Characterization of porphobilinogen deaminase from rat liver

Porphobilinogen deaminase (porphobilinogen ammonia-lyase, EC 4.3.1.8) was isolated from rat liver. The final preparation was homogeneous according to polyacrylamide gel electrophoresis and immunodiffusion criteria. Electrophoresis of the native enzyme revealed a single band of activity which was distributed into three bands after incubation with porphobilinogen. When electrophoresed under denaturing condition it displayed a single polypeptide band with a molecular weight of 42,000 confirmed by exclusion chromatography and by sucrose density gradient centrifugation. The enzyme showed a pH optimum of 7.5 both in 0.1 M sodium phosphate and 0.05 M Tris-HCl buffer, when assayed at 37 degrees C. An isoelectric point of 4.9 for the native purified protein was found. Hepatic porphobilinogen deaminase was remarkably heat-stable showing maximum activity at 55-60 degrees C with one break in the Arrhenius plot. The kinetic behaviour of the purified enzyme followed the typical Michaelis-Menten kinetics with values of Km = 17 microM and Vmax = 29.4 units power mg in 0.1 M phosphate buffer at 37 degrees C. The amino acid composition was determined, showing that the enzyme had a low content of sulphur-containing amino acids and a considerable number of acidic residues per mol of polypeptide chain. Reagents known to interact with sulphydryl groups have small effect on rat liver enzyme activity.     

Microheterogeneity of the glycoprotein subunit of the (sodium + potassium)-activated adenosine triphosphatase from the electroplax of Electrophorus electricus.

Isoelectric focusing of purified Na,K-ATPase on polyacrylamide gels resolved the protein into ten bands. The catalytic and glycoprotein subunits were separated by sodium dodecyl sulfate gel filtration. Isoelectric focusing of the isolated glycoprotein subunit showed that it accounted for nine of the ten bands. Part of this microheterogeneity can be attributed to variations in sialic acid content in individual bands, since removal of all of the sialic acid by neuraminidase treatment reduced the number of bands to four. It is suggested that the microheterogeneity of the glycoprotein subunit is due to post-translational modifications of oligosaccharides on a common polypeptide backbone.     

Isolation,purification and some properties of a lectin from the winged bean (Psophocarpus tetragonolobus)

We have isolated by affinity chromatography a lectin from the seeds of the winged bean (Psophocrapus tetragonolobus) which agglutinated human (group A, B and O), sheep and rabbit, but not mouse erythrocytes. A molecular weight of 41,000 was obtained from gel filtration, and on sodium dodecyl sulphate polyacrylamide gel electrophoresis a single polypeptide chain of molecular weight 35,000 was seen both before and after reduction. Isoelectric focussing of the lectin on polyacrylamide gel gave a single band with a calculated isoelectric point of 4.0. The lectin was found to be rich in acidic amino acids; cysteine was not detected. Carbohydrate analysis revealed no covalently bound sugars.Abbreviations PBS phosphate-buffered saline - SDS sodium dodecyl sulphate - PAGE polyacrylamide gel electrophoresis - WBL winged bean lectin - HPLC high performance liquid chromatography     

Structural basis for salinity-induced alteration in oxygen binding by haemocyanin from the estuarine amphipod Chaetogammarus marinus (L.)

In the estuarine amphipod Chaetogammarus marinus, differences in O(2) binding by haemocyanins (Hc) could be related to natural and salinity-related quantitative variation in just one polypeptide subunit (band 2) and not to variations in any of the other seven bands present. Band 2 was always present, irrespective of salinity treatment, and naturally makes up 6%-36% of the total Hc. However, low salinity exposure (S=4 per thousand for 48-49 h, T=15 degrees C) was accompanied by an increase in the prevalence of greater concentrations of band 2 (i.e., range, 20%-36% of total Hc present) and a concomitant increase in Hc-O(2) affinity (half saturation [P(50)] decreased from 1.38 to 1.12 kPa at pH=7.81, T=15 degrees C). A similar salinity-related mechanism (that is, one band altering) has been shown previously for the blue crab Callinectes sapidus, although the functional consequences were different. In contrast with C. marinus, an increase in the proportion of one polypeptide subunit in C. sapidus resulted in a decrease in Hc-O(2) affinity. This study has confirmed that between-individual variation (quantitative rather than qualitative) in just one Hc subunit may have functional consequences, although the significance of such variation is difficult to interpret.     

