Unique precursor structure of an extracellular protease, aqualysin I, with NH2- and COOH-terminal pro-sequences and its processing in Escherichia coli

Aqualysin I is a subtilisin-type serine protease which is secreted into the culture medium by Thermus aquaticus YT-1, an extremely thermophilic Gram-negative bacterium. The nucleotide sequence of the entire gene for aqualysin I was determined, and the deduced amino acid sequence suggests that aqualysin I is produced as a large precursor, consisting of at least three portions, an NH2-terminal pre-pro-sequence (127 amino acid residues), the protease (281 residues), and a COOH-terminal pro-sequence (105 residues). When the cloned gene was expressed in Escherichia coli cells, aqualysin I was not secreted. However, a precursor of aqualysin I lacking the NH2-terminal pre-pro-sequence (38-kDa protein) accumulated in the membrane fraction. On treatment of the membrane fraction at 65 degrees C, enzymatically active aqualysin I (28-kDa protein) was produced in the soluble fraction. When the active site Ser residue was replaced with Ala, cells expressing the mutant gene accumulated a 48-kDa protein in the outer membrane fraction. The 48-kDa protein lacked the NH2-terminal 14 amino acid residues of the precursor, and heat treatment did not cause any subsequent processing of this precursor. These results indicate that the NH2-terminal signal sequence is cleaved off by a signal peptidase of E. coli, and that the NH2- and COOH-terminal pro-sequences are removed through the proteolytic activity of aqualysin I itself, in that order. These findings indicate a unique four-domain structure for the aqualysin I precursor; the signal sequence, the NH2-terminal pro-sequence, mature aqualysin I, and the COOH-terminal pro-sequence, from the NH2 to the COOH terminus.     

Molecular cloning and sequencing of cDNA for yeast porin, an outer mitochondrial membrane protein: a search for targeting signal in the primary structure.

We have cloned a full-length cDNA for yeast porin, the major outer mitochondrial membrane protein from Saccharomyces cerevisiae, and determined its nucleotide sequence. The primary structure of the protein, deduced from the nucleotide sequence, consisted of 283 amino acid residues and its NH2-terminal sequence, Met-Ser-Pro-Pro-Val-Tyr-Ser, coincided with that determined by Edman degradation for yeast porin, except that the initiator methionine was missing in the mature protein. The deduced sequence had an overall polarity index of 46.3%, a value which falls in the normal range for soluble proteins. An evaluation of hydropathy of the protein indicated that the NH2-terminal one third was relatively hydrophilic and the rest of the molecule was rather hydrophobic. An interesting finding was that the NH2-terminal region of yeast porin (consisting of some 50 amino acid residues) shows structural features that resemble those of the corresponding portion of 70-kd protein, which is also a yeast outer mitochondrial membrane protein. We postulate that this NH2-terminal sequence, like that of 70-kd protein, is required for targeting the porin to the outer mitochondrial membrane.     

Primary structure of bovine matrix Gla protein, a new vitamin K-dependent bone protein

The complete amino acid sequence of bovine bone matrix Gla protein (MGP) was determined by automatic sequence analysis of the intact protein and of peptides isolated from tryptic and BNPS-skatole digests. This 79-residue, vitamin K-dependent protein contains a single disulfide bond and 4.8 gamma-carboxyglutamate (Gla) residues, one each at positions 37, 41, 48, and 52, and 0.8 Gla and 0.2 Glu at position 2. There is sufficient sequence homology between MGP and bone Gla protein (BGP) to indicate that these two bovine bone proteins arose by gene duplication and subsequent divergent evolution. Although MGP has a very low solubility in water compared to BGP, there is no hydrophobic domain in MGP which could account for its insolubility, and the overall fraction of hydrophobic residues is 32% for MGP compared to 43% for BGP. MGP is the first vitamin K-dependent protein to be discovered which has several non-gamma-carboxylated residues to the NH2-terminal side of its Gla residues. The presence of NH2-terminal Glu residues between the putative targeting domain for the gamma-carboxylase in the MGP leader sequence and the mid-molecule Gla residues suggests that the gamma-carboxylase may have additional, as yet unrecognized, specificity requirements which determine the susceptibility of Glu residues for gamma-carboxylation.     

