Human liver N-acetylglucosamine-6-sulphate sulphatase. Purification and characterization.

Human N-acetylglucosamine-6-sulphate sulphatase was purified at least 50,000-fold to homogeneity in 78% yield from liver with a simple three-step four-column procedure, which consists of a concanavalin A-Sepharose/Blue A-agarose coupled step, chromatofocusing and Cu2+-chelating Sepharose chromatography. In all, four forms were isolated and partially characterized. Forms A and B, both with a pI greater than 9.5 and representing 30% and 60% respectively of the recovered enzyme activity, were separated by hydroxyapatite chromatography of the enzyme preparation obtained from the Cu2+-chelating Sepharose step. Both forms A and B had native molecular masses of 75 kDa. When analysed by SDS/polyacrylamide-gel electrophoresis, form A consists of a single polypeptide of molecular mass 78 kDa, whereas form B contained 48 kDa and 32 kDa polypeptide subunits. Neither form A nor form B was taken up from the culture medium into cultured human skin fibroblasts. The two other forms (C and D), with pI values of 5.8 and 5.4 respectively, represented approx. 7% and 3% of the total recovered enzyme activity. The native molecular masses of forms C and D were 94 kDa and approx. 75 kDa respectively. Form C contained three polypeptides with molecular masses of 48, 45 and 32 kDa. N-Acetylglucosamine-6-sulphate sulphatase activity was measured with a radiolabelled disaccharide substrate derived from heparin. The development of this substrate enabled the isolation and characterization of N-acetylglucosamine-6-sulphate sulphatase to proceed efficiently. Forms A, B and C had pH optima of 5.0, Km values of 11.7, 14.2 and 11.1 microM respectively and Vmax. values of 105, 60 and 53 nmol/min per mg of protein respectively. The molecular basis of the multiple forms of this sulphatase is not known. It is postulated that the differences in structure and properties of the four enzyme forms are due to differences in the state of processing of a large subunit.     

Characterization of acetylcholine receptor subunits in developing and in denervated mammalian muscle

We have used subunit-specific antibodies to identify and to characterize partially the alpha, beta, gamma, and delta subunits of rat skeletal muscle acetylcholine receptor (AChR) on immunoblots. The alpha subunit of rat muscle is a single band of 42 kDa, whereas the beta subunit has an apparent molecular mass of 48 kDa. Both alpha and beta subunits are glycosylated and contain one or more N-linked oligosaccharide chains that are sensitive to endoglycosidase H digestion. The gamma and delta subunits, on the other hand, each appear as doublets on immunoblots, with apparent molecular masses of 52 kDa (gamma), 48 kDa (gamma') and 58 kDa (delta), 53 kDa (delta'), respectively. In each case, the two bands are structurally related and the lower band is probably the partial degradation product of the corresponding upper band. Each of the four gamma and delta polypeptides is N-glycosylated and contains both endoglycosidase H-sensitive and endoglycosidase H-resistant oligosaccharides. When the AChRs purified from embryonic, neonatal, adult, and denervated adult rat muscles were compared, no differences in the mobilities of alpha, beta, or delta subunits on sodium dodecyl sulfate gels were detected among them, either with or without endoglycosidase treatment. The gamma subunits, which were present in AChRs purified from neonatal, embryonic, or denervated rat muscles, were also identical; no gamma subunit was detected, however, in AChRs of normal adult rat muscle.     

Phosphorylation of numatrin and other nuclear proteins by cdc2 containing CTD kinase cdc2/p58

