Molecular Mass Estimates Research Articles

Micellar enzymology: methodology and technique

The methodological procedures employed both for serving investigations in the field of micellar enzymology and developed directly in micellar enzymology and being of general significance, such as an estimation of molecular masses and sizes of biocatalysts, titration of active sites of enzymes, chemical modification of proteins (enzymes), conjugation and nanogranulation, are reviewed. Potentialities of using and informativity of various techniques are analyzed.

Purification, Characterization and Thermostability of Ribulose 1,5-Bisphosphate Carboxylase-Oxygenase from Barley Leaves

Abstract The enzyme ribulose-1,5-bisphosphate carboxylase-oxygenase (rubisco) and its functional subunits from barley (Hordeum vulgare L.) leaves were purified to homogeneity by activitydirected sequencial steps of chromatography. Based on the molecular mass estimation by SDS-PAGE, the large subunit (LS) had an apparent molecular weight of ca. 55 kDa, whereas the small subunit (SS) was ca. a 14 kDa polypeptide chain. The N-terminal sequences, established by automated Edman degradation analysis of the purified subunits, showed very close sequence homologies (52-92%) with the subunits of other rubisco enzymes reported from several photosynthetic species. In order to establish the chemical heterogeneity in the rubisco from barley, the amino acid composition of purified native enzyme was analyzed and the results systematically compared with other known type-I rubisco enzymes from spinach, maize, tobacco and pea. Major differences have been observed in the amino acid composition of barley rubisco, the concentration of cysteine, serine, threonine, isoleucine, leucine, arginine and tryptophan residues were found quite variable as compared to other higher plants. The thermostability of the native rubisco was also investigated using circular dichroism and fluorescence spectroscopy. The critical ( Tc) and melting ( Tm) temperatures were determined to be 60 °C and 57 °C, respectively, and at this temperature the enzyme not only retains its structural integrity but also its enzymatic activity. Results of these studies were discussed in the light of structural and functional adaptation of this bifunctional enzvme in C3 and C4 plants to their environments.

A novel lysine 2,3-aminomutase encoded by the yodO gene of Bacillus subtilis: characterization and the observation of organic radical intermediates

The yodO gene product of Bacillus subtilis has been cloned and overexpressed in Escherichia coli and purified. The nucleotide sequence encodes a protein of 471 amino acids with a calculated molecular mass of 54071 Da. The translated amino acid sequence is more than 60% identical to that of the lysine 2,3-aminomutase from Clostridium subterminale SB4. Analytical HPLC gel-permeation chromatography leads to an estimate of an over all molecular mass of 224000+/-21000 Da, which corresponds to a tetrameric protein. The purified protein contains iron, sulphide and pyridoxal 5'-phosphate (PLP) and displays an optical absorption band extending to 700 nm, suggesting the presence of an iron-sulphide cluster. After reductive incubation with L-cysteine anaerobically, the protein catalyses the transformation of L-lysine into beta-lysine in the presence of S-adenosylmethionine (AdoMet) and sodium dithionite. The K(m) value for L-lysine is estimated to be 8.0+/-2.2 mM. The iron-sulphur centre is stable in air,allowing aerobic purification. EPR spectroscopy at 10 K of the purified enzyme revealed an EPR signal similar to that of the [4Fe-4S](3+) cluster observed in the clostridial lysine 2, 3-aminomutase. Incubation with cysteine under anaerobic conditions converts the iron-sulphur centre into the EPR-silent [4Fe-4S](2+). Unlike the clostridial enzyme, the fully reduced [4Fe-4S](+) could not be characterized by further reduction with dithionite in the presence of AdoMet, although both dithionite and AdoMet were required to activate the enzyme. Upon addition of L-lysine, dithionite and AdoMet to the reduced enzyme and freezing the solution to 77 K, the EPR spectrum revealed the presence of an organic free-radical signal (g=2.0023), which displayed multiple hyperfine transitions very similar to the spectrum of the beta-lysine-related radical in the mechanism of the clostridial lysine 2,3-aminomutase. Experiments with isotopically substituted L-lysine and lysine analogues verified the association of spin density with the carbon skeleton of lysine. The data indicate that the protein encoded by the yodO gene of B. subtilis is a novel lysine 2,3-aminomutase. The E. coli homologue of clostridial lysine 2,3-aminomutase was also expressed in E. coli and purified. This protein contained ironand sulphide but not PLP, it did not display lysine 2,3-aminomutase activity, and addition of PLP did not induce 2,3-aminomutase activity.