The oligodendrocyte-myelin glycoprotein belongs to a distinct family of proteins and contains the HNK-1 carbohydrate

The complete primary structure of the human oligodendrocyte-myelin glycoprotein (OMgp), a glycophospholipid-linked membrane protein of oligodendrocytes and central nervous system myelin, has been determined. The deduced amino acid sequence predicts a polypeptide of 433 amino acids which includes a 17-amino acid leader sequence. OMgp consists of four domains: (a) a short cysteine-rich motif at the NH2 terminus; (b) a series of tandem leucine-rich repeats (LRs) present in several other proteins where they may play roles in adhesion; (c) a serine/threonine-rich region that contains probable attachment sites for O-linked carbohydrates; and (d) a hydrophobic COOH-terminal segment that is likely to be cleaved concomitant with the attachment of lipid during biosynthesis of OMgp. OMgp shares the first three of its four domains with the platelet glycoprotein Ib, which is responsible for the initial adhesion of platelets to the exposed subendothelium during hemostasis. Together with glycoprotein Ib and several other proteins, OMgp belongs to a family of proteins that contain both an NH2-terminal cysteine-rich motif and an adjacent series of LRs. In addition, we report that a subpopulation of OMgp molecules contains the HNK-1 carbohydrate, which has been shown to mediate interactions among cells in the central nervous system.     

Homology of the precursor of pulmonary surfactant-associated protein SP-B with prosaposin and sulfated glycoprotein 1.

The precursor of pulmonary surfactant-associated protein, SP-B, is composed of an NH2-terminal domain of 30 residues (a-type domain) and three tandem repeats of about 90 residues (b-type domain); biophysically active mature SP-B corresponds to the second b-type repeat. Consensus sequences constructed for the b-type repeats were used to search the data base for homologous sequences, and the search has revealed that prosaposin and sulfated glycoprotein 1 show a remarkable homology with these repeats. The domain organizations of the latter proteins, however, differ from that of SP-B precursor inasmuch as they contain four tandem copies of the b-type domain and a-type domains are present both in the NH2-terminal and COOH-terminal parts of the proteins. The implications of the homology of saposins and SP-B for their structure and function are discussed.     

Amino-terminal sequence and structure of monoclonal antibody immunopurified cytochromes P-450

Six hepatic cytochromes P-450 were isolated from 3-methylcholanthrene-treated animals by immunopurification with monoclonal antibodies. The purified cytochromes P-450 include 57- and 56-kDa polypeptides from Sprague-Dawley rats, 57- and 56-kDa polypeptides from C57BL/6 mice, a 56-kDa polypeptide from DBA/2 mice, and a 53-kDa polypeptide from guinea pigs. These isozymes were structurally compared by peptide mapping using both sodium dodecyl sulfate--polyacrylamide gel electrophoresis and high-pressure liquid chromatography and by amino acid and NH2-terminal sequence analyses. The 57-kDa polypeptides from rats and mice have similar but nonidentical peptide maps and amino acid compositions and are about 80% homologous in their NH2-terminal amino acid sequence. The 56-kDa polypeptides from rats and both mice strains have very similar peptide maps and amino acid compositions and identical NH2-terminal sequences. The NH2-terminal sequence of the mice 56-kDa polypeptides corresponds to that reported for the mouse P1-450 isozyme except that we identified two additional residues, proline and serine, at the NH2 terminus in the 57-kDa polypeptide from C57BL/6 mice that were not deduced from the cDNA sequence of the mouse P1-450 isozyme. The guinea pig 53-kDa polypeptide has a distinct peptide map relative to the other polypeptides studied and an NH2-terminal sequence with only partial homology to the 56- and 57-kDa polypeptides from rats and mice. This report shows the varying degree of structural relatedness among the isozymes examined and demonstrates the suitability and advantage of immunopurified cytochromes P-450 for sequencing and structural studies.     

Molecular cloning, sequencing, deletion, and overexpression of a methionine aminopeptidase gene from Saccharomyces cerevisiae.