Numatrin is a nuclear matrix phosphoprotein whose synthesis and abundance were shown to be regulated during the cell cycle in mitogen-stimulated lymphocytes (Feuerstein, N., and Mond, J. (1987) J. Biol. Chem. 262, 11389-11397). We examined the effect of (a) CTD-kinase, which contains the cdc2 catalytic component (p34) in a complex with a p58 subunit (cdc2/p58) and (b) the M phase-specific histone H1 kinase, which contains the cdc2 kinase in association with a p62 subunit (cdc2/p62), on phosphorylation of numatrin. We show that both cdc2 kinase complexes can phosphorylate numatrin. However, cdc2/p58 at conditions that caused a similar effect to cdc2/p62 on phosphorylation of histone H1 (dpm/micrograms of substrate/micrograms of enzyme) was found to have a 5-25-fold higher catalytic activity in the phosphorylation of numatrin. Analysis of the tryptic phosphopeptide map of numatrin phosphorylated by these cdc2 kinase complexes showed that both kinase complexes phosphorylated two major identical peptides, but minor additional peptides were differentially phosphorylated by each of these kinases. This indicates that under certain experimental conditions cdc2/p58 and cdc2/p62 may express some differences in their catalytic activity. In vitro phosphorylation by CTD kinase of a whole nuclear protein extract from murine fibroblasts showed that numatrin is the most prominent substrate for CTD kinase in this nuclear extract. CTD kinase cdc2/p58 was found to induce significantly the phosphorylation of five other discrete nuclear substrates. Particularly, two nuclear proteins at 75 kDa/pI approximately 6.5 and 85 kDa/pI approximately 5.3, which were not Coomassie Blue stainable, were found to be markedly phosphorylated by CTD kinase. The results of this study call for further study of the role of CTD kinase cdc2/p58 in the phosphorylation of numatrin under physiological conditions and to further characterization of the other nuclear substrates for CTD kinase.     

Isolation and characterization of a novel 530-kDa protein complex (PC530) capable of associating with the 20S proteasome from starfish oocytes

A novel protein complex called PC530 was purified concomitantly with proteasomes from oocytes of the starfish, Asterina pectinifera, by chromatography with DEAE-cellulose, phosphocellulose, Mono Q, and Superose 6 columns. The molecular mass of this complex was estimated to be 530 kDa by Ferguson plot analysis and about 500 kDa by Superose 6 gel filtration. Since the 1500-kDa proteasome fractions contain the PC530 subunits as well as the 20S proteasomal subunits, and also since the purified PC530 and the 20S proteasome were cross-linked with a bifunctional cross-linking reagent, it is thought that PC530 is able to associate with the 20S proteasome. The PC530 comprises six main subunits with molecular masses of 105, 70, 50, 34, 30, and 23 kDa. The 70-kDa subunit showed a sequence similarity to the S3/p58/Sun2/Rpn3p subunit of the 26S proteasome, whereas the other subunits showed little or no appreciable similarity to the mammalian and yeast regulatory subunits. These results indicate that starfish oocytes contain a novel 530-kDa protein complex capable of associating with the 20S proteasome, which is distinctly different from PA700 or the 19S regulatory complex in molecular size and subunit composition.     

The protein phosphatases involved in cellular regulation. 2. Purification, subunit structure and properties of protein phosphatases-2A0, 2A1, and 2A2 from rabbit skeletal muscle

