Purification and Characterization of an Extracellular Non-Aspartyl Acid Protease (pumAe) from <Emphasis Type="Italic">Ustilago maydis</Emphasis>

The proteinase pumAe was purified to homogeneity from haploid U. maydis FB1 growing on acid mineral medium. The purification procedure consisted of ammonium sulfate fractionation and gel filtration chromatography, resulting in a 7.7% recovery and a 15.1-fold increase in specific activity. The molecular weight of the enzyme was estimated to be 72 kDa and 74 kDa by gel filtration chromatography and SDS-PAGE, respectively. Enzymatic activity was optimal at pH 4.0 and at 45°C toward hemoglobin, and the pI was determined to be 5.5. The effects of six protease inhibitors on pumAe were tested, and no inhibitory effect was observed. The pure enzyme degraded gelatin and albumin, but casein and collagen were not degraded. The K_m value was 3.5 M, and the V_max value was 11430 mol h^–1 mg^–1 for Suc-R-P-F-H-L-L-V-Y-MCA.     

The contribution of size-fractionated plankton to biomass and primary production of phytoplankton in saline–alkaline ponds

Primary productivity, biomass and chlorophyll-a of size fractionated phytoplankton (<0.22 m, <3 m, <8 m, <10 m, <40 m, <64 m, <112 m and <200 m) were estimated in 6 ponds and 5 experimental enclosures. The results showed that the planktonic algae less than 10 m are important in the biomass and production of phytoplankton in saline–alkaline ponds. The production of size fractionated phytoplankton corresponding to <112 m, <10 m and <3 m in saline–alkaline ponds were 10.5 ± 6.6 , 8.6 ± 5.4 and 0.33 ± 0.1 mgC l^–1 d^–1, respectively. Mean community respiration rate was 1.80 ± 0.73, 1.69 ± 0.90 and 1.38 ± 1.12 mgC l^–1 d^–1, respectively. The average production of phytoplankton corresponding to micro- (10–112 m), nano- (3–10 m) and pico- (<3 m) were 1.61, 8.30 and 0.33 mgC l^–1 d^–1, respectively. The ratio of those to the total phytoplankton production was 15%, 79% and 3%, respectively. The mean respiration rate of the different size groups was 0.11, 0.31 and 1.38 mgC l^–1 d^–1; the ratio of those to total respiration of phytoplankton was 6%, 17% and 77%, respectively. The production of size-fractionated phytoplankton corresponding to <200 m, <10 m and <3 m in enclosures was 2.19 ± 1.63, 2.08 ± 1.75 and 0.22 ± 0.08 mgC l^–1 d^-1, respectively. Mean community respiration rates were 1.25 ± 1.55, 1.17 ± 1.42 and 0.47 ± 0.32 mgC l^–1 d^–1, respectively. The average production of phytoplankton corresponding to micro- (10–200 m), nano- (3–10 m) and pico- (<3 m) plankton was 0.11, 1.86 and 0.22 mgC l^–1 d^–1, respectively. The ratio of those to the total production of phytoplankton was 5%, 85% and 10%, respectively. The mean respiration rate of different size groups were 0.08, 0.72 and 0.46 mgC l^–1 d^–1, the ratio of those to total respiration of phytoplankton was 6%, 57% and 37%, respectively. The concentrations of chlorophyll-a of the phytoplankton in the corresponding size of micro- (10–112 m), nano- (3–10 m) and pico- (<3 m) plankton in the experimental ponds were 19.3, 98.2 and 11. 9 g l^–1, respectively. The ratio of those to the total chlorophyll-a was 15%, 76% and 9%, respectively. The concentrations of chlorophyll-a of phytoplankton micro- (10–200 m), nano- (3–10 m) and pico- (<3 m) plankton in enclosures were 1.7, 34.3 and 3.0 g l^–1, respectively. The ratio of those to the total chlorophyll-a was 4%, 88% and 8%, respectively.     

