Immunochemical mapping of alpha-2 interferon

A panel of five monoclonal antibodies, designated U1-U5, produced by murine hybridoma clones has been raised to recombinant interferon (IFN) alpha-2, and one monoclonal antibody, designated U6, has been raised to a mixture of cyanogen bromide fragments of IFN alpha-2. These antibodies have been characterized with respect to (1) neutralization of IFN antiviral and antiproliferative activities, (2) binding to four cloned IFN alpha subtypes (alpha-1, alpha-2, alpha-4, and alpha-7) that are naturally occurring and to two novel products of recombinant DNA technology (delta-4 alpha-1 and delta-4 alpha-2/alpha-1 hybrid), and (3) binding to three cyanogen bromide fragments of IFN alpha-2. Four of the six monoclonal antibodies inhibited IFN antiviral activity. In conjunction with the previously reported monoclonal antibodies III/21 [Arnheiter, H., Thomas, R. M., Leist, T., Fountoulakis, M., & Gutte, B. (1981) Nature (London) 294, 278-280] and NK-2 [Secher, D. S., & Burke, D. C. (1980) Nature (London) 285, 446-450], eight unique epitopes have been described. Analysis of cross-reactivity patterns with IFN alpha fragments and subtypes indicated that monoclonal antibodies U1 and NK-2, which neutralized both antiviral and antiproliferative activities, and U2, which was nonneutralizing in these assays, were directed to distinct epitopes located in a polypeptide consisting of the amino-terminal 15 amino acid residues linked to residues 60-110 by a disulfide bond. The epitope recognized by U1 was determined to reside, at least in part, between residues 5 and 15. Competitive binding studies indicated that neutralizing monoclonal antibody U3, which did not bind to any of the cyanogen bromide fragments, was directed to an epitope partially overlapping that of NK-2. Epitopes to which neutralizing monoclonal antibodies U3, U4, and U5 and nonneutralizing antibody U6 were directed were readily distinguished by cross-reactivity with IFN alpha subtypes. The nonneutralizing monoclonal antibody U6 was determined to be directed to an epitope between residues 22 and 58. The fact that delta-4 alpha-1 and the delta-4 alpha-2/alpha-1 hybrid were active in an antiviral assay indicated a lack of direct functional significance for the first four amino-terminal amino acid residues and the Cys1-Cys98 disulfide bond. However, reduction with 2-mercaptoethanol of IFN alpha-2 altered the integrity of four of the eight epitopes. These data support a critical role for disulfide linkages in maintaining the native conformation of IFN alpha-2 and provide a potential basis for predicting the location of functionally important domains.     

Amino acid sequence of human plasma apolipoprotein C-II from normal and hyperlipoproteinemic subjects

The complete amino acid sequence of human plasma apolipoprotein C-II isolated from normal individuals and a subject with type V hyperlipoproteinemia has been determined. Apo-C-II contains 79 amino acids with the following amino acid composition: Asp4, Asn1, Thr9, Ser9, Glu7, Gln7, Pro4, Gly2, Ala6, Val4, Met2, Ile1, Leu8, Tyr5, Phe2, Lys6, Arg1, Trp1. Cleavage of apo-C-II by cyanogen bromide produced three peptides designated as CB-1 (9 residues), CB-2 (51 residues), and CB-3 (19 residues). Two peptides, CT-1 (50 residues) and CT-2 (29 residues), which overlapped the cyanogen bromide peptides, were obtained by tryptic digestion of citraconylated apo-C-II at the single arginine residue. The amino acid sequence of apo-C-II was obtained by the automated phenyl isothiocyanate degradation of intact apo-C-II and the peptides produced by cleavage of apo-C-II by cyanogen bromide and trypsin. Phenylthiohydantoins were identified by high performance liquid chromatography and chemical ionization-mass spectrometry. The amino acid sequence of apo-C-II from the normal subject was identical with the apo-C-II isolated from the hyperlipoproteinemic subject.     