Submillimetre imaging of NGC 3079

Submillimetre mapping observations of the active edge-on spiral galaxy NGC 3079 are presented. These maps at 850 and 450 μm were made with the Submillimetre Common User Bolometer Array (SCUBA) at the James Clerk Maxwell Telescope (JCMT). The source structure at these wavelengths consists of a central unresolved source embedded in diffuse disc emission, similar to that displayed at 1.2 mm. The disc emission is fitted with two optically thin, isothermal dust models which give temperatures of 12 and 31 K, similar to those derived previously by Braine et al. The core component is well described by a single-temperature fit (∼32 K). The combined dust mass from these observations, using the same mass absorption coefficient as Devereux & Young (1990) is 3.5×108 M⊙, of which ∼90 per cent resides in the cold component of the galactic disc. The effect of the cold dust component detected by SCUBA is thus to reduce the global gas-to-dust mass ratio from ∼1400 found in the above study to 85, very similar to the Galactic level. Calculations using the models of Draine & Lee and/or alternative molecular gas mass estimates yield gas-to-dust mass ratios in the range 60–190. The data presented here, together with previously published 1.2-mm mapping observations and IRAS data, are inconsistent with detections made with the Infrared Space Observatory (ISO). In particular, the latter give an excess of flux at 200 and 180 μm relative to that predicted by our simple model fits (approximately a factor of 2–3).

Plasma Membrane Plasticity of Xenopus laevis Oocyte Imaged with Atomic Force Microscopy

Proteins are known to form functional clusters in plasma membranes. In order to identify individual proteins within clusters we developed a method to visualize by atomic force microscopy (AFM) the cytoplasmic surface of native plasma membrane, excised from Xenopus laevis oocyte and spread on poly-L-lysine coated glass. After removal of the vitelline membrane intact oocytes were brought in contact with coated glass and then rolled off. Inside-out oriented plasma membrane patches left at the glass surface were first identified with the lipid fluorescent marker FM1-43 and then scanned by AFM. Membrane patches exhibiting the typical phospholipid bilayer height of 5 nm showed multiple proteins, protruding from the inner surface of the membrane, with heights of 5 to 20 nm. Modelling plasma membrane proteins as spherical structures embedded in the lipid bilayer and protruding into the cytoplasm allowed an estimation of the respective molecular masses. Proteins ranged from 35 to 2,000 kDa with a peak value of 280 kDa. The most frequently found membrane protein structure (40/μm²) had a total height of 10 nm and an estimated molecular mass of 280 kDa. Membrane proteins were found firmly attached to the poly-L-lysine coated glass surface while the lipid bilayer was found highly mobile. We detected protein structures with distinguishable subunits of still unknown identity. Since X. laevis oocyte is a generally accepted expression system for foreign proteins, this method could turn out to be useful to structurally identify specific proteins in their native environment at the molecular level.

Purification and characterization of a natural agglutinin from the serum of the hermit crab Diogenes affinis

