Purification of phospholipid methyltransferase from rat liver microsomal fraction.

Phospholipid methyltransferase, the enzyme that converts phosphatidylethanolamine into phosphatidylcholine with S-adenosyl-L-methionine as the methyl donor, was purified to apparent homogeneity from rat liver microsomal fraction. When analysed by SDS/polyacrylamide-gel electrophoresis only one protein, with molecular mass about 50 kDa, is detected. This protein could be phosphorylated at a single site by incubation with [alpha-32P]ATP and the catalytic subunit of cyclic AMP-dependent protein kinase. A less-purified preparation of the enzyme is mainly composed of two proteins, with molecular masses about 50 kDa and 25 kDa, the 50 kDa form being phosphorylated at the same site as the homogeneous enzyme. After purification of both proteins by electro-elution, the 25 kDa protein forms a dimer and migrates on SDS/polyacrylamide-gel electrophoresis with molecular mass about 50 kDa. Peptide maps of purified 25 kDa and 50 kDa proteins are identical, indicating that both proteins are formed by the same polypeptide chain(s). It is concluded that rat liver phospholipid methyltransferase can exist in two forms, as a monomer of 25 kDa and as a dimer of 50 kDa. The dimer can be phosphorylated by cyclic AMP-dependent protein kinase.     

Short-chain and medium-chain aliphatic-ester synthesis in Saccharomyces cerevisiae.

In the yeast Saccharomyces cerevisiae, the enzymes which catalyse the synthesis of ethyl acetate, ethyl n-hexanoate and isoamyl acetate were partly resolved from a fraction containing slowly sedimenting lipoproteins released during cell disruption with glass beads. Solubilization with detergents and fractionation by affinity chromatography have demonstrated the presence of at least three, and probably four, ester synthases which differ in their catalytic properties. Isoamyl-acetate synthase was solubilized and extensively purified to apparent homogeneity by successive chromatographies on various columns. On the basis of its specific activity in cell-free extracts, the enzyme was purified 19,000-fold with a 5% activity yield. As judged by SDS/PAGE, it consists of a single polypeptide chain with a molecular mass of 57 +/- 3 kDa and its apparent pI is 5.5. The enzyme acetylates isoamyl alcohol, ethanol and 12-DL-hydroxystearic acid from acetyl-CoA but is unable to use n-hexanoyl-CoA as a cosubstrate. This enzyme, defined as an acetyl-CoA: O-alcohol acetyltransferase, could be the product of one of the anaerobically induced genes in S. cerevisiae.     

Methylcobalamin:homocysteine methyltransferase from Methanobacterium thermoautotrophicum. Identification as the metE gene product.

Methanobacterium thermoautotrophicum is a methane-forming archaeon that grows on H2 and CO2 as sole carbon and energy source. Cell extracts of the methanogen were found to contain methylcobalamin: homocysteine methyltransferase activity which was purified 3000-fold to a specific activity of approximately 500 U.mg-1 protein. SDS/PAGE revealed the presence of a polypeptide with an apparent molecular mass of 34 kDa. Via its N-terminal amino acid sequence, the 34-kDa polypeptide was identified as the metE gene product. The metE gene was heterologously expressed in Escherichia coli. The overproduced protein was recovered in the inclusion body fraction and was found to be inactive. The protein could be partially solubilized by unfolding in 8 M urea and then refolding. The solubilized protein had a specific activity of 450 U.mg-1. It exhibited first-order kinetics with respect to methylcobalamin concentration and Michaelis-Menten kinetics with respect to L-homocysteine concentration (apparent Km 0.1 mM). The enzyme was specific for L-homocysteine as methyl acceptor. Methylcobalamin could be substituted with methylcobinamide as methyl donor.     

