Identification of a bone sialoprotein receptor in osteosarcoma cells

Bone sialoprotein (BSP) is an extracellular matrix glycoprotein associated with the mineral bone matrix. The amino acid sequence of BSP contains an Arg-Gly-Asp (RGD) sequence which confers to the protein cell binding properties (Oldberg, A., Franzén, A., and Heineg?rd, D. (1988) J. Biol. Chem. 263, 19430-19432). When BSP was used as an affinity matrix to isolate a cell surface receptor from rat osteosarcoma cells, a protein composed of polypeptides similar in size to those of a previously characterized vitronectin receptor was obtained. This putative BSP receptor, like the vitronectin receptor, bound also to an affinity matrix made of an RGD-containing heptapeptide. Moreover, similar patterns of inhibition of cell attachment to BSP and vitronectin was obtained with variant RGD-containing peptides, with BSP and with vitronectin. Finally, an anti-vitronectin receptor antiserum immunoprecipitated a receptor identical in size to the receptor bound to a BSP affinity matrix. These results show that BSP is recognized by an RGD-directed receptor and that both vitronectin and BSP can bind to this receptor.     

A rat osteogenic cell line (UMR 106-01) synthesizes a highly sulfated form of bone sialoprotein

The rat osteosarcoma cell line (UMR 106-01) synthesizes and secretes relatively large amounts of a sulfated glycoprotein into its culture medium (approximately 240 ng/10(6) cells/day). This glycoprotein was purified, and amino-terminal sequence analysis identified it as bone sialoprotein (BSP). [35S]Sulfate, [3H]glucosamine, and [3H]tyrosine were used as metabolic precursors to label the BSP. Sulfate esters were found on N- and O-linked oligosaccharides and on tyrosine residues, with about half of the total tyrosines in the BSP being sulfated. The proportion of 35S activity in tyrosine-O-sulfate (approximately 70%) was greater than that in N-linked (approximately 20%) and O-linked (approximately 10%) oligosaccharides. From the deduced amino acid sequence for rat BSP (Oldberg, A., Franzén, A., and Heineg?rd, D. (1988) J. Biol. Chem. 263, 19430-19432), the results indicate that on average approximately 12 tyrosine residues, approximately 3 N-linked, and approximately 2 O-linked oligosaccharides are sulfated/molecule. The carboxyl-terminal quarter of the BSP probably contains most, if not all, of the sulfated tyrosine residues because this region of the polypeptide contains the necessary requirements for tyrosine sulfation. Oligosaccharide analyses indicated that for every N-linked oligosaccharide on the BSP, there are also approximately 2 hexa-, approximately 5 tetra-, and approximately 2 trisaccharides O-linked to serine and threonine residues. On average, the BSP synthesized by UMR 106-01 cells would contain a total of approximately 3 N-linked and approximately 25 of the above O-linked oligosaccharides. This large number of oligosaccharides is in agreement with the known carbohydrate content (approximately 50%) of the BSP.     

Purification, characterization, and studies on biosynthesis of a 59-kDa bone sialic acid-containing protein (BSP) from rat mandible using a monoclonal antibody. Evidence that 59-kDa BSP may be the rat counterpart of human alpha 2-HS glycoprotein and is synthesized by both hepatocytes and osteoblasts.

