Amino acid sequence determination of the ADP,ATP carrier from beef heart mitochondria. The sequence of the C-terminal acidolytic fragment

The primary structure of the C-terminal region (94 residues) of the ADP,ATP carrier of beef heart mitochondria is described. CNBr cleavage results in a large peptide (CB1) with Mr 22 000 and several small peptides (CB2 to CB8). Peptide separation was achieved by gel chromatography with 80% formic acid or with an ethanol/formic acid mixture. The amino acid sequence of the small CNBr peptides was determined by solid-phase techniques. Hydrolysis in formic acid cleaves the carrier protein into an Mr 23 000 fragment (A1) with the blocked N-terminus and an Mr 10 000 fragment (A2) starting with proline. The alignment of two CNBr fragments was possible by degradation of A2 by solid-phase methods for 34 steps. The remaining CNBr fragments were arranged by sequencing the tryptic peptides of citraconylated A2.     

Single-chain, low molecular weight kininogen in the blood plasma of rabbits: isolation and fragmentation by porcine pancreatic kallikrein

A highly purified preparation of low molecular weight kininogen (LMrK) was isolated from the plasminogen-free rabbit blood plasma, using chromatography on DEAE-Sepharose CL-6B, gel filtration on Ultrogel AcA 34 and Sephadex G-100 as well as gradient chromatography on a hydroxylapatite column. The yield of the 320-fold purified LMrK was 16%. Trypsin released 13-14 micrograms-eq. of bradykinin (BK) from 1 mg of LMrK or 0.85-0,95 mol of BK per mol of kininogen. Rabbit LMrK consists of one polypeptide chain of Mr 69 000 and pI 4.63. Porcine pancreatic kallikrein splits off kinin from the LMrK polypeptide chain by disrupting two peptide bonds resulting in the formation of S-S-bound two chain molecule. After reduction of the S-S bonds by dithioerithritol the latter is separated into a heavy (Mr 61 000) and light (Mr 6 800) chains. A biologically active peptide was isolated from the products of CNBr cleavage of LMrK. This peptide consists of Lys-BK elongated from the C-terminal with several amino acid residues. Rabbit LMrK closely resembles human LMrK in terms of Mr, pI and location of the kinin fragment in the protein molecule.     

Localization of the high molecular weight kininogen binding site in the heavy chain of human factor XI to amino acids phenylalanine 56 through serine 86

We have previously demonstrated that a monoclonal antibody (5F7) directed against the heavy chain region of factor XI inhibits the binding of factor XI to high molecular weight kininogen (high Mr kininogen) and the surface-mediated proteolytic activation of factor XI by factor XIIa in the presence of high Mr kininogen. In order to identify the structural domain of factor XI that binds high Mr kininogen, CNBr-digested factor XI was passed over a 5F7 antibody affinity column. One of two CNBr peptides that bound to this 5F7 affinity column inhibited binding of 125I-factor XI to high Mr kininogen, as did intact factor XI. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate of an inhibitory peptide purified by high performance liquid chromatography revealed an Mr of 10,000-15,000. Gas-phase sequencing of this peptide revealed the following amino-terminal sequence: X-X-Val-Thr-Gln-Leu-Leu-Lys-Asp-Thr. These data together with the amino acid composition of the isolated peptide indicate that both the epitope recognized by antibody 5F7 and at least a portion of the high Mr kininogen binding site are contained within the amino-terminal portion of factor XI comprising residues Glu-1 through Met-102. Further cleavage of this peptide with o-iodosobenzoic acid at a tryptophanyl peptide bond revealed that an Mr 5,000 peptide (with the amino-terminal sequence Trp-Phe-Thr-Cys-Val-Leu) bound to a high Mr kininogen affinity column and inhibited binding of 125I-factor XI to high Mr kininogen. Finally, a synthetic peptide comprising residues Phe-56 through Ser-86 inhibited 125I-factor XI binding to high Mr kininogen. These experiments strongly suggest that the high Mr kininogen binding site is contained within the domain in the heavy chain region of factor XI comprising residues Phe-56 through Ser-86.     

Lipopolysaccharide-sensitive serine-protease zymogen (factor C) of horseshoe crab hemocytes. Identification and alignment of proteolytic fragments produced during the activation show that it is a novel type of serine protease