The carboxyl-terminal region of human interferon gamma is important for biological activity: mutagenic and NMR analysis.

Deletion of nine amino acids from the carboxyl terminus of human IFN gamma (residues 138--146; LFRGRRASQ) resulted in a 7-fold increase in specific antiviral activity. Similar increases in receptor binding affinity were seen. Deletion of residues 136 and 137 (QM) had little additional effect, but removal of Ser135 resulted in a sharp drop in antiviral activity. Further removal of residues 133 and 134 (KR) lowered antiviral activity to 1% of the peak value. Comparison of the proton NMR spectra of selected deletions down to residue 132 showed that there was no significant change in the core protein structure. Deletions down to residue 125 had the same antiviral activity as those to 132, but changes could now be seen in the aromatic proton NMR spectrum of this shorter derivative. Substitution of the homologous murine sequence between residues 124 and 130 (human SPAAKTG; murine LPESSLR) resulted in only a small decrease in antiviral activity, further suggesting that the precise sequence in this region was not critical for activity. Ser135 was substituted with a number of other amino acids with little or no change in activity. The importance of the residues between 131 and 134 for biological activity was corroborated by mutagenesis, although some substitutions in this region were tolerated.     

Monoclonal antibodies specific for different regions of human apolipoprotein A-I. Characterization of an antibody that does not bind to a genetic variant of apoA-I (Glu----136 Lys)

Three monoclonal mouse hybridoma antibodies, designated 2AI, 4AI, and 5AI, specific for human plasma apolipoprotein A-I (apoA-I) were characterized. In an enzyme-linked immunosorbent assay (ELISA) each of the antibodies reacted with purified apoA-I and with A-I in normal human serum. Immunoblotting of apoA-I subjected to isoelectric focusing revealed that the three antibodies reacted with all the charge isomorphs of apoA-I and with proapoA-I. Using a solid phase competitive displacement assay, the antigenic determinant for antibody 5AI could be localized to cyanogen bromide fragment 3 of apoA-I (residues 113-148), while the epitope for antibody 4AI resided in cyanogen bromide fragment 4. Dot blot experiments and data obtained by the competitive displacement assay revealed that antibody 2AI reacts with high affinity with CNBr fragment 2 but that it also reacts with lower affinity with fragments 1 and 4. The antibody 5AI did not bind to a genetic variant of apoA-I (Glu----136 Lys), demonstrating that the substitution of a single amino acid in human apoA-I can cause the loss of an antigenic determinant.     

Characterization of the subunits of beta-conglycinin

Four subunits of beta-conglycinin were purified from soybean cultivar CX 635-1-1-1, and were designated alpha, alpha', beta, and beta' in accordance with nomenclature proposed by Thanh and Shibasaki [(1977) Biochim. Biophys. Acta 490, 370-384]. Of these subunits, beta' has not previously been reported or characterized. Consistent with the low levels of methionine in these proteins, cyanogen bromide cleavage of alpha', alpha, and beta' subunits produced only a few fragments. The beta subunit contains no methionine and was not cleaved by cyanogen bromide. The NH2-terminal amino acid sequences of the alpha and alpha' subunits are homologous, and each has valine at its amino terminus. The beta subunit has a very different NH2-terminal sequence from those of the alpha and alpha' subunits, and has leucine at its amino terminus. The NH2-terminal sequence of the beta' subunit could not be determined, as it appeared to be blocked to Edman degradation. Although alpha and alpha' subunits have similar NH2-terminal sequences, they differ in the number of methionine residues and so yielded different numbers of cyanogen bromide fragments. Two cyanogen bromide fragments (CB-1 and CB-2) were purified from the alpha subunit. CB-1 originated from the NH2-terminal end of the subunit. The amino acid sequence of CB-2 was identical to that predicted from the nucleotide sequence of cDNA clone pB36. The insert in pB36 encoded 216 amino acids from the COOH-terminal end of the alpha subunit and contained a 138-bp trailer sequence which was followed by a poly-(A) tail. Maps showing the relative positions of methionine residues and carbohydrate moieties in the alpha and alpha' subunits were drawn, based on primary sequence data, and the size and carbohydrate content of the CNBr fragments derived from the subunits.     