A natural agglutinin from the serum of the hermit crab Diogenes affinis was purified to homogeneity by a single-step affinity chromatography using N-acetylglucosamine-coupled Sepharose 6B. The purified serum agglutinin (PSA) showed a strong affinity for rat RBC, and its hemagglutinating (HA) activity was specifically dependent on Ca 2+ and reversibly sensitive to EDTA. PSA in active form has a molecular mass estimate of 185 kDa and is composed of four non-identical subunits (51, 49, 42 and 39 kDa) cross-linked by interchain disulfide bonds. The homogeneity of PSA was corroborated by immunodiffusion and immunoelectrophoretic analyses using rabbit antiserum raised against the agglutinin. The antibodies in this antiserum appear to be specific for RBC-binding sites of the agglutinin molecules as revealed by the ability of the antiserum to neutralize HA activities of both whole serum and PSA of D. affinis. In HA-inhibition assays performed with several carbohydrates and glycoproteins, PSA showed a distinct and unique specificity for acetyl group in carbohydrates independently of the presence of this group on C-2 or C-5 and its stereochemical arrangement in the axial or equatorial orientation. Besides, this agglutinin appears to recognize the terminal N- and O- acetyl groups in the oligosaccharide chain of glycoconjugates. The HA activity of D. affinis agglutinin was also susceptible to inhibition by lipopolysaccharides from diverse Gram-negative bacteria, which might indicate a significant in vivo role of this humoral agglutinin in the host immune response against bacterial infections.

Oxaloacetate Decarboxylase fromPseudomonas stutzeri:Purification and Characterization

Oxaloacetate decarboxylase (OXAD), the enzyme that catalyzes the decarboxylation of oxaloacetate to pyruvic acid and carbon dioxide, was purified 245-fold to homogeneity fromPseudomonas stutzeri.The three-step purification procedure comprised anion-exchange chromatography, metal-chelate affinity chromatography, and biomimetic-dye affinity chromatography. Estimates of molecular mass from sodium dodecyl sulfate–polyacrylamide gel electrophoresis and native high-performance gel-filtration liquid chromatography were, respectively, 63 and 64 kDa, suggesting a monomeric protein. OXAD required for maximum activity divalent metal cations such as Mn2+and Mg2+but not monovalent cations. The enzyme is not inhibited by avidin, but is competitively inhibited by adenosine 5′-diphosphate, acetic acid, phosphoenolpyruvate, malic acid, and oxalic acid. Initial velocity, product inhibition, and dead-end inhibition studies suggested a rapid-equilibrium ordered kinetic mechanism with Mn2+being added to the enzyme first followed by oxaloacetate, and carbon dioxide is released first followed by pyruvate. Inhibition data as well as pH-dependence profiles and kinetic parameters are reported and discussed in terms of the mechanism operating for oxaloacetate decarboxylation.

Identification, purification and properties of a β-1,3-glucan-specific lectin from the serum of the cockroach, Blaberus discoidalis which is implicated in immune defence reactions

A lectin specific for laminarin, a β-1,3-glucan, agglutinating baker’s yeast and enhancing prophenoloxidase activation by laminarin, has been purified from the cockroach, Blaberus discoidalis, serum. Purification involved gel filtration with Bio-gel P300 and affinity chromatography on blue Sepharose CL-6B and laminarin-Sepharose 4B. The purified lectin has a molecular mass estimate of 520 kDa determined by gel filtration, and approximately 80 and 82 kDa by SDS-PAGE, under non-reducing and reducing conditions, respectively. After isoelectric focusing the lectin focused as a single band at pH 4.9. The purified lectin was stained by the periodic acid/Schiff’s reagent showing that it is a glycoprotein, and was deglycosylated by endo-β- N-acetylglucosaminidase F. Amino acid composition analysis showed the protein is similar to previously purified β-1,3-glucan binding proteins from other invertebrates. In electron micrographs by negative staining, the protein formed large aggregates with ‘Y’-shaped ‘structural units’ ca. 79×65 nm. Immunological tests confirmed that this lectin is not related to any other lectins previously purified from the same insect. This protein appears to be part of the hexamerin family of proteins. This is one of the first reports of a hexamerin-like molecule with lectin activity.

Determination of Diffusion Coefficients of Proteins by Flow Injection Analysis and Its Application to Estimation of Molecular Masses of Proteins

A Flow Injection Analysis (FIA) method for determination of molecular diffusion coefficients (Dm) of proteins, in a helically coiled tube, was reported. The coiling effect of the tube that causes secondary flow was avoided through using a Taylor-Aris and Golay's dispersion equation, modified by the velocity profile factor, K, and the modified equation was used for direct determination of diffusion coefficients. The experimental values of Dm were comparable with the literature values. The linear relationship between the Relative Molecular Mass (RMM, Mr) and the Molecular Diffusion Coefficient (Dm) was verified and a two-point calibration FIA method for estimation of Relative Molecular masses of proteins was suggested. Relative Molecular masses of two protein samples, hemoglobin and glucose oxidase were then estimated by this method. The values of RMM were 6.88x104 for hemoglobin and 16.0x104 for glucose oxidase.