Protein phosphatases-2A0, 2A1 and 2A2 have been purified to homogeneity from rabbit skeletal muscle. Approximately 1 mg of phosphatase-2A0 and 2A1, and 0.5 mg of phosphatase-2A2, was isolated from 4000 g muscle within 10 days. Protein phosphatases-2A0 and 2A1 each comprised three subunits, termed A, B' and C (2A0) or A, B and C (2A1), while phosphatase-2A2 contained only two subunits, A and C. The A and C components of phosphatases-2A0, 2A1 and 2A2 had indistinguishable mobilities on sodium dodecyl sulphate/polyacrylamide gels and identical peptide maps. By these criteria, the C component was also identical to the catalytic subunit of phosphatase-2A purified from ethanol-treated muscle extracts. The electrophoretic mobilities of the B and B' subunits were slightly different, and their peptide maps were distinct. The molecular masses of the native enzymes determined by sedimentation equilibrium centrifugation were 181 +/- 6 kDa (2A0), 202 +/- 6 kDa (2A1) and 107 +/- 5 kDa (2A2), while those of the subunits estimated by sodium dodecyl sulphate/polyacrylamide gel electrophoresis were 60 kDa (A), 55 kDa (B), 54 kDa (B') and 36 kDa (C). These values, in conjunction with molar ratios estimated by densitometric analyses of the gels, suggest that the subunit structures of the enzymes are AB'C2 (2A0), ABC2 (2A1) and AC (2A2). Protein phosphatase-2A2 appears to be derived from 2A0 and/or 2A1 during purification through degradation or dissociation of the B' and/or B subunits. Protein phosphatases-2A0, 2A1 and 2A2 were the only phosphorylase phosphatases in rabbit skeletal muscle that were activated by the basic proteins, protamine (A0.5 = 0.25 microM), histone H1 (A0.5 = 0.3 microM) and polylysine (A0.5 = 0.04 microM). Activation by protamine varied over 5-20-fold for phosphatase-2A0 and 5-7-fold for phosphatases-2A1 and 2A2. The dephosphorylation of glycogen synthase was activated by basic proteins in a similar manner to the phosphorylase phosphatase activity. The isolated C subunit was also stimulated by histone H1 and protamine, but 5-10-fold higher concentrations were required, and with phosphorylase as substrate, maximum activation was only about 2-fold. Activation by basic proteins appears to involve their interaction with the A and/or C subunits, but not with the B or B' subunits, or substrates phosphorylase and glycogen synthase.     

Subunit composition of the H+-ATPase complex from anaerobic bacterium Lactobacillus casei

The H+-ATPase complex has been isolated from the membranes of the anaerobic bacterium Lactobacillus casei by two independent methods. 1. The crossed-immunoelectrophoresis of the 14C-labelled ATPase complex against antibodies to a highly purified soluble ATPase has been used. The subunit composition of the complex has been established by autoradiography. The soluble part of L. casei ATPase, in contrast to coupling factor F1-ATPases of aerobic bacteria, chloroplasts and mitochondria which include two kinds of large subunit (alpha and beta), consists of one kind of large subunit with a molecular mass of 43 kDa. Moreover, a minor polypeptide of 25 kDa has been found in the soluble ATPase. Factor F0 of L. casei ATPase complex consists of a 16-kDa subunit and two subunits with molecular masses less than 14 kDa. 2. A dicyclohexylcarbodiimide-sensitive ATPase complex has been isolated from L. casei membranes by treating them with a mixture of octyl glucoside and sodium cholate. The complex, purified by centrifugation on a sucrose density gradient, contains the main subunits with molecular masses of 43 kDa, 25 kDa and 16 kDa and a dicyclohexylcarbodiimide-binding subunit with a molecular mass less than 14 kDa.     

Subunit constitution of carbonic anhydrase from Chlamydomonas reinhardtii

Carbonic anhydrase purified from the cell surface of Chlamydomonas reinhardtii was inactivated by treatment with dithiothreitol. This treatment caused dissociation of the holoenzyme into 35-kDa (A) and 4-kDa (B) subunits as revealed by SDS/PAGE. The 35-kDa subunit was further separated into two components A1 (35 kDa) and A2 (36.5 kDa) by SDS/PAGE using a gradient gel. These two components have the same amino acid sequence up to at least the 10th amino acid from the N-terminus. The molecular masses were estimated at 76 kDa and 35 kDa for the holoenzyme and the large subunit, respectively, and the molar ratio of the former to the latter at 1:2, by using the techniques of low-angle laser light-scattering photometry and precision differential refractometry combined with gel-filtration HPLC. The molar ratio of the 35-kDa/4-kDa subunits was estimated at 1:1 the gel-filtration HPLC monitored with precision differential refractometry. Atomic-absorption spectrophotometry revealed that the holoenzyme contains two atoms of zinc. These results suggest that the holoenzyme is a heterotetramer composed of two large subunits (A1 and A2) and two small subunits (B).     

3D imaging of the 58 kDa cell binding subunit of the Helicobacter pylori cytotoxin.