The Isolation,Purification, and Some Properties of NAD-Dependent Isocitrate Dehydrogenase from the Organic Acid–Producing Yeast Yarrowia lipolytica

The NAD⁺-dependent isocitrate dehydrogenase of the organic acid–producing yeast Yarrowia lipolytica was isolated, purified, and partially characterized. The purification procedure included four steps: ammonium sulfate precipitation, acid precipitation, hydrophobic chromatography, and gel-filtration chromatography. The enzyme was purified 129-fold with a yield of 31% and had a specific activity of 22 U/mg protein. The molecular mass of the enzyme was found to be 412 kDa. The enzyme consists of eight identical subunits with a molecular mass of about 52 kDa. The K _m for NAD⁺ is 136 M, and that for isocitrate is 581 M. The effect of some intermediates of the citric acid cycle and nucleotides on the enzyme activity was studied. The role of isocitrate dehydrogenase (NAD⁺) in the overproduction of citric and keto acids is discussed.     

Purification and Characterization of S-Adenosyl-L-Methionine Nicotinic Acid-N-Methyltransferase from Leaves of Glycine max

Trigonelline (TRG), which act as a cell cycle regulator and a compatible solute in response to salinity and water-stress, is the N-methyl conjugate of nicotinic acid the formation of which is catalyzed by S-adenosyl-L-methionine nicotinic acid-N-methyltransferase. The enzyme was purified 2650-fold from soybean (Glycine max L.) leaves with a recovery of 4 %. The purification procedure included ammonium sulfate (45 – 60 %) precipitation, linear gradient DEAE-Sepharose chromatography, adenosine-agarose affinity chromatography, hydroxyapatite chromatography and gel filtration (Sephacryl-S-200). The purified enzyme preparation showed a major band with a molecular mass of 41.5 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis that is related to the enzyme activity. The native enzyme had a molecular mass of about 85 kDa as estimated by gel filtration. The K_m values for S-adenosyl-L-methionine and nicotinic acid were 31 and 12.5 M, respectively. The purified enzyme showed optimum activity at pH 6.5 and temperature of 40 – 45 °C. High concentration of dithiothreitol (10 mM) and glycerol (20 %) stabilize the enzyme during purification and storage. Hg²⁺ strongly inhibits enzyme activity.     

Characterization of 2,3-butanediol-forming and valine-sensitive α-acetolactate synthase of Enterobacter cloacae

-Acetolactate synthase (-ALS) of Enterobacter cloacae ATCC 27613 was purified to homogeneity by ammonium sulphate precipitation, Sephadex G-200 gel filtration and hydroxyapatite affinity chromatography. The molecular weight of the enzyme was found to be 60 kDa by SDS–polyacrylamide gel electrophoresis and 200 kDa by gel filtration through Sephadex G-200, showing that the enzyme is a homotrimer. The K _m and V _max of the enzyme were 20 mM and 200 mol min^–1 mg (protein)^–1 respectively. The enzyme was optimally active at pH 6.0–8.0, 37 °C and showed concentration-dependent sensitivity to cofactors viz. FAD, NADP and NADPH and branched chain amino acids: leucine, isoleucine and valine. Substances like sodium formate, sodium acetate and sodium propionate, sugars and the selected intermediates of glycolytic pathway inhibited the enzyme. Glycerol, BSA and pyruvate-TPP stabilized the -ALS. The enzyme showed the properties of both a catabolic as well as an anabolic -ALS.     