A monoclonal antibody was raised against a mineralized tissue-specific sialoprotein containing no phosphorus using partially purified noncollagenous bone matrix proteins from rats as antigen. Then the sialoprotein was purified by high performance liquid chromatography from rat mandibulae using the monoclonal antibody as a marker. The sialoprotein (59-kDa bone sialoprotein (BSP)) with a molecular weight of 59,000 contained 1.4% sialic acid but no detectable phosphorus. Immunohistochemical studies with the antibody showed that the protein was specific to mineralized tissues such as bone and dentin, and present in osteoblasts, osteocytes, and bone matrix. No other soft tissues, such as the cartilage, liver, kidney, and periosteum, were stained. However, Western blot analysis showed that plasma contained immunoreactive 59-kDa BSP. The quantitative amino acid composition of 59-kDa BSP resembled that of human alpha 2-HS glycoprotein (alpha 2-HSG) (Lee, C.-C., Bowman, B.H., and Yang, F. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 4403-4407; Kellermann, J., Haupt, H., Auerswald, E.-A., and Muller-Esterl, W. (1989) J. Biol. Chem. 264, 14121-14128) and rat 64-kDa protein (Franzén, A., and Heineg?rd, D. (1985) in The Chemistry and Biology of Mineralized Tissues (Butler, W.T., ed), p. 132, EBSCO Media, Birmingham, AL). Amino acid sequence analyses of the amino-terminal region and four peptide fragments of 59-kDa BSP revealed that about 50% of the amino acids were homologous with those of human alpha 2-HSG, which is known to be synthesized by the liver, transported in the bloodstream, and incorporated into calcified tissues. But when newborn rat calvaria, primary cultures of osteoblast-rich cells, and adult rat hepatocytes were incubated with radioactive leucine, immunoreactive 59-kDa BSP was detected in their conditioned medium by fluorography. Several characteristics, including the amino acid sequence, suggest that 59-kDa BSP may be the rat counterpart of human alpha 2-HSG. However, rat 59-kDa BSP is a single peptide and synthesized by both osteoblasts and hepatocytes, whereas human alpha 2-HSG is known to be a heterodimer and to be synthesized by the liver.     

Delineation of the hydroxyapatite-nucleating domains of bone sialoprotein

Bone sialoprotein (BSP) is a highly modified, anionic phosphoprotein that is expressed almost exclusively in mineralizing connective tissues and has been shown to be a potent nucleator of hydroxyapatite (HA). Two polyglutamic acid (poly[E]) regions, predicted to be in an alpha-helical conformation and located in the amino-terminal half of the molecule, are believed to be responsible for this activity. Using a prokaryotic expression system, full-length rat BSP was expressed and tested for HA nucleating activity in a steady-state agarose gel system. The unmodified protein is less potent than native bone BSP, indicating a role for the post-translational modifications in HA nucleation. Site-directed mutagenesis of the poly[E] regions in full-length BSP was performed, replacing the poly[E] with either polyaspartic acid (poly[D]) or polyalanine (poly[A]) to examine role of charge and conformation, respectively, in HA nucleation. Replacement of single domains with either poly[A] or poly[D] did not alter nucleating activity nor did replacement of both domains with poly[D]. Replacement of both domains with poly[A], however, significantly decreased nucleating activity. In addition, two recombinant peptides, each encompassing one of the two poly[E] domains, were expressed and tested for nucleating activity. Whereas the peptide encompassing the second poly[E] domain was capable of nucleating HA, the first domain peptide showed no activity. The conformation of the wild-type and mutated proteins and peptides were studied by circular dichroism and small angle x-ray scattering, and no secondary structure was evident. These results demonstrate that a sequence of at least eight contiguous glutamic acid residues is required for the nucleation of HA by BSP and that this nucleating "site" is not alpha-helical in conformation.     

Amino acid sequence of beta-galactosidase. VIII. Sequence of the NH2-terminal segment, CNBr peptides 1 to 9, residues 1 to 377.

The amino acids in 9 cyanogen bromide peptides have been placed in sequence starting from the NH2 terminus. The peptides account for residues 1 to 377 of the whole protein and include the largest (CNBr7, 119 residues) and the smallest (CNBr1, 2 residues) of the cyanogen bromide peptides. This region contains only 3 of the 20 lysine residues in the polypeptide chain. A high proportion of charged groups are present (28 of 66 arginine, 28 of 60 glutamic acid, and 24 of 65 aspartic acid residues).     

Human recombinant CuZn-superoxide dismutase. Amino acid sequence and location of the disulfide bond

The complete amino acid sequence of recombinant human Cu-Zn superoxide dismutase (CuZnSOD) is presented. The S-carboxymethylated protein was cleaved at lysine residues (with Achromobacter protease I) to provide a set of nine non-overlapping fragments accounting for 90% of the sequence. These fragments were then overlapped and aligned, and the sequence was completed by using peptides generated by cleavage at glutamic acid residues (with S. aureus V8 protease) and at arginine (with clostripain). The recombinant protein contains a single disulfide bond between cysteine residues 57 and 146. The primary sequence of recombinant human CuZnSOD is identical to that predicted by its cDNA sequence.     

