated from a British salt mine.

Denner, Ewald B. M.↗

NASA NTRS1994

The evolution of energy-transducing systems. Studies with archaebacteria

N-ethylmaleimide (NEM) inhibits the ATPase of H. saccharovorum in a nucleotide protectable manner. The bulk of C-14 NEM is incorporated into subunit one. Cyanogen bromide cleavage of labeled subunit one indicated that NEM bound to a peptide of a Mr of about 8,900. Thus, Cys 262 (H. salinarium numbering) may be the NEM binding site. Cyanogen bromide fragments have been submitted for sequencing. To prove the presence of three Cys residues in subunit one, alkaline cleavage following treatment with NTCB was carried out. Thiol reagents such as p-chloro mercuri phenyl sulfonate also inhibited the ATPase. However, this inhibition was not nucleotide-protectable, suggesting a different location and role for the PCMS-sensitive Cys. The proteolipid which was extracted with chloroform/methanol from the membranes of H. saccharovorum cross-reacted with an antiserum against subunit c (the DCCD-binding protein) of Escherichia coli. Following labeling of membranes from H. saccharovorum with C-14 DCCD under conditions, which inhibited ATP synthesis, the isotope was incorporated into one protein of Mr of about 6,500. Thus, the proteolipid of H. saccharovorum and the DCCD-labeled peptide may be identical. If so, these results suggest that the proteolipid is a component of the membrane sector of an archaeal F-type ATP synthase.

Stan-Lotter, Helga↗

NASA NTRS1993

The evolution of energy-transducing systems: Studies with archaebacteria

N-ethylmaleimide (NEM) inhibits the ATPase of H. saccharovorum in a nucleotide protectable manner. The bulk of 14C-NEM is incorporated into subunit 1. Inhibition kinetics indicated a single binding site. To determine the sequence around this site, cyanogen bromide peptides of NEM-labeled ATPase enzyme were prepared and separated on Tris-Tricine gels. Autoradiography indicated that the NEM binding site is probably located in a fragment of Mr 10-12 K. This result will be confirmed by N-terminal sequencing of the peptide. Since the cysteinyl residue, to which NEM is bound, may be located at the C-terminal end, purification and proteolytic treatment of the 10 K peptide will be required. One inhibitor of V-type ATPases, fluoresceinisothiocyanate (FITC) inhibited also the ATPase of H. saccharovorum. Preliminary results indicated protection against inhibition by nucleotides. Localization of the binding sited to the major subunits is in progress. An extraction procedure for the membrane sector of the ATPase complex of H. saccharovorum yielded a preparation which was enriched in a peptide of Mr 5 500. Experiments to test the immunological crossreaction with subunit c from the Escherichia coli F-type ATPase and the labeling with 14C-DCCD are currently carried out. Polyclonal antiserum to the smaller of the major subunits of the ATPase from H. saccharovorum (subunit ll) reacts in Western blots strongly with the alpha and beta subunits of the F1 ATPase of E. coli, suggesting highly conserved regions on both types of ATPases. To elucidate further the regions of homology, cyanogen bromide peptides of the beta subunits were prepared for sequence analysis.

Stan-Lotter, Helga↗

NASA NTRS1993

Comparison of membrane ATPases from extreme halophiles isolated from ancient salt deposits

Halophilic microorganisms were isolated from Triassic and Permian salt deposits. Two were rods and grew as red colonies; another was a coccus and produced pink colonies. The rods lysed in solutions that lacked added sodium chloride. Growth of all isolates was inhibited by aphidicolin and their bulk-proteins were acidic as judged from isoelectric focusing. Therefore, these organisms were tentatively identified as extreme halophiles. Whole cell proteins patterns of the isolates following gel electrophoresis were distinct and differed from those of representative type strains of halophilic bacteria. The membrane ATPases from the rods were similar to the enzyme from Halobacterium saccharovorum with respect to subunit composition, enzymatic properties and immunological cross-reaction, but differed slightly in amino acid composition. If the age of the microbial isolated is similar to that of the salt deposits, they can be considered repositories of molecular information of great evolutionary interest.

Stan-Lotter, Helga↗

NASA NTRS1993

On the isolation of halophilic microorganisms from salt deposits of great geological age

From salt sediments of Triassic or Permian age from various locations in the world halophilic microorganisms were isolated. Molecular characteristics of several of the isolates suggested they belong to the archaebacteria. One group appears to represent novel strains; several properties of one such isolate, strain BIp, are described here. The existence of viable microorganisms in ancient sediment would have great implications with respect to our notions on evolution, the research for life in extraterrestrial environments, and the longterm survival of functional biological structures. Of crucial importance is thus the question if these microorganisms existed in the salt since the time of deposition or invaded at some later date. Some suggestions to address these issues experimentally are discussed.