Receptor-like cytokinin-binding protein(s) from barley leaves

Purified fractions of soluble proteins from barley leaves have been shown to contain specific binding sites fortrans-zeatin, a natural cytokinin. Such binding is very strong in vitro in concentrated solutions of some salts (ammonium sulfate or potassium phosphate) with optimum at pH 7–8 and temperature within the range 0–20°C. The cytokinin-binding sites have high affinity for zeatin (K_d1.5·10^–8 M) and low capacity corresponding to 1–1.5 pmol zeatin per milligram of initial soluble protein. Cytokinin binding is reversible; it is due to protein (or proteins) with molecular weight 40–45 kDa. This protein(s) does not bind³H-adenine and³H-abscisic acid. The ability of various compounds to displace³H-zeatin from its high-affinity binding sites is in strict accordance with their biological cytokinin activities. Other phytohormones as well as fusicoccin do not displace³H-zeatin from its binding sites. Specific zeatin binding is sensitive to heat, alkali, and pronase, but not to RNase treatment. The 150- to 200-fold purification of cytokinin-binding proteins was achieved by a combination of ammonium sulfate precipitation and Ultrogel AcA-54- and DEAE-cellulose chromatography. The zeatin-binding protein(s) from barley leaves is suggested to take part in cytokinin action in vivo.     

Reaction center light harvesting B875 complexes from Rhodocyclus gelatinosus: characterization and identification of quinones

Reaction center-B875 pigment-protein complexes were purified from Rhodocyclus gelatinosus. The proteic components consist of 7–8 polypeptides among which some were identified by their apparent molecular weights: the light harvesting B875 polypeptides and of 8 and 6 kDa, reaction center L (23 kDa), M (28 kDa) and H (34 kDa), cytochrome c (43 kDa). Four c-type hemes were found per reaction center. Flash-induced absorbance changes showed the presence of both Q_A and Q_B in the complex. Charge recombination times were determined to be: 1.16±0.2 (n=30) for P⁺Q_AQ_B ^- and 7–10 ms for P⁺Q_A ^- in presence of herbicides. From quinone analysis on one hand and kinetics of charge recombination on the other hand, we proposed that in the reaction center of Rhodocyclus gelatinosus Q_A is menaquinone 8 and Q_B is ubiquinone 8.     

Purification and characterization of an (S)-3-hydroxycarboxylate oxidoreductase from Clostridium tyrobutyricum

An NADP⁺ —dependent reversible 3-hydroxycarboxylate oxidoreductase present in Clostridium tyrobutyricum has been purified. As judged by gel electrophoresis the enzyme was pure after a 940-fold enrichment by four chromatographic steps. Its molecular mass was estimated to be 40–43 kDa. The enzyme was most active at pH 4.5 in the reduction of 3-oxobutyrate. Other substrates were 3-oxovalerate, 3-oxocaproate, 3-oxoisocaproate and 4-chloro-3-oxobutyrate. Except for the latter all substrates were converted enantioselectively to (S)-3-hydroxy acids in the presence of NADPH. 4-Chloro-3-oxobutyrate was reduced to the (R)-3-hydroxy acid. The specific activity of the enzyme was about 1400 mol min^–1 mg^–1 protein for the reduction of 3-oxobutyrate at pH 5.0. The Michaelis constant (K _m) values for 3-oxobutyrate, 3-oxovalerate and 3-oxocaproate were determined to be 0.22, 1.6 and 3.0 mM, respectively. The K _m values for dehydrogenation of (S)-3-hydroxybutyrate, (S)-3-hydroxyvalerate and (S)-3-hydroxycaproate were found to be 2.6, 1.1 and 5.2 mM, respectively. The identity of 43 of the first 45 N-terminal amino acid residues has been determined. So far such enzyme activities have been described in eucaryotes only.Dedicated to Prof. A. Trebst on the occasion of his 65th birthday     

Biochemical characterisation of extracellular phytase (myo-inositol hexakisphosphate phosphohydrolase) from a hyper-producing strain of Aspergillus niger van Teighem