Pathogenic strains of Helicobacter pylori produce a potent exotoxin, VacA, which intoxicates gastric epithelial cells and leads to peptic ulcer. The toxin is released from the bacteria as a high molecular mass homo-oligomer of a 95 kDa polypeptide which undergoes specific proteolytic cleavage to 37 kDa and 58 kDa subunits. We have engineered a strain of H. pylori to delete the gene sequence coding for the 37 kDa subunit. The remaining 58 kDa subunit is expressed efficiently and exported as a soluble dimer that is non-toxic but binds target cells in a manner similar to the holotoxin. A 3D reconstruction of the molecule from electron micrographs of quick-freeze, deep-etched preparations reveals the contribution of each building block to the structure and permits the reconstruction of the oligomeric holotoxin starting from individual subunits. In this model P58 subunits are assembled in a ring structure with P37 subunits laying on the top. The data indicate that the 58 kDa subunit is capable of folding autonomously into a discrete structure recognizable within the holotoxin and containing the cell binding domain.     

Identification of two messenger RNA cap binding proteins in wheat germ. Evidence that the 28-kDa subunit of eIF-4B and the 26-kDa subunit of eIF-4F are antigenically distinct polypeptides

Previous work has shown that eukaryotic initiation factor (eIF)-4B from wheat germ is a complex containing two subunits, 80 and 28 kDa, and eIF-4F from wheat germ is a complex containing two subunits, 220 and 26 kDa (Lax, S., Fritz, W., Browning, K., and Ravel, J. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 330-333). Here we show that both the 28-kDa subunit of eIF-4B and the 26-kDa subunit of eIF-4F cross-link to the 5' terminus of capped and oxidized satellite tobacco necrosis virus RNA in the absence of ATP and that the cross-linking of both polypeptides is inhibited by m7GDP. Several lines of evidence indicate that the 28-kDa and the 26-kDa cap binding proteins of eIF-4B and eIF-4F are antigenically distinct polypeptides. Rabbit polyclonal antibodies raised to intact eIF-4B or to the isolated 28-kDa subunit of eIF-4B react strongly with the 28-kDa subunit of eIF-4B on immunoblots, but show only a very weak reaction with the 26-kDa subunit of eIF-4F under the same conditions. In addition, a mouse monoclonal antibody was obtained that reacts strongly with the 26-kDa subunit of eIF-4F but does not react with the 28-kDa subunit of eIF-4B. Evidence is presented also which indicates that the higher molecular weight subunits of eIF-4B and eIF-4F are antigenically distinct. Rabbit polyclonal antibodies raised to intact eIF-4B or the isolated 80-kDa subunit inhibit eIF-4B-dependent polypeptide synthesis but do not inhibit eIF-4F-dependent polypeptide synthesis. Rabbit polyclonal antibodies raised to eIF-4F inhibit eIF-4F-dependent polypeptide synthesis but do not inhibit eIF-4B-dependent polypeptide synthesis.     

Radiation Inactivation Analysis of Progesterone Receptor in Human Uterine Cytosol

It is believed that human progesterone receptor (PR) contains a ligand binding subunit A (83 kDa) or subunit B (120 kDa) and 2 copies of heat shock proteins (hsp90) of molecular weight 90 kDa. To elucidate the mechanism of hormone binding, we employed radiation inactivation to determine its functional size. The functional masses determined in the presence of glycerol, molybdate and potassium chloride were 120 \pm 14, 124 \pm 13 and 130 \pm 20 kDa, respectively. From scatchard plot analysis, the radiation decreased the binding sites and increased the binding affinity of PR with ligand. The functional masses of PR dissolved in the three variant buffers were similar to the molecular weight of PR subunit B. The results implied that PR subunit B could bind with ligand despite hsp90 and hsp90 was not involved in the PR binding to progesterone.     

Different forms of human liver glutathione S-transferases arise from dimeric combinations of at least four immunologically and functionally distinct subunits.

