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PubMed1993

Isolation and structure determination of novel phosphatidylinositol turnover inhibitors, piericidin B5 and B5 N-oxide, from Streptomyces sp.

In the course of a screening program for inhibitors of epidermal growth factor (EGF)-induced phosphatidylinositol turnover in human epidermoid carcinoma cell line A431, we discovered two novel compounds of the piericidin family from the culture broth and mycelia of a Streptomyces strain MJ288-OF3. We named them piericidin B5 and B5 N-oxide. By NMR and mass spectrometric analyses, the molecular formulas of piericidin B5 and B5 N-oxide were determined to be C27H41NO4 (MW 443) and C27H41NO5 (MW 459), respectively. Piericidin B5 and B5 N-oxide inhibited phosphatidylinositol turnover of A431 cells with IC50S of 10.0 micrograms/ml and 1.1 micrograms/ml, respectively.

Anti-Bacterial Agents↗
PubMed1979

Observations on the biological roles of sulphatases.

Until recently little was known about the biological roles played by sulphatase enzymes, owing in part to the selection of assay substrates that were convenient but only removely related to the natural substrates. Once this was recognized the elucidation of function proceeded more rapidly. Microbial sulphatases appear to have roles to play in the nutrition of individual microorganisms whilst collectively they enable sulphur, returned to soils and waters in the form of sulphate esters, to be made available for recycling. In contrast, with one or two important exceptions, mammalian sulphatases are concerned, in association with other enzymes, with the turnover of macromolecules. Still defying understanding are the roles of sulphatases acting on adenosine 5'-phosphosulphate (APT) and 3'-phosphoadenosine 5'-phosphosulphate (PAPA). APS sulphatases have now been purified from ox-liver lysosomes and cytosol and from a strain of Comamonas terrigena. The lysosomal enzyme shows wide specificity and can hydrolyse ATP, ADP, FED and pyrophosphate. The cytosol enzyme is apparently specific and may be active only when cellular concentrations of ATP are low. The bacterial enzyme is also specific and has properties and a cellular localization that suggest the possibility of its involvement in sulphate transport.

Adenosine Monophosphate↗
PubMed2001

Enhanced biodegradation of methylhydrazine and hydrazine contaminated NASA wastewater in fixed-film bioreactor.

The aerobic biodegradation of National Aeronautics and Space Administration (NASA) wastewater that contains mixtures of highly concentrated methylhydrazine/hydrazine, citric acid and their reaction product was studied on a laboratory-scale fixed film trickle-bed reactor. The degrading organisms, Achromobacter sp., Rhodococcus B30 and Rhodococcus J10, were immobilized on coarse sand grains used as support-media in the columns. Under continuous flow operation, Rhodococcus sp. degraded the methylhydrazine content of the wastewater from a concentration of 10 to 2.5 mg/mL within 12 days and the hydrazine from approximately 0.8 to 0.1 mg/mL in 7 days. The Achromobacter sp. was equally efficient in degrading the organics present in the wastewater, reducing the concentration of the methylhydrazine from 10 to approximately 5 mg/mL within 12 days and that of the hydrazine from approximately 0.8 to 0.2 mg/mL in 7 days. The pseudo first-order rate constants of 0.137 day(-1) and 0.232 day(-1) were obtained for the removal of methylhydrazine and hydrazine, respectively, in wastewater in the reactor column. In the batch cultures, rate constants for the degradation were 0.046 and 0.079 day(-1) for methylhydrazine and hydrazine respectively. These results demonstrate that the continuous flow bioreactor afford greater degradation efficiencies than those obtained when the wastewater was incubated with the microbes in growth-limited batch experiments. They also show that wastewater containing hydrazine is more amenable to microbial degradation than one that is predominant in methylhydrazine, in spite of the longer lag period observed for hydrazine containing wastewater. The influence of substrate concentration and recycle rate on the degradation efficiency is reported. The major advantages of the trickle-bed reactor over the batch system include very high substrate volumetric rate of turnover, higher rates of degradation and tolerance of the 100% concentrated NASA wastewater. The results of the present laboratory scale study will be of great importance in the design and operation of an industrial immobilized biofilm reactor for the treatment of methylhydrazine and hydrazine contaminated NASA wastewater.