A yeast gene for a methionine aminopeptidase, one of the central enzymes in protein synthesis, was cloned and sequenced. The DNA sequence encodes a precursor protein containing 387 amino acid residues. The mature protein, whose NH2-terminal sequence was confirmed by Edman degradation, consists of 377 amino acids. The function of the 10-residue sequence at the NH2 terminus, containing 1 serine and 6 threonine residues, remains to be established. In contrast to the structure of the prokaryotic enzyme, the yeast methionine aminopeptidase consists of two functional domains: a unique NH2-terminal domain containing two motifs resembling zinc fingers, which may allow the protein to interact with ribosomes, and a catalytic COOH-terminal domain resembling other prokaryotic methionine aminopeptidases. Furthermore, unlike the case for the prokaryotic gene, the deletion of the yeast MAP1 gene is not lethal, suggesting for the first time that alternative NH2-terminal processing pathway(s) exist for cleaving methionine from nascent polypeptide chains in eukaryotic cells.     

A new repetitive protein from Xenopus laevis skin highly homologous to pancreatic spasmolytic polypeptide

A cDNA sequence has been used to derive the precursor structure of a highly repetitive protein in Xenopus laevis skin. From the sequence of a whole family of secretory proteins can be predicted containing a classical hydrophobic signal sequence at the NH2-terminal end of the precursor. The proteins contain four domains with high homology to porcine pancreatic spasmolytic polypeptide. These four cysteine-rich, presumably physiologically active domains are separated in the molecule by a repetitive element, locating two such domains to the NH2 terminus of the precursor protein and the remaining two to the COOH-terminal end. The separating spacer consists of very unusual, precise, threonine and proline-rich repeats containing 9 residues which could be targets for extensive O-glycosylation. Additionally, processing at two pairs of basic residues is suggested to liberate two polypeptides ("spasmolysins") and "spasmolysin-glycoprotein."     

Sequence and type-specific immunogenicity of the amino-terminal region of type 1 streptococcal M protein

The NH2-terminal sequence of type 1 M protein was determined by automated Edman degradation of purified polypeptide fragments extracted from whole streptococci by limited digestion with pepsin. Three polypeptide fragments were purified by slab gel electrophoresis on sodium dodecyl sulfate (SDS) polyacrylamide followed by electroelution. The purified fragments migrated as 28-, 25-, and 23.5-kDa fragments, respectively. Each of the fragments inhibited opsonization of a diluted antiserum prepared in rabbits by immunization with whole type 1 streptococci. The amino-terminal sequences of the peptide fragments were confirmed by comparison with the primary structure predicted from the nucleotide sequence of the type 1 M protein structural gene. The 28-kDa fragment contained the NH2-terminal asparagine residue of the processed type 1 M protein, whereas the NH2-terminal sequences of the 25- and 23.5-kDa peptides began at residues 27 and 36, respectively. A seven-residue periodicity with respect to polar and nonpolar residues was observed beginning at residue 22 and, therefore, the secondary structural potential of type 1 M protein is similar to that reported for other M proteins. In contrast to the other M proteins, however, identical repeats were rare, the longest sequence identity consisting of a three-amino acid acid sequence Lys-Asp-Leu at positions 30-32 repeated once at positions 65-67. A 23-residue synthetic peptide of the amino-terminus of the type 1 M protein evoked opsonic antibodies against type 1 streptococci. These results indicate that the NH2-terminal region of type 1 M protein retains the secondary structural characteristics of other M serotypes. Moreover, it contains epitopes that evoke protective immune responses. Our studies may have bearing in the development of safe and effective vaccines against group A streptococcal infections.     

Primary structure of anti-lipopolysaccharide factor from American horseshoe crab, Limulus polyphemus

The complete amino acid sequence of anti-lipopolysaccharide (LPS) factor purified from the hemocytes lysate of the American horseshoe crab, Limulus polyphemus, was determined by characterization of the NH2-terminal sequence and the peptides generated after digestion of the protein with lysyl endopeptidase, clostripain, and Staphylococcus aureus V8 protease. Upon sequencing the peptides by the automated Edman method, the following primary structure was obtained: (Sequence: in text). During the sequence analysis, two species of the protein, which differed from each other at one locus, were found and characterized. L. polyphemus anti-LPS factor was a basic protein consisting of a single polypeptide chain of 101 residues and a calculated molecular weight of 11,786 or 11,800. The hydrophobic NH2-terminal sequence and the clustering of positive charges found in the disulfide loop yielded a typical amphipathic character of this protein. Moreover, L. polyphemus anti-LPS factor showed 83% sequence identity with the Tachypleus tridentatus protein, and the sequence similar to that observed in the EF-hand structure was found to contain in the COOH terminal portions of these proteins, although its function is unknown.     