Aspergillus niger van Teighem, isolated in our laboratory from samples of rotten wood logs, produced extracellular phytase having a high specific activity of 22,592 units (mg protein)^–1 . The enzyme was purified to near homogeneity using ion-exchange and gel-filtration chromatography. The molecular properties of the purified enzyme suggested the native phytase to be oligomeric, with a molecular weight of 353 kDa, the monomer being 66 kDa. The purified enzyme exhibited maximum activity at pH 2.5 and 52–55°C. The enzyme retained 97% activity after a 24-h incubation at 55°C in the presence of 10 mM glycine, while 87% activity was retained when no thermoprotectant was added. Phytase activity was not affected by most metal ions, inhibitors and organic solvents. Non-ionic and cationic detergents (0.1–5%) stabilise the enzyme, while the anionic detergent (SDS), even at a 0.1% level, severely inhibited enzyme activity. The chaotropic agents guanidinium hydrochloride, urea, and potassium iodide (0.5–8 M), significantly affected phytase activity. The maximum hydrolysis rate (V_max) and apparent Michaelis-Menten constant (K_m) were 1,074 IU/mL and 606 M, respectively, with a catalytic turnover number of 3×10⁵ s^–1 and catalytic efficiency of 3.69×10⁸ M^–1 s^–1.     

Purification and properties of an aminopeptidase from the mid-gut gland of scallop (Patinopecten yessoensis)

An aminopeptidase was isolated from the mid-gut gland of Patinopecten yessoensis. The enzyme was purified from an acetone-dried preparation by extracting, ammonium sulfate precipitation, Hi-Load Q column chromatography, isoelectric focusing, and POROS HP2 and HQ column chromatography. The molecular weight of the enzyme was estimated to be 61 kDa by SDS-polyacrylamide gel electrophoresis and 59 kDa by gel permeation chromatography. The isoelectric point of the enzyme was 5.2 and the optimum pH was 7.0 toward leucine p-nitroanilide (Leu-pNA). The enzyme was inhibited by o-phenanthroline. The activity of the enzyme treated with o-phenanthroline was completely recovered by adding excess Zn²⁺. Relative hydrolysis rates of amino acid-pNAs and amino acid-4-methylcoumaryl-7-amides (amino acid-MCAs) indicated that the enzyme preferred substrates having Ala or Met as an amino acid residue. The enzyme had a K_m of 32.2 μM and k_cat of 29.5 s⁻¹ with Ala-pNA and a K_m of 11.1 μM and k_cat of 9.49 s⁻¹ with Ala-MCA. The enzyme sequentially liberated amino acids from the amino-termini of Ala–Phe–Tyr–Glu.     

1H-NMR spectroscopic characterization of dansyl glyco-asparagines derived from hen egg white clycoproteins

Dansyl glyco-asparagines were prepared from a partially fractionated mixture of hen egg white glycoproteins. Reverse-phase high performance liquid chromatography (HPLC) on a silica-based octadecyl column yielded ten such derivatives in a virtually pure state. The detailed structures of the glyco-asparagines were identified by 500-MHz¹H nuclear magnetic resonance (NMR) spectroscopy. Two of them were found to be of the oligomannosideN-type, four were of the intersected-hybridN-type and another four were of the intersected multi-antennaryN-type. In monogalactosylated, intersected structures the galactose residue was proved by¹H-NMR to be attached in (1–4)-linkage to the GlcNAc1-4Man1–3 branch.Dansyl glyco-asparagines turned out to be suitable derivatives for¹H-NMR spectroscopic analysis. The combination of HPLC and high-resolution NMR spectroscopy of such derivatives proved to be a powerful technique in studying the (micro-)heterogeneity of sugar chains in glycoproteins.Abbreviations dns dansyl (5-dimethylaminonaphthalene-1-sulfonyl) - ODS octadecyl-silica - WEFT water-eliminating Fourier transform - DSS sodium 4,4-dimethyl-4-silapentane-1-sulfonate - OVA ovalbumin - OVM ovomucoid - OVT ovotransferrin     

Functional analysis of the iron-stress induced CP 43′ polypeptide of PS II in the cyanobacterium Synechococcus sp. PCC 7942