Synthesis and organization of vitellogenin and vitellin molecules from the land crab Potamon potamios

We have previously reported that vitellogenin (Vg) of some female animals contained four polypeptides with molecular mass of 181, 115, 105 and 85 kDa, whereas Vg of most animals contained three polypeptides with molecular mass of 115, 105 and 85 kDa. In the present investigation, we examined whether the 181 kDa polypeptide is the precursor of 115 and 105 kDa Vg and vitellin (Vn) polypeptides. Labeling studies, using [35S]methionine on normal vitellogenic animals, showed that the radioactivity was distributed first among the 181 and 85 kDa polypeptides. SDS-PAGE analysis of purified hemolymph Vg from eyestalk ablated female animals revealed in most animals two polypeptides with an apparent molecular mass of 181 and 85 kDa. These results from in vivo experiments corroborated the view that the 115 and 105 kDa Vg and Vn polypeptides are derived from heaviest 181 kDa polypeptide. In addition it was demonstrated that hepatopancreas and ovary of Potamon potamios incubated in vitro with [35S]methionine synthesized five polypeptides with apparent molecular mass of 224, 181, 115, 105, and 85 kDa while the hepatopancreas appeared to secrete the 181, 115, 105 and 85 kDa polypeptides. The major 115, 105 and 85 kDa polypeptides were found to be components of egg Vn, while the 224 kDa polypeptide was found to be minor component of Vg and Vn from hepatopancreas and ovary extracts, respectively. We conclude that the Vn polypeptides produced by ovary are similar to those produced by hepatopancreas.     

Tissue specification and intracellular distribution of actin isoforms inVicia faba L.

Summary Two dimensional gel electrophoresis of total cell protein extracts from not expanded, and primary leaves, petioles, and roots ofVicia faba resulted in four actin isoforms at 43 kDa with pI values from 5.9 to 6.05. In contrast to root extracts, in all leaf extracts an additional immunoreactive polypeptide with a molecular mass of 51 kDa and pI 5.75 was detected. This polypeptide was present in high amounts in protein extracts of purified chloroplasts, whereas no actin isoform at 43 kDa could be demonstrated. Compared to the tissue extracts, two actin isoforms at 43 kDa with pI values of 5.9 and 6.0 were enriched, when purified plasma membranes and the membranous fraction of vacuoles were analysed. In contrast, the soluble protein fraction of the plasma membrane preparation contained only two isoactins with pI values of 5.95 and 6.05 and a molecular mass of 43 kDa. These results indicate, that the four actin isoforms at 43 kDa detected in all examined tissues ofV. faba fulfill different functions at specific intracellular compartments, for example, the anchorage of actin microfilaments to membranes.Abbreviations BSA bovine serum albumin - BCIP 5-bromo-4-chloro-3-indolyl phosphate - DDM n-decyl -D-maltopyranoside - EDTA ethylenediamine-tetraacetic acid - HG n-hexyl -D-glucopyranoside - IEF isoelectrical focusing - MES morpholinoethanesulfonic acid - 2-ME 2 mercaptoethanol - NBT nitro blue tetrazolium - pCMB p-chloromercuribenzoic acid - PVP polyvinylpyrrolidone - Tris tris (hydroxymethyl) aminomethane     

Isolation of the DNA polymerase alpha core enzyme from mouse cells

DNA polymerase alpha has been purified from mouse hybridoma cells approximately 30,000-fold using a combination of conventional and high performance liquid chromatography. In contrast to previous characterizations of mammalian DNA polymerase alpha, this enzyme has a single high molecular mass polypeptide (185 kDa) in tight association with a 68-kDa polypeptide and this structure appears to be the core DNA polymerase of the mouse cells. The biochemically purified enzyme, with a specific activity of approximately 200,000 units/mg protein, has an estimated molecular mass by gel filtration chromatography of 240 kDa and sedimentation value of 9 S, consistent with the enzyme being a heterodimer of 185 and 68 kDa. The enzyme is sensitive to both N-ethylmaleimide and aphidicolin and insensitive to ddTTP. Using an activated DNA template, the apparent Km values for the deoxynucleotide triphosphates are approximately 0.5-1 microM. The purified DNA polymerase has neither exonuclease nor primase activities and is the predominant DNA polymerase alpha activity in the mouse cells.     

Primary structure and processing of the Candida tsukubaensis alpha-glucosidase. Homology with the rabbit intestinal sucrase-isomaltase complex and human lysosomal alpha-glucosidase.