Alcaligenes↗
PubMed1965

FERMENTATION CAPACITY AS A MEASURE OF NET GROWTH OF RUMEN MICROORGANISMS.

A simple technique for measuring the rate of fermentation of rumen microorganisms is described. It allows quick preparation and handling of the rumen sample immediately after collection. The average rate of fermentation of rumen samples collected from a lactating cow fed on alfalfa hay and concentrate in the ratio of 2:1 was very similar to the rate obtained by other methods. On the assumption that when substrate is in excess, the fermentation rate is proportional to the total microbial cells, the method was used to estimate the net growth of rumen microorganisms. The maximal fermentation rate of subsamples, taken at the beginning and after 1 hr of incubation of a sample, was measured. The results indicate a net average growth of 8% per hr, or 192% per day, in approximate agreement with rumen turnover time. The highest net growth does not necessarily coincide with the highest gas-production rate in the rumen, in part because the bicarbonate concentration in the rumen contents varies. In a cow fed on hay and concentrate, the net growth was lowest before feeding and immediately after feeding.

Animals↗
PubMed1997

Potential responses of soil organic carbon to global environmental change.

Recent improvements in our understanding of the dynamics of soil carbon have shown that 20-40% of the approximately 1,500 Pg of C stored as organic matter in the upper meter of soils has turnover times of centuries or less. This fast-cycling organic matter is largely comprised of undecomposed plant material and hydrolyzable components associated with mineral surfaces. Turnover times of fast-cycling carbon vary with climate and vegetation, and range from <20 years at low latitudes to >60 years at high latitudes. The amount and turnover time of C in passive soil carbon pools (organic matter strongly stabilized on mineral surfaces with turnover times of millennia and longer) depend on factors like soil maturity and mineralogy, which, in turn, reflect long-term climate conditions. Transient sources or sinks in terrestrial carbon pools result from the time lag between photosynthetic uptake of CO2 by plants and the subsequent return of C to the atmosphere through plant, heterotrophic, and microbial respiration. Differential responses of primary production and respiration to climate change or ecosystem fertilization have the potential to cause significant interrannual to decadal imbalances in terrestrial C storage and release. Rates of carbon storage and release in recently disturbed ecosystems can be much larger than rates in more mature ecosystems. Changes in disturbance frequency and regime resulting from future climate change may be more important than equilibrium responses in determining the carbon balance of terrestrial ecosystems.

Journal Article↗
PubMed1973

Microbial phospholipid synthesis as a marker for microbial protein synthesis in the rumen.

Phosphate uptake into intracellular inorganic phosphorus and cellular phospholipids and the relationship between cell growth and phospholipid synthesis were studied with suspensions of washed ruminal bacteria in vitro with (33)P-phosphorus. It was shown that ruminal bacteria accumulated inorganic phosphate at a low rate when incubated without substrate. Upon the addition of substrate, the rate of inorganic phosphorus uptake into the cells increased markedly, and phospholipid synthesis and cell growth commenced. There was a highly significant relationship (r = 0.98; P < 0.01) between phospholipid synthesis and cell growth. The specific activity of the intracellular inorganic phosphorus did not equilibrate with phosphorus medium. When ruminal contents from sheep fed a high or low protein diet were incubated in vitro, the rate of (33)P incorporation into microbial phospholipids was higher for the high protein diet. Since there was a high relationship between phospholipid synthesis and growth, rumen contents were collected before and various times after feeding and incubated with (33)P-phosphorus in vitro. The short-term, zero time approach was used to measure the rate of microbial phospholipid synthesis in whole rumen contents. In these studies the average specific activity of the intracellular inorganic phosphorus was used to represent the precursor pool specific activity. Microbial phospholipid synthesis was then related to protein (N x 6.25) synthesis with appropriate nitrogen-to-phospholipid phosphorus ratios. Daily true protein synthesis in a 4-liter rumen was 185 g. This represents a rate of 22 g of protein synthesized per 100 g of organic matter digested. These data were also corrected for ruminal turnover. On this basis the rate of true protein synthesis in a 4-liter rumen was 16.1 g of protein per 100 g of organic matter digested. This value represents a 30-g digestible protein-to-Mcal digestible energy ratio which is adequate for growing calves and lambs.