Primary structure of bovine conglutinin, a member of the C-type animal lectin family

We report the complete amino acid sequence of bovine conglutinin obtained by structural characterization of peptides derived from the protein by various chemical and enzymatic fragmentation methods. The protein consists of 351 amino acid residues including 55 apparent Gly-X-Y repeats with two interruptions. This 171-residue-long collagenous domain separates a short noncollagenous NH2-terminal region of 25 residues from the 155-residue-long globular COOH terminus revealing the structural relation of conglutinin with mannose-binding proteins, pulmonary surfactant-associated proteins, and a complement component C1q. Eight hydroxylysine residues were found in the collagenous domain. All of these hydroxylysine residues which occupy a Y position in a Gly-X-Y triplet are possible glycosylation sites since no phenylthiohydantoin amino acid was identified in automated Edman degradation cycles corresponding to these sites. The noncollagenous COOH domain of conglutinin, on the other hand, contains a carbohydrate recognition domain which shares substantial sequence homology with C-type animal lectins. Conglutinin has the greatest sequence similarity with mannose-binding proteins and pulmonary surfactant-associated proteins.     

Primary structure of rat liver mannan-binding protein deduced from its cDNA sequence

cDNA clones encoding rat liver mannan-binding protein (MBP), a lectin specific for mannose and N-acetylglucosamine, were isolated from a rat liver cDNA library carried in lambda gt 11, by screening with affinity purified polyclonal rabbit anti-rat liver MBP antibodies. The nucleotide sequence of the cDNA determined by the dideoxy method revealed the complete amino acid sequence of the MBP (226 residues). The NH2-terminal residue of the MBP, glutamic acid, was preceded by a predominantly hydrophobic stretch of 18 amino acids, which was assumed to be a signal peptide. Near the NH2-terminal, there was a collagen-like domain, which consisted of 19 repeats of the sequence Gly-X-Y. Here, X and Y were frequently proline and lysine. Three proline and lysine residues were hydroxylated, and one of the latter appeared to link to galactose. Computer analysis of several lectins for sequence homology suggested that the COOH-terminal quarter of the MBP is associated with the calcium binding as well as carbohydrate recognition.     

cDNAs encoding large venom proteins from the parasitoid wasp Pimpla hypochondriaca identified by random sequence analysis

Venom from the parasitoid wasp Pimpla hypochondriaca contains numerous proteins, has potent in vitro anti-haemocytic properties, and disrupts host encapsulation responses. By sequencing 500 cDNAs randomly isolated from a venom gland library, we have identified 60 clones that encode proteins containing potential secretory signal sequences. To identify cDNAs encoding particular venom proteins, N-terminal amino acid sequences were determined for large (>30 kDa) venom proteins that had been separated using a combination of gel filtration and SDS-PAGE. We describe five of these cDNAs, which encoded residues that matched with the N-terminal sequences of previously undescribed venom proteins. cDNAs vpr1 and vpr3 encoded related proteins of approximately 32 kDa that were found in widely different fractions of gel filtration-separated venom. Neither vpr1 nor vpr3 were closely related to any other protein in the GenBank database, suggesting that they are highly specialised venom components. vpr2 encoded a 57-kDa polypeptide that was similar to a Drosophila protein, of unknown function, which lacks a signal sequence. A fourth clone, tre1, encoded a 61-kDa protein with extensive sequence similarity to trehalases. The 76-kDa sequence encoded by lac1 contained three regions which were very similar to histidine-rich copper-binding motifs, and could be aligned with the laccase from the fungus Coprinus cinereus. This study represents a significant step towards a holistic view of the molecular composition of a parasitoid wasp venom.     