2-[(4″-Hydroxy-3′-methoxy)-phenoxy]-4-(4″-hydroxy-3″-methoxy-phenyl)-8-hydroxy-6-oxo-3-oxabicylo[3.3.0]-7-octene: unusual product of the soybean lipoxygenase-catalyzed oxygenation of curcumin

Racemic (1 SR,2 SR,4 SR,5 SR)-2-[(4′-hydroxy-3′-methoxy)-phenoxy]-4-(4″-hydroxy-3″-methoxy-phenyl)-8-hydroxy-6-oxo-3-oxabicyclo[3.3.0]-7-octene ( 2, C 21H 20O 8) was isolated as major product of soybean lipoxygenase action on curcumin ( 1, C 21H 20O 6). The structure of 2 was elucidated by HPLC-APCI-MS and tandem MS, 1H, 13C, DEPT, H,H-COSY, H,C-HMQC, H,C-HMBC and phase sensitive 2D NOESY NMR techniques. For kinetic studies the rate of substrate degradation was followed spectrophotometrically at 430 nm, and the rate of oxygen consumption was measured polarographically. As evaluated by both methods, K m for 1 was about four times higher than that obtained for linoleic acid (as the best substrate for soybean lipoxygenase); V max was reduced five-fold. Lipoxygenase-mediated oxygenation of 1 was confirmed by the following criteria: (i) curcumin did not react with inactivated lipoxygenase; (ii) the enzymatic reaction was strongly inhibited by inhibitors such as BHA, deferoxamine and HgCl 2; (iii) oxygen consumption (measured polarographically) and curcumin degradation (measured photometrically) were shown to occur simultaneously at a ratio of 0.8 to 1, suggesting insertion of oxygen into 1 by lipoxygenase; (iv) molecular mass estimation by APCI-MS showed a shift of 32 in molecular mass from 1 ( M r 368) to 2 ( M r 400) being equivalent to an insertion of dioxygen. Curcumin meets none of the common features for lipoxygenase substrates and, therefore, may represent a new type of substrates for this enzyme.

Application of size exclusion chromatography matrix-assisted laser desorption/ionization time-of-flight to the determination of molecular masses in polydisperse polymers

The determination of molecular mass (MM) data for polydisperse polymers by size exclusion chromatography matrix assisted laser desorption/ionization time-of-flight (SEC/MALDI-TOF) involves the fractionation of samples through an analytical SEC. Selected fractions are then analysed by MALDI-TOF and the mass spectra of these nearly monodisperse samples allow the determination of Mn and Mw averages. To test the reliability of the molecular mass estimates by the SEC/MALDI-TOF method, a sample of polymethylmethacrylate (PMMA), two samples of polydimethylsiloxane (PDMS), and four samples of copolyesters, all polydisperse, were analysed. The results show that the molecular mass values of PMMA fractions obtained by MALDI-TOF are coincident with those obtained using the SEC calibration plots obtained with anionic PMMA standards. In the case of the two polydimethylsiloxanes (PDMS1 and PDMS2: linear and cyclic, respectively), two slightly differing SEC calibration plots were obtained, reflecting the different structures of the polymer chains of the two samples. The SEC traces of four copolyesters were obtained in tetrahydrofuran and CHCl3. Data on MM, MM distribution solvent effects and copolymer composition are reported. © 1998 John Wiley & Sons, Ltd.

