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In an attempt to isolate mRNA-binding proteins we fractionated Xenopus oocyte lysate by oligo(dT)-cellulose chromatography. A 20 kDa protein was the major component of the eluate. cDNA cloning revealed that this protein is a Xenopus homolog of the cold-inducible RNA-binding protein (CIRP) which was originally identified in mammalian cells as a protein that is overexpressed upon a temperature downshift. This Xenopus protein, termed here xCIRP2, is highly expressed in ovary, testis and brain in adult Xenopus tissues. In oocytes it is predominantly localized in the cytoplasm. By biochemical fractionation we provide evidence that xCIRP2 is associated with ribosomes, suggesting that it participates in translational regulation in oocytes. Microinjection of labeled mRNA into oocytes followed by UV cross-linking of the oocyte lysate led to identification of two major RNA-binding activities. Immunoprecipitation of the RNA-binding proteins demonstrated that one is xCIRP2 and that the other contains FRGY2. FRGY2, which is one of the principal constituents of mRNA storage particles involved in translational masking of maternal mRNA, has an RNA-binding domain conserved to those of bacterial cold shock proteins. Possible implications of the highly abundant expression in oocytes of cold shock RNA-binding proteins of both eukaryotic and prokaryotic types are discussed.

S-Adenosyl-L-methionine:L-methionine S-methyltransferase (MMT) catalyzes the synthesis of S-methyl-L-methionine (SMM) from L-methionine and S-adenosyl-L-methionine. SMM content increases during barley (Hordeum vulgare L.) germination. Elucidating the role of this compound is important from both a fundamental and a technological standpoint, because SMM is the precursor of dimethylsulfide, a biogenic source of atmospheric S and an undesired component in beer. We present a simple purification scheme for the MMT from barley consisting of 10% to 25% polyethylene glycol fractionation, anion-exchange chromatography on diethylaminoethyl-Sepharose, and affinity chromatography on adenosine-agarose. A final activity yield of 23% and a 2765-fold purification factor were obtained. After digestion of the protein with protease, the amino acid sequence of a major peptide was determined and used to produce a synthetic peptide. A polyclonal antibody was raised against this synthetic peptide conjugated to activated keyhole limpet hemocyanin. The antibody recognized the 115-kD denatured MMT protein and native MMT. During barley germination, both the specific activity and the amount of MMT protein increased. MMT-specific activity was found to be higher in the root and shoot than in the endosperm. MMT could be localized by an immunohistochemical approach in the shoot, scutellum, and aleurone cells but not in the root or endosperm (including aleurone).

Nitrile hydratase (NHase) from Rhodococcus sp. N-771 is a photoreactive enzyme that is inactivated by nitrosylation of the non-heme iron center and activated by photodissociation of nitric oxide (NO). To obtain structural information on the iron center, we isolated peptide complexes containing the iron center by proteolysis. When the tryptic digest of the alpha subunit isolated from the inactive form was analyzed by reversed-phase high performance liquid chromatography, the absorbance characteristic of the nitrosylated iron center was observed in the peptide fragment, Asn105-Val-Ile-Val-Cys-Ser-Leu-Cys-Ser-Cys-Thr-Ala-Trp-Pro-Ile-Leu - Gly-Leu-Pro-Pro-Thr-Trp-Tyr-Lys128. The peptide contained 0.79 mol of iron/mol of molecule as well as endogenous NO. Subsequently, by digesting the peptide with thermolysin, carboxypeptidase Y, and leucine aminopeptidase M, we found that the minimum peptide segment required for the nitrosylated iron center is the 11 amino acid residues from alphaIle107 to alphaTrp117. Furthermore, by using mass spectrometry, protein sequence, and amino acid composition analyses, we have shown that the 112th Cys residue of the alpha subunit is post-translationally oxidized to a cysteine-sulfinic acid (Cys-SO2H) in the NHase. These results indicate that the NHase from Rhodococcus sp. N-771 has a novel non-heme iron enzyme containing a cysteine-sulfinic acid in the iron center. Possible ligand residues of the iron center are discussed.

Lactosylceramide synthase is an enzyme that catalyzes the transfer of galactose from UDP-Gal to glucosylceramide, and thus participates in the biosynthesis of most glycosphingolipids in mammals. We purified this enzyme over 61,000-fold to near homogeneity with a 29. 7% yield from rat brain membrane fractions. The isolation procedure included solubilization with Triton X-100, affinity chromatography on wheat germ agglutinin-agarose and UDP-hexanolamine-agarose, and hydroxylapatite column chromatography, followed by ion exchange chromatography. The final preparation migrated as a broad band with an apparent molecular mass of 61 kDa on SDS-polyacrylamide gel electrophoresis. This apparent molecular mass was reduced to 51 kDa by N-glycanase digestion, suggesting that the enzyme has a glycoprotein nature. The enzyme required Mn2+ for its activity, and glucosylceramide was its preferred substrate. The cDNA for the enzyme was cloned from a rat brain cDNA library. The cDNA insert encoded a polypeptide of 382 amino acid residues, with a molecular weight of 44,776. The polypeptide contained eight putative glycosylation sites and a 20-amino acid residue transmembrane domain at its N terminus. Amino acid sequence homology analysis revealed that this enzyme shared 39% homology with mouse beta-1, 4-galactosyltransferase (EC 2.4.1.38), which catalyzes the transfer of Gal to beta-1,4-GlcNAc in glycoproteins.