Characterization of the cDNA encoding bullfrog, Rana catesbeiana, osteocalcin and two forms of the protein isolated from bone

A full-length cDNA clone encoding osteocalcin from the bullfrog, Rana catesbeiana (bone Gla-protein, BGP) has been isolated, and the complete coding sequence for the 100-amino-acid pre-pro-osteocalcin protein was determined. The amino acid sequence of Rana catesbeiana osteocalcin, especially the mature 49-amino acid sequence, is closer to the mammalian than to the fish, Sparus osteocalcin. Rana mature osteocalcin has a similarity of 67% with human or 59% with rat osteocalcin, and only 42% with fish mature osteocalcin. The 51-amino-acid pre-pro-peptide contains the expected hydrophobic leader sequence and the dibasic Arg-Arg sequence preceding the NH2-terminal Ser of the mature 49-amino-acid Rana osteocalcin. The pro-peptide sequence also contains the expected motif of polar and hydrophobic residues, which targets vitamin K-dependent gamma-carboxylation of three specific Glu residues at positions 17, 21, and 24 in the mature protein. At the native protein expression levels, extraction from Rana cortical bone in the presence of protease inhibitor cocktail resulted in the isolation of two distinct forms of osteocalcin, P-1 and P-2, with a 3:2 distribution. Using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and amino acid sequence analysis of the N-terminal domain, we confirmed that P-1 is the intact 49-residue osteocalcin with N-terminal SNLRNAVFG., and that P-2 lacks four amino acids from the N-terminus, (NAVFG.). These results demonstrate the existence of a form of osteocalcin lacking four N-terminal amino acids in Rana bone, and that mature Rana osteocalcins remained highly conserved in their molecular evolution, especially with respect to the conservation of the C-terminal domain (residues 14-49).     

Molecular cloning of a ubiquitously distributed microtubule-associated protein with Mr 190,000

A heat-stable microtubule-associated protein (MAP) with apparent molecular weight of 190,000 is a major non-neural MAP which distributes ubiquitously among bovine tissues (termed here MAP-U). Previously we reported that microtubule-binding chymotryptic fragments of MAP-U and tau contain a common assembly-promoting (AP) sequence of 22 amino acid residues (Aizawa, H., Kawasaki, H., Murofushi, H., Kotani, S., Suzuki, K., and Sakai, H. (1989) J. Biol. Chem. 264, 5885-5890). We isolated cDNA clones for MAP-U containing the whole coding sequence. Northern blot analysis revealed that a major species of MAP-U mRNA is 5 kilobases in length and is expressed ubiquitously among bovine tissues. Nucleotide sequence analysis revealed the complete amino acid sequence of MAP-U which consists of 1,072 amino acid residues. Analysis of the deduced amino acid sequence of MAP-U indicated that this molecule is clearly divided into two domains in terms of electrostatic charge distribution: an amino-terminal acidic domain (residues 1-640) and a carboxyl-terminal basic domain (residues 641-1072). The amino-terminal domain of MAP-U shows no significant sequence homology with other known protein sequences including neural MAPs, tau, and MAP-2. The amino-terminal domain of MAP-U contains unique 18 1/2 repeats of 14-amino acid motif which have not been observed in other MAPs. The carboxyl-terminal domain of MAP-U is further divided into three regions: a Pro-rich region (residues 641-880), an AP sequence region (residues 881-1003), and a short hydrophobic tail (residues 1004-1072). The Pro-rich region is mainly composed of five species of amino acid residues, Pro, Ala, Lys, Ser, and Thr. The AP sequence region contains four tandem repeats of AP sequences, and thus, this region is considered to play a leading role in the interaction of MAP-U with microtubules.     

The gamma-carboxy glutamic acid content of human and bovine prothrombin following warfarin treatment.

A form of prothrombin induced by Warfarin therapy, has been isolated which is adsorbed onto insoluble barium salts, but has a reduced biological activity. This protein contains, on average, seven out of a possible ten gamma-carboxy glutamic acid residues. A second form of prothrombin is also described, which is not adsorbed into barium slats, and has less than 1% the activity of the normal protein, contains only four gamma-carboxy glutamic acid residues. The significance of these results is discussed.     