Characterization of potato tuber lipoxygenase cDNAs and lipoxygenase expression in potato tubers and leaves

A collection of cDNAs was isolated from a λgt11 potato (Solanum tuberosum L.) tuber library by using a soybean lipoxygenase (LOX; linoleate:oxygen oxidoreductase, EC 1.13.11.12) sequence as a hybridization probe. These cDNA clones were classified by restriction patterns and sizes and two clones, plox1 and plox2, were selected for sequencing and further analysis. These clones were found to be virtually identical and contain full‐length cDNAs having a high degree of homology with other cloned LOX genes. plox1 contains a 2 806‐bp insert, with an open reading frame that encodes a protein of 861 amino acids (96.9 kDa), consistent with molecular mass estimates of the principal tuber LOX deduced from SDS‐PAGE. The deduced sequence contains iron‐binding domains that are conserved among LOXs. Restriction analysis of potato genomic DNA confirmed that potato LOX is comprised of a gene family with at least three members. The plox1 sequence hybridized to a transcript of 2.8 kb in gel blot analyses of total RNA extracted from potato tuber. Treatment of potato disks and leaves with methyl jasmonate or jasmonic acid increased LOX enzyme activity, as well as abundance of LOX mRNA, consistent with jasmonate's reported effect on LOX expression in plants. The fungal elicitor arachidonic acid (AA) strongly induced LOX enzyme activity in leaves and induced LOX mRNA transcripts in potato leaves and tuber disks. Infection of potato leaves with Phytophthora infestans induced LOX activity as well as LOX mRNA accumulation in both incompatible and compatible interaction types. LOX expression was compared with that of a wound‐inducible proteinase inhibitor (PINII) and a pathogenesis‐related protein (P4). Jasmonates induced PINII but not P4 transcript accumulation in potato leaves. In contrast, AA induced P4 but not PINII transcript accumulation in potato leaves. Infection with P. infestans resulted in a gene expression pattern similar to that observed with AA.

A novel UDP-sugar, UDP-3-ketoglucosamine or UDP-4-ketoglucosamine, from bovine heart muscle reduces metmyoglobin with NAD(P)H.

3-Ketoglucose and similar ketosugars have been identified in microorganisms only and little is known about their functions. UDP-sugars are widely found as an intermediate in sugar metabolism in living organisms. Yet what role UDP-sugars play, or whether they play a direct role in metabolism, is still unknown. UDP-sugars were isolated and purified from bovine heart muscle, and a UDP-sugar fraction capable of NAD(P)H-dependent catalytic reduction of metmyoglobin was detected. Subsequent identification revealed that the active UDP-sugar was UDP-3- or UDP-4-ketoglucosamine. These compounds were purified from bovine cardiac muscle by ultrafiltration, anion-exchange column chromatography and reverse-phase chromatography. They were further characterized by determination of their chemical reducing activity, by comparison with synthetic UDP-3- or UDP-4-ketoglucosamine standards using high-performance liquid chromatography, by estimation of molecular mass using fast atom bombardment mass spectrometry, and by Fourier transform infrared microspectroscopy and electron probe microanalysis. The results suggest that UDP-3- or UDP-4-ketoglucosamine reduces metmyoglobin in bovine cardiac muscle. It is important that the reducing activity displayed by this ketosugar is not the effect of UDP-3- or UDP-4-ketoglucosamine alone but depends on NAD(P)H. In other words, this action of UDP-3- or UDP-4-ketoglucosamine is catalytic.

The RNA molecule CsrB binds to the global regulatory protein CsrA and antagonizes its activity in Escherichia coli.

The RNA-binding protein CsrA (carbon storage regulator) is a new kind of global regulator, which facilitates specific mRNA decay. A recombinant CsrA protein containing a metal-binding affinity tag (CsrA-H6) was purified to homogeneity and authenticated by N-terminal sequencing, matrix-assisted laser desorption/ionization time of flight mass spectrometry, and other studies. This protein was entirely contained within a globular complex of approximately 18 CsrA-H6 subunits and a single approximately 350-nucleotide RNA, CsrB. cDNA cloning and nucleotide sequencing revealed that the csrB gene is located downstream from syd in the 64-min region of the Escherichia coli K-12 genome and contains no open reading frames. The purified CsrA-CsrB ribonucleoprotein complex was active in regulating glg (glycogen biosynthesis) gene expression in vitro, as was the RNA-free form of the CsrA protein. Overexpression of csrB enhanced glycogen accumulation in E. coli, a stationary phase process that is repressed by CsrA. Thus, CsrB RNA is a second component of the Csr system, which binds to CsrA and antagonizes its effects on gene expression. A model for regulatory interactions in Csr is presented, which also explains previous observations on the homologous system in Erwinia carotovora. A highly repeated nucleotide sequence located within predicted stem-loops and other single-stranded regions of CsrB, CAGGA(U/A/C)G, is a plausible CsrA-binding element.