Purification of two hexosaminidases from human kidney.

Hexosaminidase forms A and B were isolated from human kidney in a homogeneous state as demonstrated by electrophoretic and enzymic criteria. The enzymes were stable for at least 18 months when stored at -20 degrees C in 0.025 M-phosphate buffer, pH 6.5. The molecular weights of forms A and B were estimated by gel filtration to be 111 000 +/- 1500 and 114 000 +/- 1600 respectively. The molecular weights of hexosamidase A and B subunits were determined by using polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. Hexosaminidase A dissociated into one subunit with mol.wt. 68 000. Hexosaminidase B dissociated into three subunits with mol. wts. 100 000, 68 000 and 37000 respectively, and one protein band of mol.wt. 140 000. After treatment of hexosaminidases A and B with iodoacetic acid, the molecular weights of the carboxymethylated polypeptide subunits were also estimated. Carboxymethylated hexosaminidase A dissociated into one major subunit of mol.wt. 18 000 and two other protein bands of mol.wts. 65 000 and 100 000. Carboxymethylated hexosaminidase B dissociated into one major subunit for mol.wt. 19 000 and an additional band of mol.wt. 37 000. The Km of the enzymes for the synthetic substrate p-nitrophenyl 2-acetamido-2-deoxy-beta-D-glucopyranoside was 0.8 mM. Both enzymes were inhibited or activated by various metal ions. Double pH optima for the enzymes were found at pH 4.5 and 4.8.     

Structural and conformational changes of beta-lactoglobulin B: an infrared spectroscopic study of the effect of pH and temperature

Infrared spectra of 2.5 mM solutions of beta-lactoglobulin B were recorded as a function of pH (from pH 2 to pH 13) and as a function of temperature (from -100 degrees C to +90 degrees C). An analysis of the pH- and temperature-induced changes in the secondary structure was performed based on changes in the conformation-sensitive amide I bands of beta-lactoglobulin. Whereas the total amount of beta-structure remains constant (56-59%) between pH 2 and pH 10, the proportions of the various beta-components do change. In particular, the dimerization of the monomeric protein, induced by raising the pH from 2 to 3 , leads to an increase in the intensity of the 1636 cm-1 band (associated with antiparallel beta-sheet), at the expense of the 1626 cm-1 band (associated with exposed beta-strands). Both the thermal and alkaline denaturation of beta-lactoglobulin occur in two distinct stages. Although the spectra (i.e., the structures) after complete thermal or alkaline denaturation are clearly different, the spectrum of the protein after the first stage of thermal denaturation (at about 60 degrees C) is the same as that after the first stage of alkaline denaturation (at pH 11), suggesting a common denaturation intermediate, which probably represents a crossover point in a complex potential hypersurface.     

Identification and partial characterization of caltrin-like proteins in the reproductive tract of the guinea pig

The presence of caltrin-like proteins in reproductive tract fluid (RTF) and seminal vesicle content from male guinea pigs has been determined. Two fractions with electrophoretic mobility corresponding to Mr = 6200 (main band) and 5100 were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. Isoelectric focusing in thin-layer agarose gels revealed three bands with acidic pIs of 5.3, 6.0, and 6.2, respectively. RTF prevented the enhancement of calcium permeability induced by incubating guinea pig epididymal spermatozoa in medium for capacitation. Spermatozoa incubated for 2 h in minimal culture medium plus pyruvate and lactate containing RTF accumulated less than 30% of the 45Ca2+ accumulated by cells maintained in absence of this fluid. Calcium uptake by preincubated spermatozoa was also inhibited by RTF. Inhibition of calcium transport activity by RTF and seminal vesicle proteins was not decreased by heating the dialyzed preparations at 60 degrees C for 5 min. After this treatment, the inhibitory activity and the protein pattern were stable for 3 wk when stored at 4 degrees C. Unheated extracts lost calcium transport inhibitory activity after 2 or 3 days at 4 degrees C. In spite of the differences in pIs among the proteins from the guinea pig reproductive tract and bovine caltrin, several features indicate they may play a similar role in both species by controlling Ca2+ movement across the plasma membrane. By this mechanism, these proteins could regulate physiologic events essential for the fertilization process.     