Four immunologically distinct subunits were characterized in glutathione (GSH) S-transferases of human liver. Five cationic enzymes (pI 8.9, 8.5, 8.3, 8.2 and 8.0) have an apparently similar subunit composition, and are dimers of 26 500-Mr (A) and 24 500-Mr (B) subunits. A neutral enzyme, pI 6.8, is a dimer of B-type subunits. One of the anionic enzymes, pI 5.5, is also a dimer of 26 500-Mr subunits. However, the 26 500-Mr subunits of this anionic enzyme form are immunologically distinct from the A subunits of the cationic enzymes, and have been designated as A'. Immunoabsorption studies with the neutral enzyme, BB, and the antibodies raised against the cationic enzymes (AB) indicate that A and B subunits are immunologically distinct. Hybridization in vitro of the A and B subunits of the cationic enzymes (AB) results in the expected binary combinations of AA, AB and BB. Studies with the hybridized enzyme forms indicate that only the A subunits express GSH peroxidase activity. A' subunits have maximum affinity for p-nitrobenzyl chloride and p-nitrophenyl acetate, and the B subunits have highest activity towards 1-chloro-2,4-dinitrobenzene. The other anionic form, pI 4.5, present in liver is a heterodimer of 22 500-Mr (C) and B subunits. The C subunits of this enzyme are probably the same as the 22 500-Mr subunits present in human lung and placental GSH transferases. The distinct immunological nature of B and C subunits was also demonstrated by immunoaffinity and subunit-hybridization studies. The results of two-dimensional polyacrylamide-gel-electrophoretic analyses indicate that in human liver GSH transferases, three charge isomers of Mr 26 500 (A type), two charge isomers of Mr 24 500 (B type) and two charge isomers of Mr 22 500 (C type) subunits are present.     

Geotrichum candidum produces several lipases with markedly different substrate specificities.

We have purified and examined the substrate specificity of four lipases from two strains of the mould Geotrichum candidum, ATCC 34614 and CMICC 335426. We have designated the lipases I and II (ATCC 34614), and A and B (CMICC 335426). The enzymes are monomeric and have similar molecular masses and pI. Thus, lipases I and II have native molecular masses of 50.1 kDa and 55.5 kDa, and pI of 4.61 and 4.47, respectively. Lipases A and B are very similar to lipases I and II with native molecular masses of 53.7 kDa and 48.9 kDa, and pI of 4.71 and 4.50, respectively. Treatment with endo-beta-N-acetylglucosaminidase caused a reduction in molecular mass of approximately 4.5 kDa for all four lipases, indicating that these enzymes are glycosylated. Western blotting shows that the lipases are related. However, lipase B from CMICC 335426 shows a remarkable specificity for unsaturated substrates with a double bond at position 9 (cis configuration), and this specificity is not exhibited by the other three lipases. No lipase of this unique specificity has previously been purified to homogeneity. Structural studies using these four lipases should allow insight into the molecular basis of this remarkable specificity.     

Sunflower seed protein: size and charge heterogeneity in subunits of the globulin fraction

The subunit heterogeneity of the globulin fraction of sunflower seeds was investigated by two dimensional electrophoresis, using isoelectric focusing in the first dimension and sodium dodecyl sulphate polyacrylamide gel electrophoresis in the second dimension. Under non reducing conditions, intermediary subunits B, C and D (molecular weight 54 000, 48 000 and 40 000, respectively) were focused within a pI range 5.4-6.0 but intermediary subunits A (molecular weight 60 000) focused within a pI range 6.3-6.8. Under reducing conditions the electrophoretic patterns show that intermediary subunits consist in large "acidic" and small "basic" subunits linked by disulphide bonds. The large subunits of B species are more acidic and less heterogeneous than the corresponding subunits of the A species. These results confirm that helianthinin had a "legumin-type" structure.     

Multiple forms of the 20 S multicatalytic and the 26 S ubiquitin/ATP-dependent proteases from rabbit reticulocyte lysate.