The Galactic Distribution of OB Associations in Molecular Clouds

Molecular clouds account for half of the mass of the interstellar medium interior to the solar circle and for all current star formation. Using cloud catalogs of two CO surveys of the first quadrant, we have fitted the mass distribution of molecular clouds to a truncated power law in a similar manner as the luminosity function of OB associations in the companion paper to this work. After extrapolating from the first quadrant to the entire inner Galaxy, we find that the mass of cataloged clouds amounts to only 40% of current estimates of the total Galactic molecular mass. Following Solomon & Rivolo, we have assumed that the remaining molecular gas is in cold clouds, and we normalize the distribution accordingly. The predicted total number of clouds is then shown to be consistent with that observed in the solar neighborhood where cloud catalogs should be more complete. Within the solar circle, the cumulative form of the distribution is -->c(>M)=105[(M -->u/M) -->0.6-1], where -->c is the number of clouds, and Mu = 6 ? 106 M? is the upper mass limit. The large number of clouds near the upper cutoff to the distribution indicates an underlying physical limit to cloud formation or destruction processes. The slope of the distribution corresponds to d -->c/dMM -->?1.6, implying that although numerically most clouds are of low mass, most of the molecular gas is contained within the most massive clouds. The distribution of cloud masses is then compared to the Galactic distribution of OB association luminosities to obtain statistical estimates of the number of massive stars expected in any given cloud. The likelihood of massive star formation in a cloud is determined, and it is found that the median cloud mass that contains at least one O star is ~105 M?. The average star formation efficiency over the lifetime of an association is about 5% but varies by more than 2 orders of magnitude from cloud to cloud and is predicted to increase with cloud mass. O stars photoevaporate their surrounding molecular gas, and even with low rates of formation, they are the principal agents of cloud destruction. Using an improved estimate of the timescale for photoevaporation and our statistics on the expected numbers of stars per cloud, we find that 106 M? giant molecular clouds (GMCs) are expected to survive for about 3 ? 107 yr. Smaller clouds are disrupted, rather than photoionized, by photoevaporation. The porosity of H II regions in large GMCs is shown to be of order unity, which is consistent with self-regulation of massive star formation in GMCs. On average, 10% of the mass of a GMC is converted to stars by the time it is destroyed by photoevaporation.

Evaluation of the uncertainty of molecular mass measurement by electrospray ionization mass spectrometry

Electrospray ionization (ES) is a novel method used in mass spectrometry (MS) for producing gas-phase ions from substances in solutions. Common practices for molecular mass estimation from ES spectra summarize the spectrum as a single peak giving no estimate of uncertainty or treat each peak as an independent molecular mass measurement. ES-MS data analysis showed that each peak in an ES spectrum does not always provide an independent measure of molecular mass. Underestimation of measurement uncertainty is a possible result. An elementary time series method, the Yule-Walker equations, was applied to molecular mass estimation from ES data.

ISO-SWS Observations of NGC 1068

We present a first analysis of 2.4-45µm spectra of NGC 1068 obtained with the Short Wavelength Spectrometer SWS on board the Infrared Space Observatory ISO. The measured fine-structure line fluxes can be fit successfully by a simple photoionization model invoking an EUV bump in the ionizing continuum, similar to the case of the Circinus galaxy. Difference are observed between the [OIV] 26µm NLR line profile and optical NLR line profiles which may indicate significant extinction to part of the NLR. We detect pure rotational transitions of molecular hydrogen that must be emitted by molecular gas spanning a wide range of temperatures. The unusual strength of the fundamental S(0) 28µm rotational transition is evidence for a large (> 1.5 × 109 M⊙) gas mass at temperatures nea r 100 K. Either most of the gas in the circumnuclear region of NGC 1068 is warm or previous molecular mass estimates based on CO observations were too low. Strong mid-infrared continuum from the circumnuclear warm dust is prominent in our spectrum. The weak PAH emission detected at the edges of the 9.7µm silicate absorption should be considered in interpretations of the silicate feature.