Kinetic analysis of the individual steps of protein splicing for the Pyrococcus abyssi PolII intein

Protein splicing involves the excision of an intervening polypeptide, the intein, from flanking polypeptides, the exteins, concomitant with the specific ligation of the exteins. The intein that interrupts the DNA polymerase II DP2 subunit in Pyrococcus abyssi can be overexpressed and purified as an unspliced precursor, which allows for a detailed in vitro kinetic analysis of the individual steps of protein splicing. The first order rate constant for splicing of this intein, which has a non-canonical Gln at its C terminus, is 9.3 x 10(-6) s(-1) at 60 degrees C. The rate constant for splicing increases 3-fold with substitution of Asn for the C-terminal Gln. The pseudo first order rate constant of dithiothreitol-dependent N-terminal cleavage is 1 x 10(-4) s(-1). The first order rate constant of C-terminal cleavage is 1.2 x 10(-5) s(-1) with Gln at the C-terminal position, 2.8 x 10(-4) s(-1) with Asn, and decreases significantly with mutation of the penultimate His of the intein to Ala. N-terminal cleavage is most efficient between pH 7 and 7.5 and decreases at both more acidic and alkaline pH values, whereas C-terminal cleavage and splicing are both efficient over a broader range of pH values.     

Characterization of a membrane-associated glycoprotein complex implicated in cell adhesion to fibronectin

We have characterized a 140-kDa glycoprotein complex purified by a monoclonal antibody and implicated in cell adhesion to the extracellular molecule fibronectin. Three major polypeptide components were purified by monoclonal antibody JG22E, which had apparent molecular weights of 155,000 (band 1), 135,000 (band 2), and 120,000 (band 3). In two-dimensional gel electrophoresis, each subunit migrated as either a broad band or a series of spots at acidic isoelectric points. After treatment with neuraminidase, the spots became focused around pH 6.2 (band 1), pH 5.6 (band 2), and pH 5.3 (band 3). These three major bands were compared by two-dimensional peptide mapping in a series of pairwise combinations and were found to be distinct proteins. In sucrose gradients, these proteins co-migrated as a complex sedimenting at approximately 8.4 S either before or after affinity purification, whereas separated subunits migrated at 4.7 to 5.8 S. Amino acid analysis revealed no detectable hydroxyproline and a composition characterized by a substantial number of cysteine residues compared to the average protein. Our results suggest that a noncovalent complex of structurally distinct glycoproteins is involved in adhesive interactions of fibronectin with cells.     

Purification and characterization of the hypoxanthine-guanine phosphoribosyltransferase from Saccharomyces cerevisiae.

1. Hypoxanthine-guanine phosphoribosyltransferase (EC 2.4.2.8) from Saccharomyces cerevisiae was purified 9400-fold by affinity chromatography giving rise to an electrophoretically homogeneous preparation. 2. The molecular weight of the enzyme was determined by gel filtration with Sephadex G-100 and by sodium dodecylsulfate gel electrophoresis. Both methods reveal a molecular weight of 51,000. 3. The enzyme requires Mg2+ and has its pH optimum at 8.5. 4. Isoelectric focussing as well as gel electrophoresis of the purified extract reveals a single band which exhibits enzyme activity. The isoelectric point of the enzyme is 5.1. 5. The enzyme displays Michaelis-Menten kinetics with apparent Michaelis constants for hypoxanthine, guanine and phosphoribosylpyrophosphate of 23 microns, 18 microns, and 50 microns respectively.     

RAPID CONCURRENT AUTOMATED FLUORIMETRIC ASSAY OF NORADRENALINE, DOPAMINE, 3, 4-DIHYDROXYPHENYLACETIC ACID, HOMOVANILLIC ACID AND 3-METHOXYTYRAMINE IN MILLIGRAM AMOUNTS OF NERVOUS TISSUE AFTER ISOLATION ON SEPHADEX G10

Abstract— Brain tubulin subunits were separated by a combination of isoelectric focusing and electrophoresis in the presence of sodium dodecyl sulfate (SDS) using a two-dimensional polyacrylamide slab gel technique. Isoelectric focusing separated tubulin subunits into two major groups of bands, such that the more acidic group corresponded to the α subunit and the less acidic group corresponded to the β subunit. In addition, isoelectric focusing resolved the β subunit into two subspecies which differed slightly in isoelectric properties but were the same apparent molecular weight. The a subunit was resolved into many subspecies that appear to differ from each other by both apparent molecular weight and isoelectric properties.     