We have used native gel electrophoresis followed by fluorogenic peptide overlay to identify multiple forms of rabbit reticulocyte multicatalytic protease (MCP) or 20 S protease, and two forms of rabbit 26 S ubiquitin/ATP-dependent protease. An abundant, fast-migrating 20 S complex (20 SF) possesses modest ability to hydrolyze the fluorogenic peptide succinyl-Leu-Leu-Val-Tyr-4-methyl-coumaryl-7-amide. In contrast, two minor, slower migrating species cleave the peptide at high rates. A unique 30-kDa polypeptide is associated with one of the active MCPs, and a 160-kDa subunit is associated with the other. Two electrophoretically distinct 26 S proteases can also be isolated from rabbit reticulocyte lysate. The faster migrating form, 26 SF, is more resistant to inactivation by ATP depletion. Despite the differential response to nucleotides and the distinctive electrophoretic mobilities of 26 SF and 26 SS, we have not identified any subunit differences between the two enzymes. In addition to active 26 S proteases, we have discovered and purified a proteolytically inactive particle that contains subunits characteristic of the 26 S protease (e.g. molecular masses between 30 and 110 kDa). Incubation of this protein complex with purified MCP and ATP results in the formation of the 26 S proteases.     

Phosphorylase kinase from chicken skeletal muscle. Quaternary structure, regulatory properties and partial proteolysis

Phosphorylase kinase has been purified from white and red chicken skeletal muscle to near homogeneity, as judged by sodium dodecyl sulphate (SDS) gel electrophoresis. The molecular mass of the native enzyme, estimated by chromatography on Sepharose 4B, is similar to that of rabbit skeletal muscle phosphorylase kinase, i.e. 1320 kDa. The purified enzyme both from white and red muscles showed four subunits upon polyacrylamide gel electrophoresis in the presence of SDS, corresponding to alpha', beta, gamma' and delta with molecular masses of 140 kDa, 129 kDa, 44 kDa and 17 kDa respectively. Based on the molecular mass of 1320 kDa for the native enzyme and on the molar ratio of subunits as estimated from densitometric tracings of the polyacrylamide gels, a subunit formula (alpha' beta gamma' delta)4 has been proposed. The antiserum against the mixture of the alpha' and beta subunits of chicken phosphorylase kinase gave a single precipitin line with the chicken enzyme but did not cross-react with the rabbit skeletal muscle phosphorylase kinase. The pH 6.8/8.2 activity ratio of phosphorylase kinase from chicken skeletal muscle varied from 0.3 to 0.5 for different preparations of the enzyme. Chicken phosphorylase kinase could utilize rabbit phosphorylase b as a substrate with an apparent Km value of 0.02 mM at pH 8.2. The apparent V (18 mumol min-1 mg-1) and Km values for ATP at pH 8.2 (0.20 mM) were of the same order of magnitude as that of the purified rabbit phosphorylase kinase b. The activity of chicken phosphorylase kinase was largely dependent on Ca2+. The chicken enzyme was activated 2-4-fold by calmodulin and troponin C, with concentrations for half-maximal activation of 2 nM and 0.1 microM respectively. Phosphorylation with the catalytic subunit of cAMP-dependent protein kinase (up to 2 mol 32P/mol alpha beta gamma delta monomer) and autophosphorylation (up to 8 mol 32P/mol alpha beta gamma delta monomer) increased the activity 1.5-fold and 2-fold respectively. Limited tryptic and chymotryptic hydrolysis of chicken phosphorylase kinase stimulated its activity 2-fold. Electrophoretic analysis of the products of proteolytic attack suggests some differences in the structure of the rabbit and chicken gamma subunits and some similarities in the structure of the rabbit red muscle and chicken alpha'.     

Chemical modification of charged amino acid moieties alters the electrophoretic mobilities of neurofilament subunits on SDS/polyacrylamide gels

The increase in the mobilities of neurofilament subunits on SDS-PAGE after dephosphorylation was reversed upon boiling in urea or trifluoroacetylation of lysine epsilon-amino groups. Trifluoroacetylation of native and dephosphorylated neurofilaments also resulted in an overall increase in the phosphorylation of the three subunits by the catalytic subunit of cyclic-AMP-dependent protein kinase. The gel-electrophoretic mobility of neurofilament subunits was also shown to be influenced by carboxylic amino acid residues, as neutralization of these moieties by glycinamidation increased the mobilities of all three subunits on SDS-PAGE. Neurofilament subunits that were both glycinamidated and dephosphorylated had apparent molecular masses of approximately 60 kDa, 112 kDa and 138 kDa. The major sites of these changes in the two largest subunits were shown to be the carboxy-terminal tail domains, which are known to contain high percentages of glutamate. Since interspecies differences in the apparent molecular masses of neurofilament subunits were shown to persist after glycinamidation and dephosphorylation, they appear to be due to differences in polypeptide chain length, rather than glutamate content.     