Amino acid and cDNA sequence of bovine phosducin, a soluble phosphoprotein from photoreceptor cells

Vertebrate photoreceptor cells contain a soluble phosphoprotein, phosducin, which complexes with the beta, gamma subunits of the GTP-binding protein, transducin. Light-induced changes in cyclic nucleotide levels modulate the phosphorylation of phosducin by protein kinase A. The complete amino acid sequence of purified phosducin from bovine retinas was determined by Edman degradation from overlapping polypeptides derived from enzymatic digestion by trypsin and Staphylococcus aureus V8 protease or from chemical degradation by cyanogen bromide. Excluding the unidentified group which blocks the NH2 terminus, phosducin contains 245 amino acids with a calculated molecular weight of 28,185 and isoelectric point of pH 4.5. Phosducin is enriched with acidic and sulfur-containing amino acids, having 32 glutamic acid, 16 aspartic acid, 9 methionine, and 5 cysteine residues. It also contains 24 serine and 8 threonine residues, of which only serine 73 is located within a consensus phosphorylation sequence (-RKMS(P)QV-) for cyclic nucleotide-dependent protein kinase. Secondary structure analysis predicts the presence of 62% alpha-helix, 22% beta-sheet, and 16% random coil, with eight turns. Computer-aided searches of protein data banks revealed no apparent homology to any sequenced protein except that coded by a MEKA cDNA clone (Kuo, C-H., Akiyama, M., and Miki, N. (1989) Mol. Brain Res. 6, 1-10) which deviates from the confirmed phosducin sequence in the last 15 amino acids. Sequence analysis of a cDNA clone for bovine retinal phosducin confirmed that the MEKA clone deviation resulted from an unidentified cDNA guanosine nucleotide, a shifted reading frame and a premature stop codon.     

Sequencing of bovine enamelin ("tuftelin") a novel acidic enamel protein

Enamelins are a major group of 28-70-kDa acidic proteins rich in aspartic acid, glutamic acid, serine, and glycine found in developing and mature extracellular enamel; a unique and highly mineralized ectodermal tissue covering vertebrate teeth. They have been associated with the mineralization and structural organization of this tissue. In an attempt to elucidate the primary structure of enamelin, a 2674-base pair cDNA isolated from a bovine ameloblast-enriched, lambda Zap 2 expression library, was sequenced. The identity and localization of the deduced protein was confirmed by amino acid composition, enzyme-linked immunosorbent assay, Western blotting, indirect immunohistochemistry, and high resolution protein-A gold immunocytochemistry. The immunological techniques were employed using antibodies directed against synthetic peptides corresponding to the protein sequence deduced from the cloned cDNA sequence. The results reveal the deduced protein to be a novel acidic enamel protein. It contains 389 amino acids and has a calculated molecular weight of 43,814. Its amino acid composition is similar to that of "tuft" proteins (enamel matrix protein fragments remaining in the mature tissue). It contains one potential N-glycosylation site and 5 cysteine residues. Southern hybridization of the cloned cDNA with genomic bovine DNA indicated the existence of a single gene with one or more introns.     

Matrix sialoprotein of developing bone

Using nondegradative isolation procedures, we purified and characterized a glycoprotein from fetal calf bone that is rich in sialic acid. This bone sialoprotein (BSP) has an apparent Mr = 70,000-80,000 and stains with Alcian blue and Stains All on sodium dodecyl sulfate gels but does not stain with Coomassie blue without prior treatment with neuraminidase. This glycoprotein contains 50% protein, 12% sialic acid, 7% glucosamine, and 6% galactosamine. Fetal calf BSP is rich in glutamate (19%), aspartate (15.4%), and glycine (11.8%) but, in contrast to osteonectin and the bone proteoglycan, has relatively low amounts of leucine (4.3%). Antisera raised against fetal calf BSP localized the glycoprotein by indirect immunofluorescence to developing bone trabeculae with an overall tissue distribution identical with that of osteonectin. On competition enzyme-linked immunosorbent assay analysis, BSP was 11.5% (+/-2.4%, S.E.) of mineral-bound (guanidine-EDTA-soluble) calf bone protein. Immunoreplicas (Western blots) of calf bone extracts suggest that more than 95% of the antigenicity resided in the Mr = 70,000-80,000 region with the remaining cross-reactivity in Alcian blue positive, Mr = approximately 20,000 and approximately 30,000 bands. Brief treatment of the Mr = 70,000-80,000 species with trypsin produced lower molecular weight, Alcian blue-staining products of similar size. No BSP was detected in guanidine extracts of various soft or unmineralized connective tissues, but dentin contained small amounts (0.4%) of the protein. Rat and fetal human bone were also observed to contain a sialoprotein with similar properties and a certain degree of cross-reactivity with the bovine BSP.     