The horseshoe crab clotting factor, factor C, present in the hemocytes is a serine-protease zymogen activated with lipopolysaccharide. It is a two-chain glycoprotein (Mr = 123,000) composed of a heavy chain (Mr = 80,000) and a light chain (Mr = 43,000) [T. Nakamura et al. (1986) Eur. J. Biochem. 154, 511-521]. In our continued study of this zymogen, we have now also found a single-chain form of factor C (Mr = 123,000) in the hemocyte lysate. The heavy chain had the NH2-terminal sequence of Ser-Gly-Val-Asp-, consistent with that of the single-chain factor C, indicating that the heavy chain is derived from the NH2-terminal part of the molecule. The light chain had an NH2-terminal sequence of Ser-Ser-Gln-Pro-. Incubation of the two-chain zymogen with lipopolysaccharide resulted in the cleavage of a Phe-Ile bond between residues 72 and 73 of the light chain. Concomitant with this cleavage, the A (72 amino acid residues) and B chains derived from the light chain were formed. The complete amino acid sequence of the A chain was determined by automated Edman degradation. The A chain contained a typical segment which is similar in sequence to a family of repeats in human beta 2-glycoprotein I, complement factors B, protein H, C4b-binding protein, and coagulation factor XIII b subunit. The NH2-terminal sequence of the B chain was Ile-Trp-Asn-Gly-. This chain contained the serine-active site sequence-Asp-Ala-Cys-Ser-Gly-Asp-Ser-Gly-Gly-Pro-. These results indicate that horseshoe crab factor C exists in the hemocytes in a single-chain zymogen form and is converted to an active serine protease by hydrolysis of a specific Phe-Ile peptide bond.     

Identification of the peptide bond cleaved during activation of human Clr

CNBr cleavage of unreduced proenzyme Clr yielded fragment CP2b, isolated by gel filtration and highpressure gel permeation chromatography. This fragment (˜ M_τ 55000) comprised at least 4 disulphidelinked peptides, which were separated by gel filtration after reduction and alkylation. Peptide CP2bRA4, overlapping the A- and B-chain regions in proenzyme Clr was digested by V8 staphylococcal protease, and the digest separated by reversed-phase HPLC. N-terminal sequence analysis of peptide CP2bRA4SP9 established that Clr activation involves the cleavage of a single Arg-Ile bond, located in the sequence: Gln-Arg-Gln-Arg-Ile-Ile-Gly-Gly     

Partial amino acid sequence of porcine elastase II. Active site and the activation peptide regions

The partial amino acid sequence of porcine elastase II, isolated from crude trypsin Type II, was determined. The enzyme consists of two polypeptide chains, a light chain composed of 11 residues, and a heavy chain (Mr = 23 500) with four intrachain disulfide bridges; the two chains are held together by one interchain disulfide bond. Elastase II was fragmented into several peptides by chemical cleavages with CNBr at the two methionine residues, 99 and 180 (chymotrypsinogen numbering), and with hydroxylamine at the peptide bond following DIP-Ser195. About 50% of the sequence was determined and the positions of 120 amino acids were located, including the light chain residues and most of the active site residues. The partial sequence shows 64% difference between porcine elastase II and elastase I and only 26% difference between porcine elastase II and bovine chymotrypsin B.     

Primary structure of single-chain pro-urokinase

Single-chain pro-urokinase is an inactive proenzyme form of human urokinase with a single-chain structure and a Mr of 50,000 and converted to the active two-chain form by catalytic amounts of plasmin. It was isolated from culture fluid of human kidney cells and subjected to chemical (CNBr) and proteolytic (lysyl endopeptidase) degradation. The resulting peptides were separated by reverse-phase high performance liquid chromatography and subjected to automated sequence analysis. Amino acid sequence of 85% of the 411 residues recovered in 17 peptides were found to be consistent with those of the A chain (157 amino acids) and B chain (253 amino acids) of human urokinase reported by Günzler and co-workers (Günzler, W. A., Steffens, G.J., Otting, F., Kim, S.-M., A., Frankus, E., and Flohé, L. (1982) Hoppe-Seyler's Z. Physiol. Chem. 363, 133-141; 1155-1165; Steffens, G.J., Günzler, W.A., Otting, F., Frankus, E., and Flohé, L. (1982) Hoppe-Seyler's Z. Physiol. Chem. 363, 1043-1058). It revealed the presence of Lys at position 158 in single-chain pro-urokinase through which the two polypeptide chains of human urokinase are unified into one molecule. In addition, firm evidence was found that upon activation by plasmin single-chain pro-urokinase is cleaved at the Lys-Ile bond between residues 158 and 159, resulting in the formation of a two-chain urokinase molecule held together by one disulfide linkage. These results indicate that the cleavage at the Lys-Ile bond between residues 158 and 159 is responsible for conformational change, appearance of enzyme activity and reduction of its high affinity for fibrin.     