Animal Feed↗
PubMed2003

Successional changes of Collembola and soil microbiota during forest rotation.

Dynamic approaches to forest ecosystems are surprisingly rare. Here we report about successional changes in collembolan community structure and microbial performances during forest rotation. The study was carried out in a chronosequence of four spruce forest stands (5-, 25-, 45-, and 95 years old; Tharandter forest, Germany). CO2 release significantly increased after clear-cutting and the amount of C stored in the organic layer subsequently declined. The early phase of forest rotation was characterized by a very active decomposer microflora, stimulation of both fungi and bacteria as well as by a high abundance of surface-oriented Collembola. In addition, collembolan species turnover was accelerated. While the biomass of fungi further increased at intermediate stages of forest rotation, the metabolic activity of the microflora was low, the functional diversity of bacteria declined and the collembolan community became impoverished. Euedaphic species dominated during this stage of forest development. These changes can be explained by both reduction in microhabitat diversity and depletion of food sources associated with an accumulation of recalcitrant soil organic matter. Results of the General Regression Model procedure indicate a shift from specific associations between collembolan functional groups and microbiota at the early stage of forest rotation to a more diffuse pattern at intermediate stages. Though the hypothesis that Collembola are relatively responsive to changes in environmental conditions is confirmed, consistently high community similarity suggests a remarkable persistence of some components of microarthropod assemblages. Our study provides evidence for substantial ecosystem-level implications of changes in the soil food web during forest rotation. Moreover, correlations between bacterial parameters and Collembola point to the overarching impact of differences in the composition of the microbial community on microarthropods.

Animals↗
PubMed1988

Human kidney cathepsins B and L. Characterization and potential role in degradation of glomerular basement membrane.

Cathepsins B and L were purified from human kidney. SDS/polyacrylamide-gel electrophoresis demonstrated that cathepsins B and L, Mr 27000-30000, consist of disulphide-linked dimers, subunit Mr values 22000-25000 and 5000-7000. The pH optimum for the hydrolysis of methylcoumarylamide (-NHMec) substrates (see below) is approx. 6.0 for each enzyme. Km and kcat. are 252 microM and 364s-1 and 2.2 microM and 25.8 s-1 for the hydrolysis of Z-Phe-Arg-NHMec (where Z- represents benzyloxycarbonyl-) by cathepsins B and L respectively, and 184 microM and 158 s-1 for the hydrolysis of Z-Arg-Arg-NHMec by cathepsin B. A 10 min preincubation of cathepsin B (40 degrees C) or cathepsin L (30 degrees C) with E-64 (2.5 microM) results in complete inhibition. Under identical conditions Z-Phe-Phe-CHN2 (0.56 microM) completely inhibits cathepsin L but has little effect on cathepsin B. Incubation of glomerular basement membrane (GBM) with purified human kidney cathepsin L resulted in dose-dependent (10-40 nM) GBM degradation. In contrast, little degradation of GBM (less than 4.0%) was observed with cathepsin B. The pH optimum for GBM degradation by cathepsin L was 3.5. Cathepsin L was significantly more active in degrading GBM than was pancreatic elastase, trypsin or bacterial collagenase. These data suggest that cathepsin L may participate in the lysosomal degradation of GBM associated with normal GBM turnover in vivo.