Stan-Lotter, Helga↗

NASA NTRS1993

Comparison of Membrane ATPases from Extreme Halophiles Isolated from Ancient Salt Deposits

Halophilic microorganisms were isolated from Triassic and Permian salt deposits. Two were rods and grew as red colonies; another was a coccus and produced pink colonies. The rods lysed in solutions that lacked added sodium chloride. Growth of all isolates was inhibited by aphidicolin and their bulk proteins were acidic as judged from isoelectric focusing. Therefore, these organisms were tentatively identified as extreme halophiles. Whole cell proteins patterns of the isolates following gel electrophoresis were distinct and differed from those of representative type strains of halophilic bacteria. The membrane ATPases from the rods were similar to the enzyme from Halobacterium saccharovorum with respect to sub unit composition. enzymatic properties and immunological cross-reaction, but differed slightly in amino acid composition. If the age of the microbial isolated is similar to that of the salt deposits, they can be considered repositories of molecular information of great evolutionary interest.

Stan-Lotter, Helga↗

NASA NTRS1993

On the Isolation of Halophilic Microorganisms from Salt Deposits of Great Geological Age

From salt sediments of Triassic or Permian ace from various locations in the world halophilic microorganisms were isolated. Molecular characteristics of several of the isolates suggested they belong to the archaebacteriae. One group appears to represent novel strains; several properties or one such isolate, strain BIp, are described here. The existence of viable microorganisms in ancient sediments would have great implications with respect to our notions on evolution, the search for life in extraterrestrial environments and the long- term survival of functional biological structures. Of crucial importance is thus the question if these microorganisms existed in the salt since the time of deposition or invaded at some later date. Some suggestions to address these issues experimentally are discussed.

Stan-Lotter, Helga↗

NASA NTRS1992

The evolution of energy-transducing systems. Studies with an extremely halophilic archaebacterium

The F-type ATPases are found in remarkably similar versions in the energy-transducing membranes of eubacteria, chloroplasts, and mitochondria. Thus, it is likely that they have originated early in the evolution of life, which is consistent with their function as key enzymes of cellular metabolism. The archaebacteria are a group of microorganisms which, as shown by molecular sequencing and biochemical data, have diverged early from the main line of prokaryotic evolution. From studies of members of all three major groups of archaebacteria - the halophiles, methanogens, and thermoacidophiles - it emerged that they possess a membrane ATPase which differs from the F-ATPases. The goal of this project was a comparison of the ATPase from the halophilic archaebacterium Halobacterium saccharovorum with the well-characterized F-type ATPases on the molecular level. Amino acid sequences of critical regions of the enzyme were to be determined, as well as immunoreactions of single subunits in the search for common epitopes. The results were expected to allow a decision about the nature of archaebacterial ATPases, their classification as one of the known or, alternatively, novel enzyme complexes, and possibly deduction of events during the early evolution of energy-transducing systems.

Stan-Lotter, Helga↗

NASA NTRS1992

Nucleotide-protectable labeling of sulfhydryl groups in subunit I of the ATPase from Halobacterium saccharovorum

The membrane ATPase from Halobacterium saccharovorum was purified as described by Hochstein et al. (1987) and was incubated with C-14 labeled N-ethylmaleimide (NEM), with and without adenine nucleotides, to determine the effect of nucleotides on the enzyme labeling. It was found that NEM incorporates into the 87,000-Da subunit (subunit I) of the enzyme and that the conditions for enzyme modification are similar to those which result in the inhibition of the enzyme activity. The presence of ATP, ADP, and AMP was found to reduce both the inhibitor incorporation and enzyme inhibition. It was shown that the reaction involves a modification of thiol groups.

Sulzner, Michael↗

NASA NTRS1992

Nucleotide-Protectable Labeling of Sulfhydryl Groups in Subunit I of the ATPhase from Halobacterium Saccharovorum

A membrane-bound ATPase from the archaebacterium Halobacterium saccharovorum is inhibited by N-ethyl-maleimide in a nucleotide-protectable manner. When the enzyme was incubated with N-[C-14]jethylmaleimide, the bulk of radioactivity was as- sociated with the 87,000-Da subunit (subunit 1). ATP, ADP, or AMP reduced incorporation of the inhibitor. No charge shift of subunit I was detected following labeling with N-ethylmaleimide, indicating an electroneutral reaction. The results are consistent with the selective modification of sulfhydryl groups in subunit I at or near the catalytic site and are further evidence of a resemblance between this archaebacterial ATPase and the vacuolar-type ATPases.