Isolation of an intrinsic antenna chlorophyll a-protein from the photosystem I reaction center complex of the thermophilic cyanobacterium Synechococcus sp

A chlorophyll-protein was isolated from a Synechococcus P700-chlorophyll a-protein complex free from small subunits (CP1-e) by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis after treatment with 2% 2-mercaptoethanol and 2% SDS. In contrast to CP1-e which, when electrophoresed under denaturating conditions, showed two polypeptide bands of 62 and 60 kDa, the chlorophyll-protein contained only the 60-kDa polypeptide and hence is called CP60. The yield of CP60 was maximal with 1-2% SDS and 2-4% sulfhydryl reagents because the chlorophyll-protein was denatured at higher concentrations of the reagents. The absorption spectrum of CP60, which retained more than half of the chlorophyll alpha molecules originally associated with the 60-kDa subunit of the photosystem I reaction center complex, showed a red band maximum at 672 nm and a small absorption band around 700 nm at liquid nitrogen temperature. CP60 emitted a fluorescence band at 717 to 725 nm at 77 degrees K. The temperature dependence of the far red band of CP60 was essentially the same as that of CP1-e between 77 and 273 degrees K. No photoresponse of P700 was detected in CP60. The results suggest that the two polypeptides resolved by SDS-gel electrophoresis from CP1-e are apoproteins of two distinct chlorophyll-proteins and that CP60 represents a chlorophyll-bearing 60-kDa subunit functioning as an intrinsic antenna protein of the photosystem I reaction center complex. It will also be shown that the temperature dependence of the far red fluorescence band is not related to the photosystem I photochemistry.     

The interrelation between high- and low-molecular-weight forms of GM1-beta-galactosidase purified from porcine spleen

1) Two forms of acid beta-galactosidase [EC 3.1.23] with different molecular weights catalyzing the hydrolysis of GM1-ganglioside and p-nitrophenyl-beta-D-galactoside were separated and purified from porcine spleen. 2) The apparent molecular weights were 400,000-600,000 and 70,000-74,000 for the high (termed Am form) and low (termed A1 form) molecular weight forms, respectively. 3) On examination by sodium dodecyl sulfate (SDS)/polyacrylamide gel electrophoresis, both forms of the enzyme had a common protein band of molecular weight 63,000, and the Am form showed three additional protein bands with molecular weights of 31,000, 21,000, and 20,000. 4) Both forms of the enzyme had similar catalytic functions with regard to pH-optimum, Km, substrate specificity and sensitivity to substrate analogues and other substances such as detergents, bovine serum albumin (BSA) and NaCl. 5) Both forms of the enzyme were fairly stable upon preincubation at 45 degrees C at acidic pH (pH 4.5), but lost their activities at neutral pH (pH 7.0). 6) The A1 form was a monomer at neutral pH (pH 7.0) and formed a dimer at acidic pH (pH 4.5). However, most of the Am form could not be converted to a dimeric form on gel filtration at acidic pH.     

Isoelectric focusing of Campylobacter jejuni flagellin: microheterogeneity and restricted antigenicity of charged species with a monoclonal antibody

Abstract Isoelectric focusing analysis was performed on flagellins purified from serologically different isolates of Campylobacter jejuni . C. jejuni flagellin exhibits charge heterogeneity with 5 acidic bands demonstrated (p I 4.7–5.7). When immunoblotting was performed with polyclonal human sera, all charged species reacted. However, monoclonal antibody 10–44, specific for a common epitope of Campylobacter sp. flagellin, exhibited binding to predominantly 1 of the 6 bands.     

Purification and characterization of human transcortin.

Human transcortin was purified to apparent homogeneity from plasma by a two-step procedure involving affinity and hydroxyapatite chromatography. The affinity gel incorporated denatured bovine serum albumin as the spacer and cortisol hemisuccinate as the ligand. Although isolated transcortin showed a propensity for spontaneous polymerization according to a geometric progression (1, 3, 9) only one band was observed on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Cortisol-binding activity of the isolated protein gave an apparent association constant of 2.5 X 10(8) M-1 at 4 degree C in equilibrium dialysis. Isoelectric focusing of purified native transcortin showed six discrete bands, five between pH 3.75 and 4.15 and another, possibly desialylated, at pH 6.15. Desialylated transcortin also gave six bands on isoelectric focusing, with pI values ranging from 4.90 to 6.30.     