Basement Membrane↗
PubMed1990

Genetic heterogeneity of Porphyromonas (Bacteroides) gingivalis by genomic DNA fingerprinting.

This study describes the use of total genomic DNA fingerprinting with the use of restriction endonucleases to characterize clinical isolates of Porphyromonas gingivalis (Bacteroides gingivalis) obtained from patients with periodontitis or with root-canal infections. The majority of independent isolates had a unique DNA fingerprint, indicating extensive genetic heterogeneity within this species. Twenty-nine distinct DNA fingerprints were found among the 33 isolates investigated. This is in contrast to biotyping and serotyping, where only one type and three types, respectively, have been reported. The observed heterogeneity indicates that DNA fingerprinting is a sensitive measure of genetic dissimilarity between P. gingivalis isolates and is able to characterize individual isolates. These results have ecological implications, indicating that there is considerable natural diversity in the global population of P. gingivalis, and that there are likely to be relatively large numbers of genetically distinct clonal lines. Furthermore, DNA fingerprinting is a sensitive and powerful tool for longitudinal and cross-sectional epidemiological studies. This technique provides far greater discrimination between isolates than either biotyping or serotyping, and will be most helpful in, for example, the analysis of distribution of clonal lines within one periodontal patient, or the analysis of the transmission to and turnover of strain populations within a patient population, since the probability of two strains with the same DNA fingerprint being found by chance is small.

Bacteroides↗
PubMed1981

Rates of microbial transformation of polycyclic aromatic hydrocarbons in water and sediments in the vicinity of a coal-coking wastewater discharge.

To facilitate predictions of the transport and fate of contaminants at future coal conversion facilities, rates of microbial transformation of polycyclic aromatic hydrocarbons were measured in stream water and sediment samples collected in the vicinity of a coal-coking treated wastewater discharge from November 1977 through August 1979. Six radiolabeled polycyclic aromatic hydrocarbons were incubated with sediment and water samples; CO(2), cell-bound C, and polar transformation products were isolated and quantified. Whereas CO(2) and bound C were major transformation products in sediment assays, soluble polar C dominated transformation in water samples. Mean rate constants (measured at 20 degrees C) in sediments collected downstream from the effluent outfall were 7.8 x 10 h (naphthalene), 1.6 x 10 h (anthracene), and 3.3 x 10 h [benz(a)anthracene], which corresponded to turnover times of 13, 62, and 300 h, respectively. No unequivocal evidence for transformation of benzo(a)pyrene or dibenz(a,h)anthracene was obtained. Only naphthalene and anthracene transformations were observed in water samples; rate constants were consistently 5- and 20-fold lower, respectively, than in the corresponding sediment samples. The measured rate constants for anthracene transformation in July 1978 sediment samples were not related to total heterotroph numbers. In late July 1978, the effluent was diverted from the primary study area; however, no differences were observed either in transformation rate constants or in the downstream/upstream sediment rate constant ratio. These results are consistent with the hypothesis that continuous inputs of polycyclic aromatic hydrocarbons result in an increased ability within a microbial community to utilize certain polycyclic aromatic hydrocarbons. However, because transformation rates remained elevated for more than 1 year after removal of the polycyclic aromatic hydrocarbon source, microbial communities may shift only slowly in response to changes in polycyclic aromatic hydrocarbon concentrations.

Journal Article↗
PubMed1996

Acetogenic capacities and the anaerobic turnover of carbon in a kansas prairie soil.