The complete amino acid sequences of two lysine-specific zinc metalloendopeptidases (EC 3.4.24), Grifola frondosa metalloendopeptidase (GFMEP) and Pleurotus ostreatus metalloendopeptidase (POMEP), from the fruiting bodies of these two edible mushrooms have been established based on the sequence information of the peptides generated from the reduced and alkylated GFMEP and POMEP by proteolytic digestions using GFMEP, trypsin, and other proteinases as well as by several chemical cleavages. From the sequences, it was found that GFMEP and POMEP were polypeptides composed of 167 and 168 amino acid residues, from which their molecular weights were calculated to be 18,040.5 and 17,921.3 in accord with the observed (M+H)+ values of 18,028 and 17,927, respectively, as determined by matrix-assisted laser desorption ionization-time of flight mass spectrometry. Two disulfide bonds in GFMEP were found to link Cys5 to Cys75 and Cys77 to Cys97. An unusual post-translational modification of GFMEP was corroborated to be a partial attachment of a single mannose to Thr42. Comparison of the sequences revealed that overall identity between the enzymes was 61.3%. Although a highly homologous sequence was not found in sequence data bases except for a consensus zinc-binding sequence, HEXXH, both metalloendopeptidases somewhat resembled a family of metalloproteinases categorized as deuterolysin. These proteases together with GFMEP and POMEP do not have conserved third and/or fourth liganding amino acid residues seen in metzincin or thermolysin superfamily proteins and belong to a novel zinc metalloendopeptidase superfamily.

Primary structural information of a plant aldehyde oxidase (AO), which was purified from maize coleoptiles using indole-3-acetaldehyde as a substrate, was obtained by sequencing a series of cleavage peptides, permitting the cloning of the corresponding cDNA (zmAO-1). The complete nucleotide sequence was determined; the deduced amino acid sequence encodes a protein of 1358 amino acid residues of Mr 146,681, which is consistent with the size of the AO monomeric subunit. There is a significant similarity with AO from mammals and xanthine dehydrogenases from various sources. The maize AO polypeptide contains consensus sequences for iron-sulfur centers and a putative molybdopterin cofactor-binding domain. In addition, another cDNA (zmAO-2), highly homologous to zmAO-1 at both the nucleotide and amino acid sequence levels, was cloned. zmAO-2 would encode a protein of 1349 amino acid residues of Mr 145,173 and has molecular characteristics similar to those of zmAO-1. zmAO-1 was expressed at a high level in roots, which was closely correlated with immunoblotting results using antiserum raised against the purified maize AO protein, whereas zmAO-2 was expressed at a higher level in coleoptiles than in roots. We propose each zmAO may have a unique function during plant development.

Rat platelets secrete two types of phospholipases upon stimulation; one is type II phospholipase A2 and the other is serine-phospholipid-selective phospholipase A. In the current study we purified serine-phospholipid-selective phospholipase A and cloned its cDNA. The final preparation, purified from extracellular medium of activated rat platelets, gave a 55-kDa protein band on SDS-polyacrylamide gel electrophoresis. [3H]Diisopropyl fluorophosphate, an inhibitor of the enzyme, labeled the 55-kDa protein, suggesting that this polypeptide possesses active serine residues. The cDNA for the enzyme was cloned from a rat megakaryocyte cDNA library. The predicted 456-amino acid sequence contains a putative short N-terminal signal sequence and a GXSXG sequence, which is a motif of an active serine residue of serine esterase. Amino acid sequence homology analysis revealed that the enzyme shares about 30% homology with mammalian lipases (lipoprotein lipase, hepatic lipase, and pancreatic lipase). Regions surrounding the putative active serine, histidine, and aspartic acid, which may form a "lipase triad," were highly conserved among these enzymes. The recombinant protein, which we expressed in Sf9 insect cells using the baculovirus system, hydrolyzed a fatty acyl residue at the sn-1 position of lysophosphatidylserine and phosphatidylserine, but did not appreciably hydrolyze phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidic acid, and triglyceride. The present enzyme, named phosphatidylserine-phospholipase A1, is the first phospholipase that exclusively hydrolyses the sn-1 position and has a strict head group specificity for the substrate.