A nonfunctional sequence converted to a signal for glycophosphatidylinositol membrane anchor attachment

The COOH terminus of decay-accelerating factor (DAF) contains a signal that directs glycophosphatidylinositol (GPI) membrane anchor attachment in a process involving concerted proteolytic removal of 28 COOH-terminal residues. At least two elements are required for anchor addition: a COOH-terminal hydrophobic domain and a cleavage/attachment site located NH2-terminal to it, requiring a small amino acid as the acceptor for GPI addition. We previously showed that the last 29-37 residues of DAF, making up the COOH-terminal hydrophobic domain plus 20 residues of the adjacent serine/threonine-rich domain (including the anchor addition site), when fused to the COOH terminus of human growth hormone (hGH) will target the fusion protein to the plasma membrane via a GPI anchor. In contrast, a similar fusion protein (hGH-LDLR-DAF17, abbreviated HLD) containing a fragment of the serine/threonine-rich domain of the LDL receptor (LDLR) in place of the DAF-derived serine/threonine-rich sequences, does not become GPI anchored. We now show that this null sequence for GPI attachment can be converted to a strong GPI signal by mutating a pair of residues (valine-glutamate) in the LDLR sequence at a position corresponding to the normal cleavage/attachment site, to serine-glycine, as found in the DAF sequence. A single mutation (converting valine at the anchor addition site to serine, the normal acceptor for GPI addition in DAF) was insufficient to produce GPI anchoring, as was mutation of the valine-glutamate pair to serine-phenylalanine (a bulky residue). These results suggest that a pair of small residues (presumably flanking the cleavage point) is required for GPI attachment. By introducing the sequence serine-glycine (comprising a cleavage-attachment site for GPI addition) at different positions in the LDLR sequence of the fusion protein, HLD, we show that optimal GPI attachment requires a processing site positioned 10-12 residues NH2-terminal to the hydrophobic domain, the efficiency anchor attachment dropping off sharply as the cleavage site is moved beyond these limits. These data suggest that the GPI signal consists solely of a hydrophobic domain combined with a processing site composed of a pair of small residues, positioned 10-12 residues NH2-terminal to the hydrophobic domain. No other structural motifs appear necessary.     

Towards a comprehensive view of the primary structure of venom proteins from the parasitoid wasp Pimpla hypochondriaca

Venom from the parasitoid wasp Pimpla hypochondriaca has potent in vivo activity against insect haemocytes and disrupts host immune responses. Using hybridisation techniques, and more recently random sequence analysis, we had previously identified cDNAs encoding 10 venom proteins from this wasp and deduced their primary structures. We have now extended the random sequence analysis and discovered a further nine cDNAs encoding proteins with predicted signal sequences. The mature proteins were calculated to have masses of between 4 and 22 kDa. Post-signal sequence residues predicted from the cDNAs matched those derived by Edman degradation from venom proteins separated using gel filtration and reverse phase chromatography, confirming that the cloned cDNAs encode proteins which are secreted into the venom sac. Proteins containing at least six cysteine residues were abundant and seven of these cysteine-rich venom proteins, cvp1-7, were identified. The sequences of some of these proteins were similar, or contained similar cysteine arrangements, to Kunitz type protease inhibitors, pacifastin, the trypsin inhibitor domain protein family, atracotoxin and omega-conotoxin, respectively, which occur in a diverse range of animals including spiders, molluscs, humans and grasshoppers. Two small venom proteins, svp1 and svp2, as well as cvp7 did not have similar sequences to proteins in the GenBank protein database suggesting they may be highly specialised venom components. The random sequencing approach has provided a rapid means of determining the primary structure of the majority of Pimpla hypochondriaca venom proteins.     