Purification and biochemical characterization of the F1-ATPase from Acidithiobacillus ferrooxidans NASF-1 and analysis of the atp operon

ATPase was purified 51-fold from a chemoautotrophic, obligately acidophilic iron-oxidizing bacterium, Acidithiobacillus ferrooxidans NASF-1. The purified ATPase showed the typical subunit pattern of the F1-ATPase on a polyacrylamide gel containing sodium dodecyl sulfate, with 5 subunits of apparent molecular masses of 55, 50, 33, 20, and 18 kDa. The enzyme hydrolyzed ATP, GTP, and ITP, but neither UTP nor ADP. The K(m) value for ATP was 1.8 mM. ATPase activity was optimum at pH 8.5 at 45 degrees C, and was activated by sulfite. Azide strongly inhibited the enzyme activity, whereas the enzyme was relatively resistant to vanadate, nitrate, and N,N'-dicyclohexylcarbodiimide. The genes encoding the subunits for the F1F(O)-ATPase from A. ferrooxidans NASF-1 were cloned as three overlapping fragments by PCR cloning and sequenced. The molecular masses of the alpha, beta, gamma, delta, and epsilon subunits of the F1 portion were deduced from the amino acid sequences to be 55.5, 50.5, 33.1, 19.2, and 15.1 kDa, respectively.     

Species-specific oocyte proteins as molecular markers for Lineus taxonomy

The yolk proteins from the eggs of five species of thenemertean genus Lineus were analysed and theresulting data were used to define L. longissimus, L. sanguineus, L. lacteus, L. ruber, and L. viridis as distinct taxa (species to date). All fivespecies have at least one large (80–60 kDa) and onemedium sized (45–30 kDa) vitellin subunit, but thereare significant differences in the number and sizes ofthe molecular subunits. The large and medium sizedvitellin components of all the species areglycosylated, except for the 145 kDa protein of L. sanguineus. Rabbit antibodies to the L. lacteus vitellin subunits cross-reacted withthe vitellin subunits in the eggs of the otherspecies. The vitellin components of these five speciesof nemertean are very similar. Two species, L.ruber and L. viridis, lay their eggsin gelatinous masses, and the electrophoretic patternsof the jelly proteins show that the physicalconsistency of the jelly depends on molecular weightsof the components. The egg mass of L. viridis contains smaller proteins than the egg massof L. ruber. The repeatablespecies-specific patterns of vitellin componentsprovide a useful complement to the usual taxonomiccriteria.     

Purification and characterization of Lip2 and Lip3 isoenzymes from a Candida rugosa pilot-plant scale fed-batch fermentation

Previous purification of a crude extracellular enzyme preparation from Candida rugosa ATCC 14830 pilot-plant fed-batch fermentations showed the presence of two lipase isoenzymes, Lip2 and Lip3, differing in their molecular masses (58 and 62 kDa, respectively). These enzymes were purified but the lipases were forming active aggregates with a molecular mass higher than 200 kDa. In this work we developed a purification method following three steps: ammonium sulfate precipitation, sodium cholate treatment and ethanol/ether precipitation, and anion exchange chromatography which allowed the sequential disaggregation of the isoenzymes. Pure and monomeric Lip2 and Lip3 were characterized according to pI, glycosylation and activity for p-nitrophenol esters and triacylglycerols of varying acyl chain. Lip3 was the best catalyst for the hydrolysis of the simple esters and triacylglycerols with short and medium acyl chains.