Sulzner, Michael↗

NASA NTRS1992

Purification and properties of an ATPase from Sulfolobus solfataricus

The paper reports properties of a sulfite-activated ATPase from Sulfolobus solfataricus, purified using ammonium sulfate precipitation, column chromatography on UltraGel and Sepharose 6B, and SDS-PAGE. The 92-fold purified enzyme had a relative molecular mass of 370,000. It could be dissociated into three subunits with respective molecular masses of 63,000, 48,000, and 24,000. The ATPase activity was found to be inhibitable by nitrate, N-ethylmaleimide (which bound predominantly to the largest subunit), and 4-chloro 7-nitrobenzofurazan, but not by azide, quercetin, or vanadate. While the ATPase from S. solfataricus shared a number of properties with the S. acidocaldarius ATPase, there were also significant differences suggesting the existence of several types of archaeal ATPases.

Hochstein, Lawrence I.↗

NASA NTRS1992

Purification and Properties of an ATPase from Sulfolobus solfataricus

A sulfite-activated ATPase isolated from Sulfolobus solfataricus had a relative molecular mass of 370,000. It was composed of three subunits whose relative molecular masses were 63,000, 48,000, and 24,000. The enzyme was inhibited by the vacuolar ATPase inhibitors nitrate and N-ethylmaleimide; 4-chloro-7-nitrobenzo-furazan (NBD-Cl) was also inhibitory. N-Ethylmaleimide was predominately bound to the largest subunit while NBD-CL was bound to both subunits. ATPase activity was inhibited by low concentrations of p-chloromercuri-phenyl sulfonate and the inhibition was reversed by cysteine which suggested that thiol groups were essential for activity. While the ATPase from S. solfataricus shared several properties with the ATPase from S. acidocaldarius there were significant differences. The latter enzyme was activated by sulfate and chloride and was unaffected by N-ethylmaleimide, whereas the S. solfataricus ATPase was inhibited by these anions as well as N-ethyimaleimide. These differences as well as differences that occur in other vacuolar-like ATPases isolated from the methanogenic and the extremely halophilic bacteria suggest the existence of several types of archaeal ATPases, none of which have been demonstrated to synthesize ATP.

Hochstein, Lawrence I.↗

NASA NTRS1992

Presence of methyl sterol and bacteriohopanepolyol in an outer-membrane preparation from Methylococcus capsulatus (Bath)

Cytoplasmic/intracytoplasmic and outer membrane preparations of Methylococcus capsulatus (Bath) were isolated by sucrose density gradient centrifugation of a total membrane fraction prepared by disruption using a French pressure cell. The cytoplasmic and/or intracytoplasmic membrane fraction consisted of two distinct bands, Ia and Ib (buoyant densities 1.16 and 1.18 g ml (exp -1), respectively) that together contained 57% of the protein, 68% of the phospholipid, 73% of the ubiquinone and 89% of the CN-sensitive NADH oxidase activity. The only apparent difference between these two cytoplasmic bands was a much higher phospholipid content for Ia. The outer membrane fraction (buoyant density 1.23-1.24 g ml (exp -1)) contained 60% of the lipopolysaccharide-associated, beta-hydroxypalmitic acid, 74% of the methylsterol, and 66% of the bacteriohopanepolyol (BHP); phospholipid to methyl sterol or BHP ratios were 6:1. Methanol dehydrogenase activity and a c-type cytochrome were also present in this outer membrane fraction. Phospholipase A activity was present in borh the cytoplasmic membrane and outer membrane fractions. The unique distribution of cyclic triterpenes may reflect a specific role in conferring outer membrane stability in this methanotrophic bacterium.

Jahnke, Linda L.↗

NASA NTRS1991

The evolution of energy-transducing systems. Studies with an extremely halophilic archaebacterium

The halobacterial ATPase was labeled with C-14-dicyclohexylcarbodiimide and subunit 2 of the enzyme was prepared by electroelution. Subunit 2 was cleaved by several chemical and enzymatic procedures for further preparation of peptides. Immunoreactions (Western blotting) of halobacterial membranes were performed with an antiserum against subunit A of the vacuolar ATPase from Neurospora crassa. A 85 K band (subunit 1) from the membranes of H saccharovorum and from two halobacterial isolates, which were isolated from Permian salt sediments, reacted strongly with the antiserum. The ATPase from the latter isolates resembled the ATPase from H saccharovorum, but had a higher content of acidic amino acids. If it can be verified that the age of the bacterial isolates is in the same range as when deposition of salt occurred, an extremely interesting system for the study of evolutionary questions would be available, since the salt-embedded bacteria presumably did not undergo mutational and selectional events.

Stan-Lotter, Helga↗