The deduced protein sequence of the human carboxypeptidase N high molecular weight subunit reveals the presence of leucine-rich tandem repeats

Human plasma carboxypeptidase N is a 280-kDa tetramer with two high molecular mass (83-kDa) glycosylated subunits which protect the two 50-kDa catalytic subunits and keep them in the circulation. An initial clone for the 83-kDa subunit was obtained by screening two lambda gt11 human liver cDNA expression libraries with antiserum specific for carboxypeptidase N or the 83-kDa subunit. The libraries were rescreened with the labeled cloned cDNA, and the largest clone obtained (2536-base pair insert) was completely sequenced. The deduced protein sequence matched the sequence of several tryptic peptides from the 83-kDa subunit but did not contain the NH2-terminal sequence. The remaining portion of the protein coding sequence was synthesized by the polymerase chain reaction, cloned, and sequenced. The composite cDNA sequence is 2870 base pairs long with an open reading frame of 1608 base pair coding for a protein of 536 amino acids (Mr = 58,762). The protein sequence contains seven potential N-linked glycosylation sites and a threonine/serine-rich region which is a potential site for attachment of O-linked carbohydrate. The most striking feature is a region (residues 68-355) containing 12 leucine-rich tandem repeats of 24 residues with the following consensus sequence: P-X-X-alpha-F-X-X-L-X-X-L-X-X-L-X-L-X-X-N-X-L-X-X-L (X = any amino acid and alpha = aliphatic amino acids, I, L, or V). This repeating pattern is found in the leucine-rich alpha 2-glycoprotein and in other proteins where it might mediate interactions with macromolecules. This region also contains five sequences with heptad repeating leucine residues comprising a leucine zipper motif. The leucine-rich domain likely constitutes an important structural or functional element in the interactions of the 83- and 50-kDa subunits to form the active tetramer of carboxypeptidase N.     

Molecular cloning of a histidine-rich Ca2+-binding protein of sarcoplasmic reticulum that contains highly conserved repeated elements

We reported previously the purification of a 165-kDa muscle-specific protein identified by virtue of its ability to bind 125I-labeled low density lipoprotein with high affinity after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Hoffmann, S. L., Brown, M. S., Lee, E., Pathak, R. K., Anderson, R. G. W., and Goldstein, J. J. (1989) J. Biol. Chem. 264, 8260-8270). The protein is located in the lumen of the sarcoplasmic reticulum, where it has no access to plasma lipoproteins. It binds to 45Ca2+ on nitrocellulose blots and stains metachromatically blue with Stains-all, a cationic dye that stains Ca2+-binding proteins. In the current paper, we have isolated a full-length rabbit cDNA clone for the 165-kDa protein. The deduced amino acid sequence reveals a 852-amino acid protein with the following structural features: 1) an NH2-terminal 27-residue putative signal sequence; 2) a highly repetitive region containing nine nearly identical tandem repeats of 29 residues, each consisting of a histidine-rich sequence HRHRGH, a stretch of 10-11 acidic amino acids, and a sequence containing 2 serines and a threonine in a negatively charged context; 3) a 13-residue stretch of polyglutamic acid; and 4) a COOH-terminal cluster of 14 closely spaced cysteine residues with the repeating pattern of Cys-X-X-Cys suggestive of a heavy metal binding domain. Histidine, aspartic acid, and glutamic acid accounted, respectively, for 13, 12, and 19% of the amino acids. The protein does not share any significant sequence homology with the cell surface low density lipoprotein receptor. Stretches of acidic amino acids are a feature of two other luminal sarcoplasmic reticulum proteins, suggesting that these may be a general feature of luminal sarcoplasmic reticulum proteins. We suggest that the histidine-rich Ca2+-binding protein described in the current study be designated HCP. The role of HCP in Ca2+ homeostasis in the sarcoplasmic reticulum of skeletal and cardiac muscle remains to be determined.     

A tripartite structure of the signals that determine protein insertion into the endoplasmic reticulum membrane

Multilineage colony stimulating factor is a secretory protein with a cleavable signal sequence that is unusually long and hydrophobic. Using molecular cloning techniques we exchanged sequences NH2- or COOH-terminally flanking the hydrophobic signal sequence. Such modified fusion proteins still inserted into the membrane but their signal sequence was not cleaved. Instead the proteins were now anchored in the membrane by the formerly cleaved signal sequence (signal-anchor sequence). They exposed the NH2 terminus on the exoplasmic and the COOH terminus on the cytoplasmic side of the membrane. We conclude from our results that hydrophilic sequences flanking the hydrophobic core of a signal sequence can determine cleavage by signal peptidase and insertion into the membrane. It appears that negatively charged amino acid residues close to the NH2 terminal side of the hydrophobic segment are compatible with translocation of this segment across the membrane. A tripartite structure is proposed for signal-anchor sequences: a hydrophobic core region that mediates targeting to and insertion into the ER membrane and flanking hydrophilic segments that determine the orientation of the protein in the membrane.     