Solution conformations of the N-terminal CNBr fragment of glycogen phosphorylase and its interaction with calmodulin

The CNBr peptides, CBPa and CBPb, corresponding to the N-terminal 1-91 amino acid residues of glycogen-phosphorylase a and b, respectively, were purified and characterized. CD, 31P-NMR and fluorescence spectroscopy were used to assess the structural organization of the cyanogen bromide peptides in solution. The cyanogen bromide peptides yielded 21% of alpha-helical structures by CD compared to a calculated value of 36.3%. These peptides interact with calmodulin which induces measurable alpha-helices in the cyanogen bromide peptides. The helix stabilizing reagent, trifluoroethanol, induces high numbers of alpha-helices in CBP, thereby demonstrating the conformation fluidity of this peptide. The dissociation constants for calmodulin and CBP estimated by fluorescence titrations were 36.0 and 29.9 nM for CBPb in the presence of Ca2+ and EGTA, respectively. The phosphorylated residue in CBPa causes a decrease in binding interactions with calmodulin and corresponding values obtained for CBPa by fluorescence titration are 56.0 and 141.0 nM, respectively. The Ser-P-14 of CBPa is titratable, yielding a pKa = 5.45 and a Hill coefficient of 1.5. A helical wheel analysis using a computer program in PC/GENE of the CBP shows that peptide stretches in the alpha-1 and alpha-2 helices are most basic and fairly amphiphilic and therefore represent the most probable segment for CaM binding. It is this structural character of these segments which presumably confer the ability to bind CaM and facilitate some of the allosteric transitions of glycogen phosphorylase.     

The amino acid sequence of the activation peptide of bovine pro-carboxypeptidase A

The amino acid sequence of the activation peptide of bovine pro-carboxypeptidase A subunit I has been determined by automated Edman degradation of the cyanogen bromide fractions derived from the precursor protein. The activation peptide contains 94 amino acid residues in a unique sequence which precedes directly the amino-terminal alanine residue of carboxypeptidase A alpha. A notable feature of the activation peptide is the presence of acidic amino acid residues immediately preceding the site of activation. The amino acid sequence of the activation peptide of bovine pro-carboxypeptidase A shows extensive similarity to those of the corresponding porcine and rat enzymes.     

Mapping the binding of monoclonal antibodies to histone H5

The binding sites of nine monoclonal antibodies along the polypeptide chain of histone H5 were mapped. Immunoblotting experiments with peptides generated from H5 by trypsin digestion, N-bromosuccinimide cleavage, and cyanogen bromide cleavage revealed that all of the monoclonal antibodies reacted with the globular region of H5 which is encompassed by amino acid residues 22-98. Within this globular segment, the epitopes could be subdivided into three regions. Monoclonals 1G11, 2E5, and 2H5 bind to residues 28-31. The close proximity of the epitopes was verified by a competitive enzyme-linked immunosorbent assay and by their binding pattern to a tryptic digest of H5. Monoclonals 4C6, 6E12, and 2E12 bind to a region encompassed by amino acids 28-53 while monoclonals 4H7, 1C3, and 3H9 bind to a region encompassed by residues 53-98. Precise localization of the epitopes in the primary sequence of H5 will allow detailed studies on the mode of binding of H5 to core particles in chromatin.     

The primary structure of staphylococcal protease.