To assess the anaerobic capacities of a temperate grassland soil, a Kansas prairie soil was incubated anaerobically as either soil-water (1:2) suspensions or as soil microcosms at 78% soil water-holding capacity. Prairie soil formed acetate and CO(inf2) as the two main initial carbonaceous products from the anaerobic turnover of endogenous organic matter. Metabolic capacities of soil suspensions and microcosms were similar. Rates of acetate formation from endogenous organic matter in soil-water suspensions incubated at 40, 30, and 15(deg)C approximated 3.3, 2.4, and 1.1 (mu)g of acetate per g (dry weight) of soil per h, respectively. Supplemental H(inf2) and CO(inf2) were subject to consumption with the apparent concomitant synthesis of acetate in both soil suspensions and soil microcosms. In soil microcosms, rates of H(inf2)-dependent acetogenesis at 30 and 55(deg)C were nearly equivalent. The uptake of supplemental H(inf2) was not coupled to methanogenesis under any condition examined. These anaerobic activities were relatively stable when soils were subjected to either aerobic drying or alternating periods of O(inf2) enrichment. On the basis of the formation of nitrogen (N(inf2)), denitrification was engaged during anaerobic incubation periods; nitrous oxide (N(inf2)O) was also formed under certain conditions. Although extended incubation of soil induced the delayed methanogenic turnover of acetate, acetate was subject to immediate turnover under either O(inf2)- or nitrate-enriched conditions. These studies support the following concepts: (i) obligately anaerobic bacteria such as acetogenic bacteria are stable to periods of aerobiosis and are active in the anaerobic microsites of oxic soils, and (ii) acetate synthesized in anaerobic microsites of oxic terrestrial soils constitutes a trophic link to both aerobic and anaerobic microbial communities.

Journal Article↗
PubMed1985

Gas metabolism evidence in support of the juxtaposition of hydrogen-producing and methanogenic bacteria in sewage sludge and lake sediments.

We developed new techniques to measure dissolved H(2) and H(2) consumption kinetics in anoxic ecosystems that were not dependent on headspace measurements or gas transfer-limited experimentation. These H(2) metabolism parameters were then compared with measured methane production rates, and estimates of H(2) production and interspecies H(2) transfer were made. The H(2) pool sizes were 205 and 31 nM in sewage sludge from an anaerobic digestor and in sediments (24 m) from Lake Mendota, respectively. The H(2) turnover rate constants, as determined by using in situ pool sizes and temperatures, were 103 and 31 h for sludge and sediment, respectively. The observed H(2) turnover rate accounted for only 5 to 6% of the expected H(2)-CO(2)-dependent methanogenesis in these ecosystems. Our results are in general agreement with the results reported previously and are used to support the conclusion that most of the H(2)-dependent methanogenesis in these ecosystems occurs as a consequence of direct interspecies H(2) transfer between juxtapositioned microbial associations within flocs or consortia.

Journal Article↗
PubMed2005

Stable isotope probing analysis of the influence of liming on root exudate utilization by soil microorganisms.

Rhizosphere microorganisms play an important role in soil carbon flow, through turnover of root exudates, but there is little information on which organisms are actively involved or on the influence of environmental conditions on active communities. In this study, a 13CO2 pulse labelling field experiment was performed in an upland grassland soil, followed by RNA-stable isotope probing (SIP) analysis, to determine the effect of liming on the structure of the rhizosphere microbial community metabolizing root exudates. The lower limit of detection for SIP was determined in soil samples inoculated with a range of concentrations of 13C-labelled Pseudomonas fluorescens and was found to lie between 10(5) and 10(6) cells per gram of soil. The technique was capable of detecting microbial communities actively assimilating root exudates derived from recent photo-assimilate in the field. Denaturing gradient gel electrophoresis (DGGE) profiles of bacteria, archaea and fungi derived from fractions obtained from caesium trifluoroacetate (CsTFA) density gradient ultracentrifugation indicated that active communities in limed soils were more complex than those in unlimed soils and were more active in utilization of recently exuded 13C compounds. In limed soils, the majority of the community detected by standard RNA-DGGE analysis appeared to be utilizing root exudates. In unlimed soils, DGGE profiles from 12C and 13C RNA fractions differed, suggesting that a proportion of the active community was utilizing other sources of organic carbon. These differences may reflect differences in the amount of root exudation under the different conditions.