Characterization of the calmodulin-binding and catalytic domains in skeletal muscle myosin light chain kinase

Limited proteolysis has been utilized to study the structural organization of rabbit skeletal muscle myosin light chain kinase. The enzyme (Mr approximately 89,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) consists of an amino-terminal, protease-susceptible region of unidentified function and a carboxyl-terminal, protease-resistant region of Mr approximately 40,000 containing the catalytic and calmodulin-binding domains. Partial digestion with trypsin produced an intermediate 56,000-dalton fragment and a stable 38,000-dalton fragment, both of which were catalytically active and calmodulin-dependent. Chymotryptic digestion yielded three catalytically active fragments of about 37,000, 36,000, and 35,000 daltons. The Mr = 37,000 fragment was calmodulin-dependent with an apparent affinity equivalent to that of the native enzyme (approximately 1 nM). The 36,000-dalton fragment was also calmodulin-dependent but had a approximately 200-fold lower apparent affinity. The Mr = 35,000 fragment was calmodulin-independent. These three chymotryptic fragments, had identical amino termini. Nineteen residues were missing from the carboxyl terminus of the calmodulin-independent chymotryptic fragment whereas only 8 or 9 carboxyl-terminal residues were missing from the calmodulin-dependent tryptic fragments. These results suggest that the 11-residue sequence (IAVSAANRFKK) in the carboxyl-terminal region of myosin light chain kinase contributes directly to the binding of calmodulin. This conclusion is in accord with data (Blumenthal, D. K., Takio, K., Edelman, A. M., Charbonneau, H., Titani, K., Walsh, K. A., and Krebs, E. G. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 3187-3191) that the carboxyl-terminal, 27-residue CNBr peptide of the native enzyme shows Ca2+-dependent, high affinity binding to calmodulin and that similar calmodulin-binding activity, although detectable in unfractionated CNBr digests of calmodulin-dependent enzyme forms, is much reduced in a CNBr digest of the calmodulin-independent, Mr = 35,000 chymotryptic fragment.     

Localization within the heavy chain sequence of tyrosyl peptides from the combining sites in a rabbit anti-3-azopyridine antibody preparation

A relatively homogeneous rabbit heavy chain was cleaved by CNBr. Fragment C-1 (the N-terminal half of the heavy chain) was isolated. Reduction and alkylation of C-1 liberated three fragments and partial sequence analysis of these isolated fragments showed that C-1 had been split on the carboxyl-side of Met 84. Similar results were obtained with another anti-hapten antibody preparation in which tyrosyl residues in the combining sites had been labeled. The labeled tyrosyl residues were found in the fragment representing residues 85–253. Since the constant region begins at about residue 120 and the sequences of the tyrosyl peptides from the combining sites are not present in published constant region sequences, these peptides appear to be derived from a variable region between residues 85 and 120.     

Separation, purification, partial characterization and comparison of the heavy and light chains of botulinum neurotoxin types A, B, and E

Clostridium botulinum produces botulinum neurotoxin (NT) in antigenically distinct forms. When isolated from bacterial cultures type E is a single chain, type B is a mixture of single and two-chain molecules, and type A is essentially a two-chain molecule (Mr approximately 150,000). Protease(s) in the cultures or trypsin nick single-chain NT to the two-chain form. The heavy (Mr approximately 100,000) and light (Mr approximately 50,000) chains of the two-chain molecule remain held together by -S-S-bond(s). The two chains are presumed to have different functions. NT binds to nerve cells via the heavy chain and then light chain enters the cell and blocks release of acetylcholine (Simpson, L. L. (1981) Pharmacol. Rev. 33, 155-188). We nicked single-chain NT to form the two-chain form with trypsin, minimizing secondary cleavages, then separated and purified the heavy and light chains using ion-exchange chromatography. The technique, with minor modifications, is a generalized method for types A, B, and E. These subunit chains (each a single band in sodium dodecyl sulfatepolyacrylamide gel electrophoresis) were analyzed for their complete amino acid compositions. The amino acid contents of the heavy and light chains agreed well with the parent two-chain molecule. This affirms that NT is composed of two chains. The two subunit chains are now usable for amino acid sequence and other studies. Comparison of the amino acid contents indicates more similarity among the light chains than the heavy chains of the three NT types, a similarity that agrees with our published partial amino acid sequences (first 13-18 residues) of these chains. Several (up to 9) different amino acid residues of the heavy chain (which is twice the size of the light chain) are present in double the number of corresponding residues in the light chain.     

Biochemical characterization of peptides from herpes simplex virus glycoprotein gC: loss of CNBr fragments from the carboxy terminus of truncated, secreted gC molecules.