Under conditions of iron-stress, the Photosystem II associated chlorophyll a protein complex designated CP 43, which is encoded by the isiA gene, becomes the major pigment-protein complex in Synechococcus sp. PCC 7942. The isiB gene, which is located immediately downstream of isiA, encodes the protein flavodoxin, which can functionally replace ferredoxin under conditions of iron stress. We have constructed two cyanobacterial insertion mutants which are lacking (i) the CP 43 apoprotein (designated isiA ^–) and (ii) flavodoxin (designated isiB ^–). The function of CP 43 was studied by comparing the cell characteristics, PS II functional absorption cross-sections and Chl a fluorescence parameters from the wild-type, isiA ^– and isiB ^– strains grown under iron-stressed conditions. In all strains grown under iron deprivation, the cell number doubling time was maintained despite marked changes in pigment composition and other cell characteristics. This indicates that iron-starved cells remained viable and that their altered phenotype suggests an adequate acclimation to low iron even in absence of CP 43 and/or flavodoxin. Under both iron conditions, no differences were detected between the three strains in the functional absorption crossection of PS II determined from single turnover flash saturation curves of Chl a fluorescence. This demonstrates that CP 43 is not part of the functional light-harvesting antenna for PS II. In the wild-type and the isiB ^– strain grown under iron-deficient conditions, CP 43 was present in the thylakoid membrane as an uncoupled Chl-protein complex. This was indicated by (1) an increase of the yield of prompt Chl a fluorescence (F_o) and (2) the persistence after PS II trap closure of a fast fluorescence decay component showing a maximum at 685 nm.Abbreviations Chl chlorophyll - CP 43, CP 47 and CP 43 Chl a binding protein complexes of indicated molecular mass - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - F_m and F_m fluorescence when all PS II reaction centers are dosed in dark- and light-acclimated cells, respectively - F_o fluorescence when all PS II reaction centers are open in dark acclimated cells - F_v variable fluorescence after dark acclimation (F_m–F_o)     

Purification of hydroxycinnamoyl-CoA:tyramine hydroxycinnamoyltransferase from cell-suspension cultures of Solanum tuberosum L. cv. Datura

A pathogen-elicitor-inducible acyltransferase [tyramine hydroxycinnamoyltransferase (THT); EC 2.3.1], which catalyzes the transfer of hydroxycinnamic acids from hydroxycinnamoyl-CoA esters to tyramine in the formation of N-hydroxycinnamoyltyramine, was purified to apparent homogeneity from cell-suspension cultures of potato (Solanum tuberosum L. cv. Datura), with a 1400-fold enrichment, a 5% recovery and a final specific activity of 208 mkat·(kg protein)^–1. Affinity chromatography on Reactive Yellow-3-Agarose using the acyl donor (feruloyl-CoA) as eluent was the decisive step in the purification sequence. The purified protein showed a native molecular mass of ca. 49 kDa. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence and in the absence of a reducing agent (2-mercaptoethanol) indicated that THT is a heterodimer in which the protein subunits (ca. 25 kDa) are non-covalently associated. The enzyme was stimulated fivefold by 10 mM Ca²⁺. The apparent K _m value for tyramine was dependent on the nature of the hydroxycinnamoyl-CoA present. Thus, the K _m value for tyramine was about tenfold greater (174 M) in the presence of 4-coumaroyl-CoA than in the presence of feruloyl-CoA (20 M).Abbreviations PAL phenylalanine ammonia-lyase - THT hydroxycinnamoyl-CoA:tyramine hydroxycinnamoyltransferase We thank the Deutsche Forschungsgemeinschaft and the Fonds der Chemischen Industrie for financial support. Further support by a grant from the Studienstiftung des Deutschen Volkes to H.H. is gratefully acknowledged.     

The GABAA receptor family in the mammalian brain

The GABA_A-benzodiazepine receptor protein from bovine brain was purified by affinity chromatography and the subunit composition examined by gel electrophoresis in sodium dodecyl sulfate. Protein staining revealed a doublet at 51–53 kDa, a band at 55 kDa, and a broad band at 57–59 kDa. The 51 and 53 kDa bands co-migrated with the ₁ and ₂ gene products identified by Western blotting with subtype-specific antibodies. These two bands were also photoaffinity labeled by [³H]flunitrazepam, as was a breakdown product at 44 kDa. Partial sequencing of proteolytic fragments of these polypeptides yielded sequences found in all clones, and identified the benzodiazepine binding site within residues 8–297 and probably between 106–297 of ₁; the 44 kDa and 31 kDa bands yielded fragments containing ₃ sequence. The native ₃ polypeptide was identified with subtype-specific antibody at 57 kDa overlapping with the two major bands photolabeled with [³H]muscimol at 55 and 58 kDa. Antisera to a -selective peptide recognized four bands at 60, 58, 57 and 55 kDa. Thus, one can identify 6–8 distinct polypeptides with the possibility of another 4–6 in purified GABA_A receptor proteins, depending on brain region, consistent with the family of gene products suggested by molecular cloning.Special issues dedicated to Dr. Eugene Roberts     