Bacteria↗
PubMed2002

Ageing and infection.

Average life expectancy throughout developed countries has rapidly increased during the latter half of the 20th century and geriatric infectious diseases have become an increasingly important issue. Infections in the elderly are not only more frequent and more severe, but they also have distinct features with respect to clinical presentation, laboratory results, microbial epidemiology, treatment, and infection control. Reasons for increased susceptibility include epidemiological elements, immunosenescence, and malnutrition, as well as a large number of age-associated physiological and anatomical alterations. Moreover, ageing may be the cause of infection but infection can also be the cause of ageing. Mechanisms may include enhanced inflammation, pathogen-dependent tissue destruction, or accelerated cellular ageing through increased turnover. In most instances, treatment of infection leads to a satisfactory outcome in the elderly. However, in palliative care situations and in patients with terminal dementia, the decision whether or not to treat an infectious disease is becoming a difficult ethical issue.

Aging↗
PubMed1990

Age-related thermal stability and susceptibility to proteolysis of rat bone collagen.

The shrinkage temperature (Ts) and the pepsin-solubilizability of collagen fibrils in bone matrix obtained from decalcified femur diaphysis from 2-, 5-, 15- and 25-month-old rats were found to decrease with age. Digestion with human fibroblast collagenase dissolved less than half of the collagen, whereas sequential treatment by pepsin followed by collagenase resulted in its complete dissolution. This result shows that collagenase and a telopeptide-cleaving enzyme, when acting in an appropriate sequence, have a great potential for the degradation of bone collagen. The 'melting' profile of the pepsin-solubilized collagen showed a biphasic transition with transition peak at 35.9 degrees C and 40.8 degrees C. With increasing age an increasing proportion of the collagen 'melted' in the transition peak at 35.9 degrees C (pre-transition), and the 'melting' temperature (Tm) of the collagen decreased in parallel with Ts in relation to age. Both Ts and Tm decreased by 3 degrees C in the age span investigated. The age-related change in Ts could therefore be accounted for by the decrease in molecular stability. The collagenase-cleavage products of the bone collagen obtained by the sequential treatment with pepsin and collagenase showed only one peak transition (at 35.1 degrees C), and the Tm for the products was independent of age. The results indicate that the pre-transition for the pepsin-solubilized collagen is due to an age-related decrease in thermal stability may have implications for the mechanical strength and turnover of the bone collagen. In contrast with bone collagen, soft-tissue collagen showed neither the age-dependency of thermal stability nor the characteristic biphasic 'melting' profile.

Aging↗
PubMed2005

Microbial dimethylsulfoxide and trimethylamine-N-oxide respiration.