The amino acid sequence of staphylococcal protease has been determined by analysis of tryptic peptides obtained from cyanogen bromide fragments. Selected peptides obtained from digests with staphylococcal protease, thermolysin, and chymotrypsin provided the information necessary to align the tryptic peptides and the cyanogen bromide fragments. The protease is a single polypeptide chain of some 250 amino acids and is devoid of sulfhydryl groups. The COOH-terminal tryptic peptide of of the protease molecule contains some 43 residues, most of which are aspartic acids, asparagines, and prolines. The amino acid sequence of this peptide was not determined. The primary structure near the active serine residue indicates that staphylococcal protease is related to the pancreatic serine proteases. However, it has little or no additional sequence homologies with these enzymes except for the regions near histidine-50 and aspartic acid - 91. These regions have striking similarities with the corresponding regions of protease B and the trypsin-like enzyme of Streptomyces griseus.     

Human apolipoprotein A-I polymorphism. Identification of amino acid substitutions in three electrophoretic variants of the Münster-3 type

Variant forms of apolipoprotein A-I (apo-A-I) have been shown to exist in the human population. One mutant form, referred to as apo-A-I-Münster-3, is one charge unit more basic than normal apo-A-I on isoelectric focusing gels. This variant has the same immunologic characteristics and molecular weight as normal apo-A-I. The apo-A-I-Münster-3 from subjects in three unrelated families (in two of which the trait has been shown to be transmitted as an autosomal co-dominant) has been analyzed by partial amino acid sequencing to define the cause of the electrophoretic abnormality. In the apo-A-I of family A, the abnormality was shown to occur in the smallest cyanogen bromide fragment, CB-2 (residues 87-112), and amino acid sequencing revealed asparagine instead of the usual aspartic acid at residue 103. Subjects with this mutant form have shown no signs of dyslipoproteinemia. The NH2-terminal cyanogen bromide fragment (CB-1, residues 1-86) from the apo-A-I of family B was shown to differ electrophoretically from normal CB-1, and amino acid sequencing revealed that a substitution of arginine for proline at residue 4 was responsible for this variant form. Analysis of the plasma lipids of one affected family B member demonstrated that the percentage of the total cholesterol that was esterified was somewhat lower than that normally observed. In a third family, family C, a variant having the same electrophoretic abnormality as the other two was determined to have an amino acid substitution at yet a different position. In this variant, histidine was found at residue 3 in the apo-A-I sequence, rather than the usual proline. In all three cases, the substitution could account for the electrophoretic abnormality. It is proposed that these three apo-A-I-Münster-3 variants be designated apo-A-I(Asp103----Asn), apo-A-I(Pro4----Arg), and apo-A-I(Pro3----His), respectively, to indicate the substitution that accounts for the abnormality in isoelectric focusing gels.     

The primary structure of Lactobacillus casei thymidylate synthetase. I. The isolation of cyanogen bromide peptides 1 through 5 and the complete amino acid sequence of CNBr 1, 2, 3, and 5.

Thymidylate synthetase from Lactobacillus casei was S-carboxymethylated and degraded by treatment with cyanogen bromide. Although the protein contains 6 methionine residues, only 5 cyanogen bromide peptides were obtained due to the presence of 1 methionine on the NH2 terminus and another adjacent to a threonine residue which was resistant to cleavage. The peptides were isolated by differential extraction, first with ammonium acetate, then pyridine acetate, and finally the residue was solubilized with 50% acetic acid. Each peptide was further purified to homogeneity by Bio-Gel chromatography. The size of the peptides from the amino to carboxyl end of the enzyme subunit was CNBr 1, 4,100; CNBr 2, 10,300; CNBr 3, 8,100; CNBr 4, 11,800; CNBr 5, 2,200. The sum of the amino acid residues of the peptides is equal to the sum of the residues in an enzyme subunit, indicating that all of the CNBr peptides have been isolated. The CNBr-resistant methionine was located in CNBr 2 and the 5-fluoro-2'-deoxyuridine 5'-monophosphate binding site in CNBr 4. The holoenzyme molecular weight, based on the residue weights of the amino acids in the two equivalent subunits, is equal to 73,176. The complete sequence of each of the CNBr peptides, except for CNBr 4, which is presented in the following paper, is described.     