Purification and characterization of aspartate aminotransferase from the thermoacidophilic archaebacterium Sulfolobus solfataricus

Aspartate aminotransferase from the archaebacterium Sulfolobus solfataricus, a thermoacidophilic organism isolated from an acidic hot spring (optimal growth conditions: 87 degrees C, pH 3.5) was purified to homogeneity. The enzyme is a dimer (Mr subunit = 53,000) showing microheterogeneity when submitted to chromatofocusing and/or isoelectric focusing analysis (two main bands having pI = 6.8 and 6.3 were observed). The N-terminal sequence (22 residues) does not show any homology with any stretch of known sequence of aspartate aminotransferases from animal and bacterial sources. The apoenzyme can be reconstituted with pyridoxamine 5'-phosphate and/or pyridoxal 5'-phosphate, each subunit binding 1 mol of coenzyme. The absorption maxima of the pyridoxamine and pyridoxal form are centered at 325 and 335 nm, respectively; the shape of the pyridoxal form band does not change with pH. The enzyme has an optimum temperature higher than 95 degrees C, and at 100 degrees C shows a half-inactivation time of 2 h. The above properties seem to be unique even for enzymes from extreme thermophiles (Daniel, R. M. (1986) in Protein Structure, Folding, and Design (Oxender, D. L., ed) pp. 291-296, Alan R. Liss, Inc., New York) and lead to the conclusion that aspartate aminotransferase from S. solfataricus is one of the most thermophilic and thermostable enzymes so far known.     

Gel-electrophoretic identification of hen brain neurotoxic esterase, labelled with tritiated di-isopropyl phosphorofluoridate.

The particulate fraction from hen brain was labelled with [3H]di-isopropyl phosphorofluoridate (DiPF) and separated by polyacrylamide-gel electrophoresis. Four radioactive protein bands (1--4) of molecular weights 155000, 92000, 60000, and 30000 were resolved. Most of the labelling of bands 2, 3 and 4 was inhibited by preincubation with Paraoxon. The residue in band 4 was sensitive to pH 5.2. Successive treatments with Paraoxon and pH 5.2 resulted in the abolition of bands 3 and 4. Bands 1 and 2 contained one and two polypeptides respectively, whose labelling was sensitive to Mipafox, but one, in band 2, was sensitive to higher concentrations of Paraoxon. The concentrations of the other two polypeptides were 6.7 and 1.95 pmol of DiPF bound/g of brain in bands 1 and 2 respectively. Both were as sensitive to Mipafox as neurotoxic esterase and were also sensitive to phenyl benzylcarbamate. 4-Nitrophenyl di-n-pentylphosphinate given in vivo inhibited neurotoxic esterase and the labelling of the band-1 polypeptide by 82% and 84% respectively, but inhibited the labelling of the band 2 polypeptide by 51%. The phosphinate in vitro produced 98% inhibition of the labelling of the band-1 polypeptide, with only 26% inhibition of the band-2 polypeptide, under conditions sufficient to inhibit neurotoxic esterase totally. Both neurotoxic esterase and the band-1 polypeptide were found in the forebrain at 1.74-fold their concentration in the rest of the brain, whereas the band-2 polypeptide was uniformly distributed. The evidence indicates that the Mipafox-sensitive polypeptide in band 1 is the [3H]DiPF-labelled active-site subunit of neurotoxic esterase. The catalytic-centre activity of the enzyme for phenyl valerate hydrolysis was found to be 2.6 x 10(5) min-1.     

Characterization of pre-messenger-RNA-containing nuclear ribonucleoprotein particles from avian erythroblasts.

Ribonucleoprotein particles have been isolated from duck erythroblast nuclei using a procedure designed to produce maximal cytoplasmic dispersion with minimal release of endogenous hydrolytic enzymes. The RNA extracted from the purified nuclear ribonucleoprotein fraction is shown to contain globin messenger RNA sequences at a concentration comparable to that present in total nuclear RNA. The polypeptide composition of this fraction revealed by electrophoresis in two dimensions is complex, consisting of at least 65 acidic species and 21 basic species. Several lines of evidence suggest that these are authentic components of nuclear ribonucleoprotein. The so-called 'core' proteins of nuclear ribonucleoprotein which were previously shown to migrate as a single band on low-pH urea gels, and as six bands on sodium dodecyl sulphate gels are here shown to be considerably more complex being resolved by two-dimensional electrophoresis into a group of 15 basic and 6 more and less neutral polypeptides. Isoelectric focusing of nuclear ribonucleoprotein under non-denaturing conditions suggests that these latter species are not uniformly distributed along the pre-messenger RNA molecule.     