Structural analysis of NADPH-cytochrome P-450 reductase from porcine hepatic microsomes. Sequences of proteolytic fragments, cysteine-containing peptides, and a NADPH-protected cysteine peptide

Detergent-solubilized NADPH-cytochrome P-450 reductase was purified from porcine hepatic microsomes and compared to the rabbit enzyme isolated under identical conditions. The porcine enzyme had an equivalent specific activity toward cytochrome c compared to the rabbit enzyme. When analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the porcine enzyme exhibited a major band at Mr = 80,000 and two additional bands at Mr = 20,000 and 60,000. The 20-kDa fragment was shown to be the COOH-terminal portion of the protein which contains a hydrophobic sequence of 28 residues homologous to the pyrophosphate-binding portion of the FAD-binding protein p-hydroxybenzoate hydroxylase. The 60-kDa fragment corresponded to the NH2-terminal portion of the protein since this peptide and the intact protein have blocked NH2 terminal. The trypsin-solubilized porcine enzyme has an NH2-terminal sequence which is homologous to the equivalent trypsin-solubilized enzymes from rat and rabbit (80% sequence homology). Eight cysteine-containing peptides were isolated from a tryptic digest of the S-carboxymethylated pig enzyme. Significant sequence homology was not found between these peptides and other flavoproteins, except for one peptide (Glu-Val-Gly-Glu-Thr-Leu-Leu-Tyr-Tyr-Gly-Cys-Arg) which exhibited partial homology with the known NADPH-binding site of glutathione reductase. When the NADPH-protected enzyme was first S-alkylated with unlabeled iodoacetate, NADPH depleted, and further alkylated with 14C-labeled iodoacetate, the above radiolabeled peptide was isolated from a tryptic digest. The equivalent peptide was also isolated by a similar procedure from rabbit liver cytochrome P-450 reductase.     

Purification and pore-forming activity of two hydrophobic polypeptides from the secretion of the Red Sea Moses sole (Pardachirus marmoratus)

A new column chromatography procedure, based on ion exchange, chromatofocusing, and reverse phase high pressure liquid chromatography was employed to isolate the two main proteinaceous, toxic, cytolytic, pore-forming factors from the secretion of the Red Sea Moses sole Pardachirus marmoratus. Pardaxin I, comprising 10% of the gland secretion proteins, was shown to be 5-10 times more toxic, cytolytic, and active in membrane pore formation than pardaxin II (8% of gland secretion proteins). Gel electrophoresis, amino acid analysis, and NH2-terminal amino acid sequence reveals a high degree of homogeneity and resemblance between the two toxins. They are rich in aspartic acid, serine, glycine, and alanine and devoid of arginine, tyrosine, and tryptophan. Their NH2-terminal residue sequence was found to be NH2-Gly-Phe-Phe. Their hydrophobicity is evident from chromatographic behavior on a hydrophobic matrix, presence of 9 successive hydrophobic residues at the NH2 terminus, and a decrease in drop size during elution of active fractions during chromatographic purification. The minimal molecular weight of pardaxin I is about 3500 as determined by sodium dodecyl sulfate gel electrophoresis and amino acid analyses. It is composed of 35 amino acids and is free of carbohydrate and sialic acid residues. Mass spectrometry of the ethyl acetate extract of the gland secretion and purified toxin reveals the presence of sterols in the secretion but their absence in the purified toxins. Pardaxin I was iodinated without affecting its chemical and pore-forming properties. It binds to liposomes of different phospholipid compositions. In hyperpolarized unilamellar liposomes, pardaxin I produced a fast, nonspecific permeabilization and in multilamellar liposomes, a slow, cation-specific pore. It is suggested that pardaxins exert their effects due to their hydrophobic and pore-formation properties.