The nucleotide sequence of a 4.39-kb DNA fragment encoding the alpha-glucosidase gene of Candida tsukubaensis is reported. The cloned gene contains a major open reading frame (ORF 1) which encodes the alpha-glucosidase as a single precursor polypeptide of 1070 amino acids with a predicted molecular mass of 119 kDa. N-terminal amino acid sequence analysis of the individual subunits of the purified enzyme, expressed in the recombinant host Saccharomyces cerevisiae, confirmed that the alpha-glucosidase precursor is proteolytically processed by removal of an N-terminal signal peptide to yield the two peptide subunits 1 and 2, of molecular masses 63-65 kDa and 50-52 kDa, respectively. Both subunits are secreted by the heterologous host S. cerevisiae in a glycosylated form. Coincident with its efficient expression in the heterologous host, the C. tsukubaensis alpha-glucosidase gene contains many of the canonical features of highly expressed S. cerevisiae genes. There is considerable sequence similarity between C. tsukubaensis alpha-glucosidase, the rabbit sucrase-isomaltase complex (proSI) and human lysosomal acid alpha-glucosidase. The cloned DNA fragment from C. tsukubaensis contains a second open reading frame (ORF 2) which has the capacity to encode a polypeptide of 170 amino acids. The function and identity of the polypeptide encoded by ORF 2 is not known.     

POLYCLONAL ANTIBODIES AGAINST IRON-SUPEROXIDE DISMUTASE FROM ESCHERICHIA COLI B CROSS-REACT WITH SUPEROXIDE DISMUTASES FROM SYMBIODINIUM MICROADRIATICUM (DINOPHYCEAE)1

Assays for superoxide dismutases (SODs) were performed using cell-free extracts of the symbiotic dinoflagellate Symbiodinium microadriaticum Freudenthal (emend Trench and Blank) after separation in undenatured polyacrylamide gels. Using appropriate inhibitors (KCN and H₂O₂) we detected the presence of Cu/Zn-, Mn-, and Fe-SODs. In immunoblot assays, polyclonal antibodies against Fe-SOD from Escherichia coli B cross-reacted with two major polypeptides in the water-soluble fraction and one polypeptide in the Triton X-100-solubilized pellet fraction. The polypeptide common to both fractions, with a relative molecular mass of 43.5 kDa, was identified as Mn-SOD. In S. microadriaticum, FeSOD, found only in the water-soluble fraction, appears to be monomeric, with a relative molecular mass of 49.5 kDa.     

Reconstitution of trimethylamine-dependent coenzyme M methylation with the trimethylamine corrinoid protein and the isozymes of methyltransferase II from Methanosarcina barkeri.

Reconstitution of trimethylamine-dependent coenzyme M (CoM) methylation was achieved with three purified polypeptides. Two of these polypeptides copurified as a trimethylamine methyl transfer (TMA-MT) activity detected by stimulation of the TMA:CoM methyl transfer reaction in cell extracts. The purified TMA-MT fraction stimulated the rate of methyl-CoM formation sevenfold, up to 1.7 micromol/min/mg of TMA-MT protein. The TMA-MT polypeptides had molecular masses of 52 and 26 kDa. Gel permeation of the TMA-MT fraction demonstrated that the 52-kDa polypeptide eluted with an apparent molecular mass of 280 kDa. The 26-kDa protein eluted primarily as a monomer, but some 26-kDa polypeptides also eluted with the 280-kDa peak, indicating that the two proteins weakly associate. The two polypeptides could be completely separated using gel permeation in the presence of sodium dodecyl sulfate. The corrinoid remained associated with the 26-kDa polypeptide at a molar ratio of 1.1 corrin/26-kDa polypeptide. This polypeptide was therefore designated the TMA corrinoid protein, or TCP. The TMA-MT polypeptides, when supplemented with purified methylcorrinoid:CoM methyltransferase (MT2), could effect the demethylation of TMA with the subsequent methylation of CoM and the production of dimethylamine at specific activities of up to 600 nmol/min/mg of TMA-MT protein. Neither dimethylamine nor monomethylamine served as the substrate, and the activity required Ti(III) citrate and methyl viologen. TMA-MT could interact with either isozyme of MT2 but had the greatest affinity for the A isozyme. These results suggest that TCP is uniquely involved in TMA-dependent methanogenesis, that this corrinoid protein is methylated by the substrate and demethylated by either isozyme of MT2, and that the predominant isozyme of MT2 found in TMA-grown cells is the favored participant in the TMA:CoM methyl transfer reaction.     