Over the last two decades, the biochemistry and genetics of dimethylsulfoxide (DMSO) and trimethylamine-N-oxide (TMAO) respiration has been characterised, particularly in Escherichia coli marine bacteria of the genus Shewanella and the purple phototrophic bacteria, Rhodobacter sphaeroides and R. capsulatus. All of the enzymes (or catalytic subunits) involved the final step in DMSO and TMAO respiration contain a pterin molybdenum cofactor and are members of the DMSO reductase family of molybdoenzymes. In E. coli, the dimethylsulfoxide reductase (DmsABC) can be purified from membranes as a complex, which exhibits quinol-DMSO oxidoreductase activity. The enzyme is anchored to the membrane via the DmsC subunit and its catalytic subunit DmsA is now considered to face the periplasm. Electron transfer to DmsA involves the DmsB subunit, which is a polyferredoxin related to subunits found in other molybdoenzymes such as nitrate reductase and formate dehydrogenase. A characteristic of the DmsAB-type DMSO reductase is its ability to reduce a variety of S- and N-oxides. E. coli contains a trimethylamine-N-oxide reductase (TorA) that is highly specific for N-oxides. This enzyme is located in the periplasm and is connected to the quinone pool via a membrane-bound penta-haem cytochrome (TorC). DorCA in purple phototrophic bacteria of the genus Rhodobacter is very similar to TorCA with the critical difference that DorA catalyses reduction of both DMSO and TMAO. It is known as a DMSO reductase because the S-oxide is the best substrate. Crystal structures of DorA and TorA have revealed critical differences at the Mo active site that may explain the differences between substrate specificity between the two enzymes. DmsA, TorA and DorA possess a "twin arginine" N-terminal signal sequence consistent with their secretion via the TAT secretory system and not the Sec system. The enzymes are secreted with their bound prosthetic groups: this take place in the cytoplasm and the biogenesis involves a chaperone protein, which is cognate for each enzyme. Expression of the DMSO and TMAO respiratory operons is induced in response to a fall in oxygen tension. dmsABC expression is positively controlled by the oxygen-responsive transcription factor, Fnr and ModE, a transcription factor that binds molybdate. In contrast, torCAD expression is not under Fnr- or ModE-control but is dependent upon a sensor histidine kinase-response regulator pair, TorSR, which activate gene expression under conditions of low oxygen tension in the presence of N- or S-oxide. Regulation of dorCDA expression is similar to that seen for torCAD but it appears that the expression of the sensor histidine kinase-response regulator pair, DorSR is regulated by Fnr and there is an additional tier of regulation involving the ModE-homologue MopB, molybdate and the transcription factor DorX. Analysis of microbial genomes has revealed the presence of dms and tor operons in a wide variety of bacteria and in some archaea and duplicate dms and tor operons have been identified in E. coli. Challenges ahead will include the determination of the significance of the presence of the dms operon in bacterial pathogens and the determination of the significance of DMSO respiration in the global turnover of marine organo-sulfur compounds.

Dimethyl Sulfoxide↗
PubMed2004

Biophysical and kinetic analysis of wild-type and site-directed mutants of the isolated and native dehydroquinate synthase domain of the AROM protein.

Dehydroquinate synthase (DHQS) is the N-terminal domain of the pentafunctional AROM protein that catalyses steps 2 to 7 in the shikimate pathway in microbial eukaryotes. DHQS converts 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) to dehydroquinate in a reaction that includes alcohol oxidation, phosphate beta-elimination, carbonyl reduction, ring opening, and intramolecular aldol condensation. Kinetic analysis of the isolated DHQS domains with the AROM protein showed that for the substrate DAHP the difference in Km is less than a factor of 3, that the turnover numbers differed by 24%, and that the Km for NAD+ differs by a factor of 3. Isothermal titration calorimetry revealed that a second (inhibitory) site for divalent metal binding has an approximately 4000-fold increase in KD compared to the catalytic binding site. Inhibitor studies have suggested the enzyme could act as a simple oxidoreductase with several of the reactions occurring spontaneously, whereas structural studies have implied that DHQS participates in all steps of the reaction. Analysis of site-directed mutants experimentally test and support this latter hypothesis. Differential scanning calorimetry, circular dichroism spectroscopy, and molecular exclusion chromatography demonstrate that the mutant DHQS retain their secondary and quaternary structures and their ligand binding capacity. R130K has a 135-fold reduction in specific activity with DAHP and a greater than 1100-fold decrease in the kcat/Km ratio, whereas R130A is inactive.

Alcohol Oxidoreductases↗
PubMed1982

Sucrase metabolism in germfree rats.

We studied degradation of the intestinal brush-border protein sucrase-isomaltase in germfree animals as well as in ex-germfree animals and conventional controls to determine the mechanism by which intestinal bacteria alter disaccharidase levels. Our results indicate that sucrase-isomaltase turnover is as rapid in germfree animals as in the other groups and are consistent with surface removal of disaccharidases by pancreatic proteases under physiological conditions. Our results also suggest that elevated disaccharidase levels in germfree animals are in large part the consequence of an increased number of mature enterocytes, which persists for at least 2 wk after exposure of animals to a conventional microbial flora.

Animals↗