Argon-laser-induced fluorescence detection in sodium dodecyl sulfate-capillary gel electrophoretic separations of proteins

The use of argon-laser-induced fluorescence detection in sodium dodecyl sulfate-capillary gel electrophoresis (SDS-CGE) separations of proteins is described. A simple, rapid (10 min) precolumn labeling reaction for a series of standard proteins (molecular masses of 20100–77000) was developed which utilizes 4-fluoro-7-nitrobenzofurazan and 4-chloro-7-nitrobenzofurazan as fluorogenic labeling reagents. Detection limits of 25 ng/ml for labeled bovine serum albumin were obtained. These detection limits compare favorably with alternate detection techniques such as Coomassie blue staining (2.5 μg/ml) and UV-Vis absorption detection in SDS-CGE (0.5 μg/ml). Conditions were identified for the labeling reactions which produce no measurable zone broadening effects for labeled proteins relative to unlabeled species. By using relative migration data from labeled proteins and molecular mass calibration plots obtained exclusively from unlabeled proteins, molecular mass estimates of labeled proteins typically accurate to within 4.5% of literature values can be obtained. The labeling reaction thus maintains the molecular mass information inherent in the separation.

6-phosphofructo-1-kinase from the lipid accumulating, non-fermentative, red yeast Rhodotorula glutinis.

6-Phosphofructo-1-kinase (PFK, EC 2.7.1.11) activity was detected in extracts of Rhodotorula glutinis grown on different nitrogen and carbon sources. The activity of PFK varied depending on the composition of the culture medium and on the state of growth. Using a carbon-limited medium containing glucose and a mixed organic/inorganic nitrogen source, maximal yield, activity and stability of PFK were found under aerobic conditions at the end of the exponential growth phase. Native Rhodotorula-PFK could be separated by polyacrylamide gel electrophoresis. Applying this method for molecular mass estimation a value of 450 +/- 90 kDa was calculated. Taking into account a molecular mass of 130 +/- 5 kDa for the subunit, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis following immunoblotting, a tetrameric structure of the native PFK is likely. Polyclonal antibodies, raised against PFK from Saccharomyces cerevisiae, were able to cross-react with Rhodotorula-PFK. Therefore, there are similarities in the primary structure of both enzymes. Fructose 2,6-bisphosphate was identified as a significant activator of Rhodotorula-PFK leading to a 10-fold activation of the enzyme: maximal activation was achieved with 5 microM fructose 2,6-bisphosphate.

6-Phosphofructo-1-kinase from the lipid-accumulating, non-fermentative, red yeast Rhodotorula glutinis

6-Phosphofructo-1-kinase (PFK, EC 2.7.1.11) activity was detected in extracts of Rhodotorula glutinis grown on different nitrogen and carbon sources. The activity of PFK varied depending on the composition of the culture medium and on the state of growth. Using a carbon-limited medium containing glucose and a mixed organic/inorganic nitrogen source, maximal yield, activity and stability of PFK were found under aerobic conditions at the end of the exponential growth phase. Native Rhodotorula-PFK could be separated by polyacrylamide gel electrophoresis. Applying this method for molecular mass estimation a value of 450 ± 90 kDa was calculated. Taking into account a molecular mass of 130 ± 5 kDa for the subunit, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis following immunoblotting, a tetrameric structure of the native PFK is likely. Polyclonal antibodies, raised against PFK from Saccharomyces cerevisiae, were able to cross-react with Rhodotorula-PFK. Therefore, there are similarities in the primary structure of both enzymes. Fructose 2,6-bisphosphate was identified as a significant activator of Rhodotorula-PFK leading to a 10-fold activation of the enzyme: maximal activation was achieved with 5 μM fructose 2,6-bisphosphate.