Carbohydrate-binding profile of a pregnancy-associated rat uterine glycoprotein

Sugar-binding proteins obtained from the peri-implantation uterine tissue have been thought in recent years to have significant roles in embryo implantation, where carbohydrate moieties of the protein are actively involved. Based on this rationale a mannose-containing glycoprotein/lectin (named uterine agglutinin or UA) was purified by Concanavalin A (Con A) affinity chromatography in a previous study. A modification of the original purification procedure to include a 33% ammonium sulfate fractionation improves the yield of the protein significantly. An alternative purification procedure by Mannan affinity matrix, indicates that apart from containing mannose, UA possesses mannose-binding properties as well.In this paper, we report some of the biochemical and more specifically, the carbohydrate-binding characteristics of UA. The protein is seen to contain mannose-6-phosphate (M-6-P)-binding sites, which is of importance since M-6-P receptors have a large number of biologically significant roles, including that of binding to growth factors.SDS-PAGE, gel filtration chromatography and alkaline PAGE indicate the homogenous nature of the protein with subunit molecular weights of 36 kDa and 19 kDa, and a native size of 64kDa. Amino acid analysis shows glycine, glutamic acid and aspartic acid to be the major constituents.UA is a glycoprotein and shows presence of N-acetyl glucosamine and galactose, apart from mannose.De nove synthesis studies in the presence of tunicamycin show that the carbohydrate moiety of the glycoprotein is attached by N-linkage to the protein. Binding characteristics of the protein is studied quantitatively in which (¹²⁵I)-labelled lectin is bound to Mannan-Sepharose affinity matrix. The sugar inhibition pattern of this binding shows -methyl mannopyranoside and M-6-P to be equally effective as inhibitors. Scatchard analysis of the binding of UA to (¹⁴C)-mannose shows a Ka of 6.43×10⁵ (M^–1) and that 1 mole of UA can bind to 8 moles of mannose. The possible role of the protein in implantation has also been discussed.Abbreviations b.w. body weight - BSA Bovine Serum Albumin - Con A Concanavalin A - cpm counts per minute - Endo H endoglycosidase H - GlcNAc N-acetyl glucosamine - Man mannose - M-6-P mannose-6-phosphate - MEM-deficient Minimum Essential Medium Eagle-deficient modification - NaBH₄ sodium borohydride - NaN₃ sodium azide - (NH₄)₂SO₄ ammonium sulphate - p.c. post coitum - PMSF phenyl methyl sulphonyl fluoride - PTA phosphotungstic acid - RCA Ricinus communis Agglutinin - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis - TCA trichloroacetic acid - UA Uterine Agglutinin - WGA Wheat-germ Agglutinin     

Changes in amino acid content of an algal feed species (Navicula sp.) and their effect on growth and survival of juvenile abalone (Haliotis rubra)