S gene product: identification and membrane localization of a lysis control protein.

The product of the bacteriophage S gene has been previously shown to be required for an essential step in triggering host cell lysis. By using two different protein labeling systems, maxicells and UV-irradiated infected cells, we identified the S gene product as an 8,500-molecular-weight polypeptide associated with the cell envelope. The apparent molecular weight is significantly less than the 11,500 predicted from the S gene sequence. We were unable to confirm two previous identifications of S gene products, an acidic 15,000-molecular-weight polypeptide found by two-dimensional gel electrophoresis of infected cells and a 5,500-molecular-weight polypeptide in purified phage particles.     

Properties of a phosphocarrier protein (HPr) extracted from intact cells of Streptococcus sanguis

Cells of Streptococcus sanguis strain Challis were incubated with sodium lauroylsarcosinate to extract surface proteins. A polypeptide of apparent molecular mass 16 kDa comprising about 12% of the extract was purified using anion-exchange chromatography. The polypeptide was shown to be a phosphocarrier protein (HPr) that could also be found in the soluble (cytoplasmic) fraction from cells broken by homogenization with glass beads. In vivo labelling of S. sanguis cells with 32Pi showed that the polypeptide carried a heat- and acid-stable phosphorylation and that during sucrose starvation the HPr became dephosphorylated. Antiserum raised to the S. sanguis HPr reacted on Western blots with HPrs from all oral streptococci tested, together with strains of S. pyogenes and S. salivarius, but not with HPrs from S. faecalis or S. bovis, nor with proteins from Staphylococcus aureus, Bacillus subtilis, Actinomyces viscosus and various lactobacilli. The S. sanguis HPr had a high content of alanine (17.2%) and was similar in overall amino acid composition to the HPrs from S. mutans an S. salivarius. The N-terminal residues (to 37) of the S. sanguis HPr showed strong sequence identity (82%) with the N-terminal sequence of S. faecalis HPr. It is suggested that HPr in S. sanguis is associated closely with the cytoplasmic membrane. Non-disruptive methods of removing cell-surface proteins from streptococci effect release of HPr and possibly other cytoplasmic components.     

The predominant form of mammalian DNA topoisomerase Iin vivo has a molecular mass of 100 kDa

DNA topoisomerase I was purified to apparent homogeneity from human HeLa cells as a single polypeptide with a molecular mass of 100 kDa, as assayed by both gel filtration column chromatography and SDS-polyacrylamide gel electrophoresis. No smaller forms of the enzyme were detected in the purified fraction. Therefore, smaller forms, which have been observed by other investigators, are likely to be the result of proteolysis during isolation and are not relevant to thein vivo activity of DNA topoisomerase I.Abbreviations 2-ME 2-Mercaptoethanol - DTT Dithiothreitol - PMSF Phenylmethylsulfonyl Fluoride - SDS Sodium Dodecyl Sulfate     

Cloning and functional expression of a human heparanase gene.

We have cloned a gene (HSE1) from a human placental cDNA library that encodes a novel protein exhibiting heparanase activity. The cDNA was identified through peptide sequences derived from purified heparanase isolated from human SK-HEP-1 hepatoma cells. HSE1 contains an open reading frame encoding a predicted polypeptide of 543 amino acids and possesses a putative signal sequence at its amino terminus. Northern blot analysis suggested strong expression of HSE1 in placenta and spleen. Transient transfection of HSE1 in COS7 cells resulted in the expression of a protein with an apparent molecular mass of 67-72 kDa. HSE1 protein was detectable in conditioned media but was also associated with the membrane fraction following cell lysis. The HSE1 gene product was shown to exhibit heparanase activity by specifically cleaving a labeled heparan sulfate substrate in a similar manner as purified native protein.