Sequential roles of receptor binding and low pH in forming prehairpin and hairpin conformations of a retroviral envelope glycoprotein

A general model has been proposed for the fusion mechanisms of class I viral fusion proteins. According to this model a metastable trimer, anchored in the viral membrane through its transmembrane domain, transits to a trimeric prehairpin intermediate, anchored at its opposite end in the target membrane through its fusion peptide. A subsequent refolding event creates a trimer of hairpins (often termed a six-helix bundle) in which the previously well-separated transmembrane domain and fusion peptide (and their attached membranes) are brought together, thereby driving membrane fusion. While there is ample biochemical and structural information on the trimer-of-hairpins conformation of class I viral fusion proteins, less is known about intermediate states between native metastable trimers and the final trimer of hairpins. In this study we analyzed conformational states of the transmembrane subunit (TM), the fusion subunit, of the Env glycoprotein of the subtype A avian sarcoma and leukosis virus (ASLV-A). By analyzing forms of EnvA TM on mildly denaturing sodium dodecyl sulfate gels we identified five conformational states of EnvA TM. Following interaction of virions with a soluble form of the ASLV-A receptor at 37 degrees C, the metastable form of EnvA TM (which migrates at 37 kDa) transits to a 70-kDa and then to a 150-kDa species. Following subsequent exposure to a low pH (or an elevated temperature or the fusion promoting agent chlorpromazine), an additional set of bands at >150 kDa, and then a final band at 100 kDa, forms. Both an EnvA C-helix peptide (which inhibits virus fusion and infectivity) and the fusion-inhibitory agent lysophosphatidylcholine inhibit the formation of the >150- and 100-kDa bands. Our data are consistent with the 70- and 150-kDa bands representing precursor and fully formed prehairpin conformations of EnvA TM. Our data are also consistent with the >150-kDa bands representing higher-order oligomers of EnvA TM and with the 100-kDa band representing the fully formed six-helix bundle. In addition to resolving fusion-relevant conformational intermediates of EnvA TM, our data are compatible with a model in which the EnvA protein is activated by its receptor (at neutral pH and a temperature greater than or equal to room temperature) to form prehairpin conformations of EnvA TM, and in which subsequent exposure to a low pH is required to stabilize the final six-helix bundle, which drives a later stage of fusion.     

Patterns of community change among ammonia oxidizers in meadow soils upon long-term incubation at different temperatures

The effect of temperature on the community structure of ammonia-oxidizing bacteria was investigated in three different meadow soils. Two of the soils (OMS and GMS) were acidic (pH 5.0 to 5.8) and from sites in Germany with low annual mean temperature (about 10 degrees C), while KMS soil was slightly alkaline (pH 7.9) and from a site in Israel with a high annual mean temperature (about 22 degrees C). The soils were fertilized and incubated for up to 20 weeks in a moist state and as a buffered (pH 7) slurry amended with urea at different incubation temperatures (4 to 37 degrees C). OMS soil was also incubated with less fertilizer than the other soils. The community structure of ammonia oxidizers was analyzed before and after incubation by denaturing gradient gel electrophoresis (DGGE) of the amoA gene, which codes for the alpha subunit of ammonia monooxygenase. All amoA gene sequences found belonged to the genus Nitrosospira. The analysis showed community change due to temperature both in moist soil and in the soil slurry. Two patterns of community change were observed. One pattern was a change between the different Nitrosospira clusters, which was observed in moist soil and slurry incubations of GMS and OMS. Nitrosospira AmoA cluster 1 was mainly detected below 30 degrees C, while Nitrosospira cluster 4 was predominant at 25 degrees C. Nitrosospira clusters 3a, 3b, and 9 dominated at 30 degrees C. The second pattern, observed in KMS, showed a community shift predominantly within a single Nitrosospira cluster. The sequences of the individual DGGE bands that exhibited different trends with temperature belonged almost exclusively to Nitrosospira cluster 3a. We conclude that ammonia oxidizer populations are influenced by temperature. In addition, we confirmed previous observations that N fertilizer also influences the community structure of ammonia oxidizers. Thus, Nitrosospira cluster 1 was absent in OMS soil treated with less fertilizer, while Nitrosospira cluster 9 was only found in the sample given less fertilizer.