Amino acid sequence of protein B23 phosphorylation site

A major phosphopeptide labeled in vivo, was identified in nucleolar protein B23 (Mr/pI = 37,000/5.1) after tryptic digestion. This peptide was purified by high performance liquid chromatography using reverse-phase (C8 and C18) columns. The phosphopeptide contains 20 amino acids including 1 phosphoserine, 7 glutamic acids, and 4 aspartic acids. The amino acid sequence is: His-Leu-Val-Ala-Val-Glu-Glu-Asp-Ala-Glu-Ser(P)-Glu-Asp-Glu-Asp- Glu-Glu-Asp-Val-Lys. This amino acid sequence is similar to that of nucleolar phosphoprotein C23 (8 consecutive amino acids were identical), and to the regulatory subunit (RII) of cAMP-dependent protein kinase (7 consecutive amino acids were identical, which is phosphorylated by casein kinase II (Carmichael, D.F., Geahlen, R.L., Allen, S.M., and Krebs, E.G. (1982) J. Biol. Chem 257, 10440-10445). The regions near these phosphorylation sites are enriched with glutamic and aspartic acids, suggesting that this acidic amino acid cluster may be essential for kinase recognition.     

The vitamin K-dependent synthesis of gamma-carboxyglutamic acid by bone microsomes

Microsomes prepared from embryonic chick bone contain a vitamin K-dependent carboxylating system which post-translationally converts glutamic acid residues in peptides to gamma-carboxyglutamic acid (gamma-CGlu). Glutamic acid residues in both endogenous chick bone microsomal protein and in the synthetic peptide Phe Leu-Glu-Glu-Val are gamma-carboxylated. These data suggest that bone cells have the capacity for de novo gamma-CGlu synthesis and may be responsible for synthesis of osteocalcin, the major gamma-CGlu protein in bone.     

Complete amino acid sequence of canine cardiac calsequestrin deduced by cDNA cloning

cDNA cloning was used to deduce the complete amino acid sequence of canine cardiac calsequestrin, the principal Ca2+-binding protein of cardiac junctional sarcoplasmic reticulum. Cardiac calsequestrin contains 391 amino acid residues plus a 19-residue amino-terminal signal sequence. The molecular weight of the mature protein, excluding carbohydrate, is 45,269. Cardiac calsequestrin is highly acidic, and a striking feature is the enrichment of acidic residues (60%) within the 63 carboxyl-terminal residues. No part of the sequence contains EF hand Ca2+-binding structures. The photo-affinity probe 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine was used to localize the Ca2+-regulated hydrophobic site to amino acid residues 192-223. The cardiac and skeletal muscle isoforms of calsequestrin (Fliegel, L., Ohnishi, M., Carpenter, M. R., Khanna, V. K., Reithmeier, R. A. F., and MacLennan, D. H. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 1167-1171), although the products of different genes, are 65% identical, are acidic, and share one glycosylation site. However, cardiac calsequestrin has several unique features. First, it has a 31-amino acid extension at its carboxyl terminus (residues 361-391), which contains 71% acidic residues and a second glycosylation site. Second, its mRNA contains a second open reading frame with the capacity to code for a 111-amino acid protein. Third, contrary to the restricted expression of the fast skeletal isoform, cardiac calsequestrin mRNA is present in both cardiac and slow skeletal muscle, but not in fast skeletal muscle. We conclude that the deduced amino acid sequence of cardiac calsequestrin is consistent with its ability to bind large amounts of Ca2+ (40 mol of Ca2+/mol of calsequestrin). The protein probably binds Ca2+ by acting as a charged surface rather than by presenting multiple discrete Ca2+-binding sites.     