SUBUNITS OF CHOLINE ACETYLTRANSFERASE (EC 2.3.1.6)

Abstract— The molecular weight of choline acetyltransferase is determined to be approx 87,000-89,000 daltons. There are 808 amino acid residues per molecule of the enzyme with a calculated mol. wt of 89,300. Peptides of ChAc were studied after tryptic digestion, cyanogen bromide and BNPS-skatole cleavages. The results of these studies suggest that the enzyme contains 6 identical subunits with 133 amino acid residues each and with a calculated molecular weight of 14,700 daltons for the monomer. This conclusion is supported by the fact that serine is the only amino terminal residue and alanine is the only carboxyl-terminal group.     

Primary structure of tyrosinase from Neurospora crassa. II. Complete amino acid sequence and chemical structure of a tripeptide containing an unusual thioether

To align the four cyanogen bromide peptides of Neurospora tyrosinase whose amino acid sequences were reported in the preceding paper, suitable methionine-containing overlap peptides were isolated. The required peptides were obtained by tryptic, peptic, and thermolytic digestion of the unmodified protein and of the maleylated derivative. From the partial sequence information of these peptides and a cyanogen bromide overlap peptide, the four cyanogen bromide fragments were aligned in the order CB3-CB1-CB4-CB2. These data establish Neurospora tyrosinase as a single-chain protein of 407 amino acids with a molecular weight of 46,000. The single cysteinyl residue 94 was found to be covalently linked via a thioether bridge to histidyl residue 96. The chemical nature of this unusual structure was elucidated by physicochemical analysis of peptides obtained from in vivo 35S, [2,5-3H]histidine, and [5-3H]histidine-labeled Neurospora tyrosinase.     

Topographic antigenic determinants on cytochrome c. Immunoadsorbent separation of the rabbit antibody populations directed against horse cytochrome.

Seven populations of site-specific antibodies were isolated from each of three sera of rabbits immunized against glutaraldehyde-polymerized horse cytochrome c. The antibodies were separated using an immunoadsorption scheme which employed the following cytochromes c: horse, beef, guanaco, rabbit, mouse testicular, pigeon, and the cyanogen-bromide cleaved fragment of the rabbit protein containing residues 1 to 65. The monovalent, antigen-binding fragments of the antibodies (Fab') gave 1:1 stoichiometries with native horse cytochrome c in fluorescence quenching assays. Cross-reactivities with heterologous cytochromes c using fluorescence quenching and a modified Farr assay demonstrated that the antigenic determinants are situated around residues 44, 60, and 89/92, four of the six amino acid sequence positions where horse and rabbit cytochromes c differ. The remaining two differences occur at residues 47 and 62. The apparent lack of immunogenicity of these two substitutions may result from the presence of the more immunogenic residues 44 and 60 nearby. Of the seven antibody populations isolated, four were shown to bind in the region of residues 89 and 92. Since several cytochromes c have amino acid sequence differences from the horse protein at either of these two residue positions, it was possible to fractionate the antibodies directed against this complex site on the basis of subtle specificity differences between them. Two antibody populations bind in the region of residue 44. One of these is specific for proline at that position, while the other antibody population also binds to cytochrome c containing glutamic acid at position 44. The remaining antibody population binds in the region of the lysine residue at position 60. Each of the seven site-specific antibody populations binds effectively to any cytochrome c having a suitable amino acid sequence in the antigenic determinant regardless of any residue differences from the immunogen outside of that area. It was also demonstrated that these seven antibody populations represent the totality of the antibodies elicited in rabbits against horse cytochrome c, since the immunoadsorbants bound all the antibodies specific for the native protein. Furthermore, the rabbit antisera contained no other antibody population that could bind to the conformationally disturbed, cyanogen bromide-cleaved fragment of horse cytochrome c containing residues 1 to 65, making it appear that there were no antibodies elicited against a "processed" form of cytochrome c.     