The growth and survival of juvenile Haliotis rubra, when fed with the diatom Navicula sp. cultured in f/2 medium containing combined nitrogen at 24.71 mg NO₃-N L^–1 (high), 12.35 mg NO₃-N L^–1 (standard) or 2.47 mg NO₃-N L^–1 (low), were compared in a 33-day trial. The alga in the low nitrogen medium contained 37% less total amino acid than that in the high and standard nitrogen media. There was a slightly greater reduction in essential amino acids (40%) compared to non-essential amino acids (35%). Juvenile abalone feeding on Navicula grown in medium with low nitrate and lower total amino acid content grew more slowly than when fed on the same species grown in standard or higher nitrogen medium with a higher amino acid content. The growth rate of juveniles was highest (43 m d^–1) in the high nitrate treatment followed (40 m d^–1) by the standard nitrate treatment and lowest (31 m d^–1) in the low nitrate treatment. The survival of the juveniles was also effected by the diet. Survival was better in the high and standard nitrogen media (88%) than the low nitrogen medium (75%). The results suggest that in order to achieve uniformity in nutritional quality of diatoms and good growth of abalone juveniles in commercial abalone nurseries, the nitrogen concentration in tanks should be monitored and additional nitrate added to provide an optimum concentration of between 2 and 12 mg NO₃-N L^–1.     

Purification and characterization of a novel imidazole dipeptide synthase from the muscle of the Japanese eel Anguilla japonica

We have purified a novel enzyme from eel white muscle which catalyzes the syntheses of imidazole dipeptides, such as carnosine (β-alanyl-l-histidine), anserine (β-alanyl-π-methyl-l-histidine), and balenine (ophidine; β-alanyl-τ-methyl-l-histidine), directly from their precursors. The enzyme was purified 1130-fold from eel muscle by a series of column chromatographies. Although eel muscle contains a large amount of carnosine and only trace amounts of anserine and balenine, the anserine synthesizing activity was by far the highest. From gel permeation chromatography, the molecular mass of the enzyme was calculated to be 275 kDa. SDS-PAGE of the purified enzyme represented a band around 43 kDa, suggesting that the native enzyme is a hexamer or heptamer. The optimal pH and temperature were around 9.5 and 60 °C, respectively. K_m values for β-alanine and π-methyl-l-histidine were 44 and 89 mM, respectively. The enzyme was greatly activated by Zn²⁺ and inhibited by EDTA. The N-terminal amino acid sequence of 25 residues of the purified enzyme showed 52% amino acid identity to 38–62 residues of zebrafish haptoglobin precursor. The purified enzyme also exhibited hydrolytic activity against these imidazole dipeptides.     

Purification, characterization, and relation to bikunin of rat urinary trypsin inhibitors

Two forms of urinary trypsin inhibitor (UTI-1 and UTI-2) were purified from pooled urine of normal male rats to apparent homogeneity by salting out, affinity chromatography, gel filtration, and reverse-phase HPLC. UTIs-1 and 2 were shown to be thermostable glycoproteins with the respective molecular weights of 22,000 and 18,000 estimated by SDS-PAGE. These inhibitors combined with bovine trypsin in a 1:1 molar ratio: the K _d values were 2.5 × 10^–10 and 2.3 × 10^–10 M, respectively. Amino acid composition and sequence analysis indicated that UTI-1 corresponded to rat bikunin of which the amino acid sequence was deduced from a rat liver cDNA clone encoding ₁-microglobulin [Lindqvist et al. (1992), Biochim. Biophys. Acta 1130, 63–67] except that the protein sequence seemed to lack C-terminal serine, and UTI-2 corresponded to UTI-1 lacking N-terminal 21 amino acid residues.     

Photoaffinity labeling and partial purification of the putative plant receptor for the fungal wilt-inducing toxin,fusicoccin