Periostin, a member of a novel family of vitamin K-dependent proteins, is expressed by mesenchymal stromal cells

The modification of glutamic acid residues to gamma-carboxyglutamic acid (Gla) is a post-translational modification catalyzed by the vitamin K-dependent enzyme gamma-glutamylcarboxylase. Despite ubiquitous expression of the gamma-carboxylation machinery in mammalian tissues, only 12 Gla-containing proteins have so far been identified in humans. Because bone tissue is the second most abundant source of Gla-containing proteins after the liver, we sought to identify Gla proteins secreted by bone marrow-derived mesenchymal stromal cells (MSCs). We used a proteomics approach to screen the secretome of MSCs with a combination of two-dimensional gel electrophoresis and tandem mass spectrometry. The most abundant Gla-containing protein secreted by MSCs was identified as periostin, a previously unrecognized gamma-carboxylated protein. In silico amino acid sequence analysis of periostin demonstrated the presence of four consensus gamma-carboxylase recognition sites embedded within fasciclin-like protein domains. The carboxylation of periostin was confirmed by immunoprecipitation and purification of the recombinant protein. Carboxylation of periostin could be inhibited by warfarin in MSCs, demonstrating its dependence on the presence of vitamin K. We were able to demonstrate localization of carboxylated periostin to bone nodules formed by MSCs in vitro, suggesting a role in extracellular matrix mineralization. Our data also show that another fasciclin I-like protein, betaig-h3, contains Gla. In conclusion, periostin is a member of a novel vitamin K-dependent gamma-carboxylated protein family characterized by the presence of fasciclin domains. Furthermore, carboxylated periostin is produced by bone-derived cells of mesenchymal lineage and is abundantly found in mineralized bone nodules in vitro.     

Molecular cloning of the 18-kDa growth-associated protein of developing brain

An 18-kDa protein (designated herein as the heparin-binding growth-associated molecule, HB-GAM), the expression of which in rat brain correlates to the rapid postnatal developmental phase, was previously isolated and suggested to have a role in the maturation and growth of brain (Rauvala, H. (1989) EMBO J. 8, 2933-2941). A protein with a similar molecular mass, similar heparin-binding properties, and the same N-terminal sequence was more recently also isolated as a mitogen for NIH 3T3 cells (Milner, P. G., Li, Y.-T., Hoffman, R. M., Kodner, C. M., Siegel, N. R., and Deuel, T. F. (1989) Biochem. Biophys. Res. Commun. 165, 1096-1103). This study reports the cloning and sequencing of the cDNA that encodes HB-GAM. The sequence that precedes the structure of the mature molecule has the characteristics of a signal sequence found in secretory proteins. The sequence of HB-GAM is a novel structure that contains 136 amino acid residues. The sequence is very rich in cationic amino acids (24% of the residues); lysine cluster sequences are found in the N-terminal and C-terminal ends of the structure. Cysteine is also abundant in the sequence (7% of the residues). The only homologous sequence found in computer searches is the retinoic acid-induced differentiation factor. The mRNA of HB-GAM detected by the cloned cDNA shows the same kind of developmental regulation as the protein; the mRNA is strongly expressed during the early postnatal growth phase of rat brain as compared with embryonic or adult tissue.     

Nucleotide sequence of the pldB gene and characteristics of deduced amino acid sequence of lysophospholipase L2 in Escherichia coli

The nucleotide sequence of the pldB gene of Escherichia coli K-12, which codes for lysophospholipase L2 located in the inner membrane, was determined. The deduced amino acid sequence of lysophospholipase L2 contains 340 amino acid residues, resulting in a protein with a molecular weight of 38,934. It is characterized by a high content of arginine residues (36 out of 340 residues). The amino acid sequence near the NH2-terminus of the protein is composed of a large number of polar or charged amino acid residues, suggesting that this region cannot be a signal peptide. The hydropathy profile of the deduced amino acid sequence of lysophospholipase L2 was studied. Most of the region was rather hydrophilic, and there was no stretch of hydrophobic amino acid region, such as might be predicted to traverse the lipid bilayer. These results are consistent with the experimental observation that lysophospholipase L2 is extracted by salt solution from the membrane fraction, and it may be classified as a peripheral membrane protein. Computer analysis showed that there is no homology in amino acid sequences between lysophospholipase L2 and other extracellular phospholipases, as well as detergent-resistant phospholipase A, which is another membrane-bound phospholipase in E. coli and whose DNA sequence was determined (Homma, H., Kobayashi, T., Chiba, N., Karasawa, K., Mizushima, H., Kudo, I., Inoue, K., Ideka, H., Sekiguchi, M., & Nojima, S. (1984) J. Biochem. 96, 1655-1664). This is the first report of the primary structure of a lysophospholipase.