Isolation and characteristics of cyanogen bromide peptides of transducin alpha and beta subunits

The alpha- and beta-subunits of the GTP-binding protein (transducin) from cattle retina were cleaved with cyanogen bromide. 21 peptides covering 90-100% of the amino acid sequence of the alpha- and beta-subunits were isolated from the hydrolyzate. Cyanogen bromide peptides complete or partial amino acid sequence was determined, the results were compared with those by Numa and coworkers [1] and Lochrie et al. [2] at the primary structure of the transducin alpha-subunit deduced from the nucleotide sequence of the cDNA. The structure by Lochrie is shown to differ much from the true structure of the alpha-subunit; probably, the investigators isolated cDNA, corresponding to the gene for some GTP-binding protein homologous to transducin, but not to the gene for the transducin alpha-subunit. The Numa's structure also contains an error. The final primary structure of the transducin alpha-subunit is given. The protein polypeptide chain consists of 349 amino acid residues and has an acetylmethionine residue as the N-terminal residue.     

The receptor-binding domain of human apolipoprotein E. Binding of apolipoprotein E fragments

To identify the domain of apolipoprotein E (apo-E) involved in binding to low density lipoprotein (LDL) receptors on cultured human fibroblasts, apo-E was cleaved and the fragments were tested for receptor binding activity. Two large thrombolytic peptides (residues 1-191 and 216-299) of normal apo-E3 were combined with the phospholipid dimyristoylphosphatidylcholine (DMPC) and tested for their ability to compete with 125I-LDL for binding to the LDL (apo-B,E) receptors on human fibroblasts. The NH2-terminal two-thirds (residues 1-191) of apo-E3 was as active as intact apo-E3 . DMPC, while the smaller peptide (residues 216-299) was devoid of receptor-binding activity. When apo-E3 was digested with cyanogen bromide (CNBr) and the four largest CNBr fragments were combined with DMPC and tested, only one fragment competed with 125I-LDL for binding to cultured human fibroblasts (CNBr II, residues 126-218). This fragment possessed binding activity similar to that of human LDL. The 125I-labeled CNBr II . DMPC complex also demonstrated high affinity, calcium-dependent saturable binding to solubilized bovine adrenal membranes. The binding of CNBr II . DMPC was inhibited by 1,2-cyclohexanedione modification of arginyl residues or diketene modification of lysyl residues. In addition, the CNBr II had to be combined with DMPC before it demonstrated any receptor-binding activity. Pronase treatment of the membranes abolished the ability of this fragment to bind to the apo-B,E receptors. This same basic region in the center of the molecule has been implicated as the apo-B,E receptor-binding domain not only by this study but also by other studies showing that 1) natural mutants of apo-E that display defective binding have single amino acid substitutions at residues 145, 146, or 158; and 2) the apo-E epitope of the monoclonal antibody 1D7, which inhibits apo-E binding, is centered around residues 139-146.     

Amino acid sequence studies on cyanogen bromide peptides of chicken caldesmon which bind to calmodulin

Three major calmodulin-binding cyanogen bromide peptides (fragments A, B, and D) were isolated from chicken gizzard muscle caldesmon and their amino acid sequences were determined. The molecular masses of fragments A, B, and D were estimated to 16, 12, and 9 kDa, respectively, by SDS-urea polyacrylamide gel electrophoresis. Fragment A was composed of 102 amino acid residues and contained homoserine at the C terminus. The amino acid sequence from the 37th residue of fragment A corresponds to the N-terminal sequence of the 15 kDa peptide which was obtained by thrombin digestion [Mornet, D., Audemard, E., & Derancourt, J. (1988) Biochem. Biophys. Res. Commun. 154, 564-571]. Thrombin 15 kDa peptide binds to F-actin but does not bind to calmodulin. Thus the N-terminal 36 residues and the C-terminal part from the 37th residue of fragment A are supposed to bind to calmodulin and F-actin, respectively. The sequences of fragments B and D were identical, but fragment D was composed of 64 amino acid residues and ended with tryptophan, whereas fragment B was of 98 or 99 amino acid residues and ended with proline. Both fragments B and D are supposed to be the C-terminal peptides of chicken caldesmon. Fragment B had heterogeneous sequences at the C-terminal region. These results can explain the reported heterogeneity of chicken caldesmon in charge and molecular mass.     