The high-affinity fusicoccin-binding protein (FCBP) was solubilized from plasma-membrane vesicles prepared from leaves of Vicia faba L. by aqueous two-phase partitioning. Conditions for the solubilization of intact FCBP-radioligand complexes were worked out. About 60–70% of the complexes can be solubilized with 50–60 mM nonanoyl-N-methylglucamide in the presence of 1 mg· ml^-1 soybean phosphatidylcholine, type IV S, and 20% (v/v) glycerol at pH 5.5. The slow dissociation of the radioligand, 9-nor-fusicoccin-8-alcohol-[³H] from the FCBP at low temperatures permits the purification of FCBP-radioligand complexes at 4–10° C by fast protein liquid chromatography on anion-exchange and gel permeation columns. The FCBP, extracted from plasma membranes with cholate and chromatographed in the presence of this detergent, gave an apparent molecular mass (M_r) of 80±20 kDa on gel permeation columns under the conditions used. By comparison of the elution profiles of the fraction most enriched in FCBP-radioligand complexes with polypeptide patterns obtained on sodium dodecyl sulfate-polyacrylamide gels, a polypeptide with an M_r of approx. 34kDa co-separated with the radioactivity profile. A second, faint band of approx. 31 kDa was sometimes also observed co-electrophoresing. Photoaffinity labeling of plasma-membrane vesicles with the new compound 9-nor-8[(3,5-[³H]-4-azidobenzoy)ethylenediamine]-fusicoccin ([³H]ABE-FC) and subsequent separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis labeled a single band with an M_r of 35±1 kDa. Labeling in this band was strongly reduced when the membranes were incubated with [³H]ABE-FC in the presence of 0.1–1 M fusicoccin. From our data, we conclude (i) that the 34-35-kDa polypeptide represents the FCBP and (ii) that in detergent extracts of plasma membranes this polypeptide is probably present as a di- or trimeric structure.Abbreviations ABE-FC [(4-azidobenzoyl)-ethylenediamine]-fusicoccin - ABE-NHS (4-azidobenzoyl)-N-hydroxysuccinimide ester - FC fusicoccin - FCBP fusicoccin-binding protein - FCol 9-norfusicoccin-8-alcohol - MAB monoclonal antibody - Mega-9(10) nonanoyl(decanoyl)-N-methylglucamide - M_r apparent molecular mass - PMSF phenylmethyl-sulfonyl fluoride - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - TCA trichloroacetic acid - Tris 2-amino-2-(hydroxymethyl)-1,3-propanediol     

A soluble calcium-binding protein from the terrestrial annelidLumbricus terrestris L.

Summary Soluble calcium-binding proteins (SCBP) considerably different from calmodulin were purified from the body wall muscle of the earthwormLumbricus terrestris. Three isoforms were obtained with similar UV absorption spectra and amino acid compositions and an apparent molecular weight close to 20 kDa. They can be distinguished by their histidine and proline content and by their peptide maps. The tissue content, as determined by quantitative ELISA varies individually from 0.1 to 0.3 mmol kg^–1. The calcium-binding property can be demonstrated by Ca²⁺-dependent electrophoretic mobility shift and⁴⁵Ca²⁺ autoradiography on nitrocellulose sheets. The apparentK _D values for the SCBP-Ca²⁺ complex is approximately 10^–7 mol l^–1 as revealed by euquilibrium and flow dialysis experiments. In the presence of 1 mmol l^–1 MgCl₂ the maximum binding capacity of SCBP was determined to be either 2 mol Ca²⁺ mol^–1 protein (SCBP₂) or 3 mol Ca²⁺ mol^–1 protein (SCBP₃). Preliminary studies concerning the functional role of SCBP indicate that it facilitates the diffusion of Ca²⁺ ions by a factor of 2 and is capable of inhibiting the ATPase of isolated body wall muscle actomyosin. The results reveal that earthworm SCBP are similar to vertebrate parvalbumin and to SCBP characterized from aquatic invertebrates.Abbreviations ABTS 2,2-azino-di-(3-ethyl)-benzothiazolinsulfonate - CN-PDE 3:5-cyclic nucleotide-phosphodiesterase - DEAE diethylaminoethyl - EGTA ethyleneglycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid - ELISA enzyme linked immuno sorbent assay - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - HPLC high performance liquid chromatography - HRP horseradish peroxydase - PAGE polyacrylamide gel electrophoresis - PBS phosphate buffered saline - P _i inorganic phosphate - PMSF phenylmethylsulfonyl fluoride - SCBP soluble calcium-binding protein - SDS sodium dodecyl sulphate - SPDP N-succininydyl-3-(2-pyridyldithio)propionate - SR sarcoplasmic reticulum - Tris tris(hydroxymethyl)-aminomethane - UV ultraviolet