Complete amino acid sequence of a cytochrome P-450 isolated from beta-naphthoflavone-induced rabbit liver microsomes. Comparison with phenobarbital-induced and constitutive isozymes and identification of invariant residues

The complete covalent structure of a cytochrome P-450, form 4, isolated from liver microsomes of beta-naphthoflavone-induced rabbits was determined. The S-carboxyamidomethylated protein was cleaved with cyanogen bromide, endoproteinase Lys-C, and trypsin before and after succinylation. Selected peptides from CNBr digests of alkylated rabbit cytochrome P-450 forms 3a and 3c were also isolated and sequenced. Form 4 exhibited microheterogeneity due to the presence of several truncated forms. The existence of multiple NH2-terminal residues for form 4 was confirmed by the isolation and sequence analysis of the corresponding tryptic peptides. The predominant form contained 514 residues, corresponding to Mr 58,030. A peptide having Gly-232 and Gln-246 replaced by Ser and Asn residues, respectively, was also found in the isozyme preparation investigated here. The amino acid sequences of form 4 and selected peptide sequences from forms 3a and 3c were compared with the primary structures of forms 2 and 3b (previously determined in this laboratory). This comparison identified some 90 invariant residues. A cysteinyl residue at position 456, earlier reported as the heme-binding cysteine 436 (Heineman, F. S., and Ozols, J. (1982) J. Biol. Chem. 257, 14988-14999), was also present in forms 4, 3a, and 3c. Other single invariant residues identified were form 4/forms 2,3b, Trp-132/121, and His 270/252. The tyrosyl residues at positions 71/62 and 365/348 were also invariant. The latter is present in the "conserved segment" of the protein, residues 363/346 to 375/359, and may be involved in the substrate binding of cytochrome P-450. Also a lysyl residue, formerly identified by other laboratories to be involved in the electron transfer between the reductase and cytochrome P-450 form 2, was invariant in all five species. This lysyl residue corresponds to Lys-402 in form 4 or Lys-384 in the other forms.     

Human milk bile-salt stimulated lipase. Sequence similarity with rat lysophospholipase and homology with the active site region of cholinesterases

To determine the active site residue, human milk bile-salt stimulated lipase (BSSL) was labelled with [3H]diisopropyl fluorophosphate (DFP). Partial sequence analysis of cyanogen bromide fragments (a total of 146 residues from 6 peptides) revealed 84% sequence identity with a putative rat lysophospholipase. Sequence analysis of a [3H]DFP-labelled peptide indicated that the active site serine was contained in the sequence Gly-Glu-Ser-Ala-Gly. In addition to similarity with rat lysophospholipase, this sequence showed homology with regions of human butyrylcholinesterase and electric ray acetylcholinesterase (68% identity). It is concluded that these proteins are members of a new supergene family.     

Purification and characterization of PSP-I and PSP-II, two major proteins from porcine seminal plasma.

Two major glycoproteins, designated PSP-I and PSP-II, were purified from porcine seminal plasma by ammonium sulfate fractionation, CM-cellulose chromatography, gel filtration on Sephadex G-75 (superfine), and reverse phase high performance liquid chromatography. These two proteins exist in several forms differing mainly in the carbohydrate moiety. The complete amino acid sequence of PSP-I has been determined by automated Edman degradation of peptides generated by proteolytic digestion and cyanogen bromide cleavage. The protein is 109 residues long and has a single glycosylation site at the asparagine residue at position 47. In addition, the N-terminal sequence of PSP-II has also been determined. PSP-I is a unique protein; a sequence homology search using the protein data base did not reveal any significant homology with other proteins. PSP-II shares 50% sequence homology with a family of zona pellucida-binding glycoproteins at the N-terminus.