A biochemical characterization of peptides from herpes simplex virus type 1 glycoprotein gC was carried out. We utilized simple micromethods, based on immunological isolation of biosynthetically radiolabeled gC, to obtain gC in pure form for biochemical study. CNBr fragments of gC were prepared, isolated, and characterized. These CNBr fragments were resolved into six peaks by chromatography on Sephacryl S-200 in 6 M guanidine hydrochloride. Only three of the CNBr fragments contained carbohydrate side chains, as judged from the incorporation of [14C]glucosamine. Radiochemical microsequence analyses were carried out on the gC molecule and on each of the CNBr fragments of gC. A comparison of this amino acid sequence data with the amino acid sequence predicted from the DNA sequence of the gC gene showed that the first 25 residues of the predicted sequence are not present in the gC molecule isolated from infected cells and allowed alignment of the CNBr fragments in the gC molecule. Glycoprotein gC was also examined from three gC mutants, synLD70, gC-8, and gC-49. These mutants lack an immunoreactive envelope form of gC but produce a secreted, truncated gC gene product. Glycoprotein gC from cells infected with any of these gC- mutants was shown to have lost more than one CNBr fragment present in the wild-type gC molecule. The missing fragments included the one containing the putative transmembrane anchor sequence. Glycoprotein gC from the gC-8 mutant was also shown, by tryptic peptide map analysis, to have lost more than five major arginine-labeled tryptic peptides arginine-labeled tryptic peptides present in the wild-type gC molecule and to have gained a lysine-labeled tryptic peptide not present in wild-type gC.     

Type VIII collagen from bovine Descemet's membrane: structural characterization of a triple-helical domain

Bovine corneal Descemet's membrane (DM) was subjected to limited pepsin digestion. Soluble native collagens were fractionated by differential salt precipitation, and a mixture of type V collagen and collagenous fragments with a chain Mr of 50,000 (50K) was obtained at a concentration of 1.5 M NaCl. Further purification of the 50K collagen by molecular sieve and high-performance liquid chromatography resulted in the isolation of two-non-disulfide-bonded polypeptides, 50K-A and 50K-B, which were susceptible to several neutral proteases, including bacterial collagenase. By the criteria of peptide mapping, amino acid composition, and N-terminal sequence analysis, 50K-A and 50K-B were structurally dissimilar, although both chains contained Gly-X-Y repeats. 50K-A and 50K-B were immunologically and structurally distinct from collagen type I, III, IV, V, and VI. Immunohistochemical studies of bovine ocular tissue showed preferential distribution of the collagen containing the 50K fragment in the DM, with a more disperse arrangement of apparently interconnecting fibrils in the corneal stroma. Type VIII collagen isolated from the culture medium of metabolically radiolabeled bovine corneal endothelial (BCE) cells and its pepsin-resistant Mr 50 000 domain(s) both cross-reacted with antisera to 50K polypeptides from the corneal DM. Additionally, the CNBr peptide maps of pepsin-resistant Mr 50 000 polypeptides of type VIII collagen isolated from BCE cells and bovine corneal DM were highly similar.(ABSTRACT TRUNCATED AT 250 WORDS)     

Human low-molecular-weight urinary urokinase. Partial characterization and preliminary sequence data of the two polypeptide chains

Low-molecular-weight urokinase (molecular weight 33100) was separated by analytical and preparative isoelectric focusing into five major subforms with isoelectric points between 8.7 and 9.6. These subforms are very similar in molecular weight, specific activity, amino acid composition and content of amino sugar and their N-terminal sequence constellation is identical. Low-molecular-weight urokinase consists of two polypeptide chains connected by a single disulfide bridge. The N-terminal region of the heavy chain (calculated Mr 30700) exhibits homology within the first 46 residues analyzed, with the known primary structure of other serine proteases. The mini chain (Mr 2426), whose complete sequence was determined, consists of 21 residues which show homology with the primary structure of the C-terminal region of the plasmin heavy chain. Based on sequence data and homology criteria with serine proteases a single-chain urokinase precursor is postulated having a peptide bond constellation between heavy and light chain region compatible with the requirements for serine protease activation.     

The covalent structure of calf skin type III collagen. I. The amino acid sequence of the amino terminal region of the alpha 1(III) chain (position 1--222).

The amino terminal 227 amino acid residues of the alpha 1(III) chain contain four CNBr peptides: alpha 1(III)CB3A (79 residues), CB3B, CB3C (6 residues each), CB7 (37 residues) and CB6 (99 residues). Fragmentation of the CNBr peptides was carried out using trypsin, chymotrypsin and the protease from Staphylococcus aureus V8. The fragments obtained were isolated by a combination of molecular sieve and ion exchange chromatography. The sequence analysis was performed according to the automated Edman degradation procedure.     

Amino acid sequences of the human kidney cathepsins H and L

The complete amino acid sequences of human kidney cathepsin H (EC 3.4.22.16) and human kidney cathepsin L (EC 3.4.22.15) were determined. Cathepsin H contains 230 residues and has an Mr of 25116. The sequence was obtained by sequencing the light, heavy and mini chain and the peptides produced by cyanogen bromide cleavage of the single-chain form of the enzyme. The glycosylated mini chain is a proteolytic fragment of the propeptide of cathepsin H. Human cathepsin L has 217 amino acid residues and an Mr of 23720. Its amino acid sequence was deduced from N-terminal sequences of the heavy and light chains and from the sequences of cyanogen bromide fragments of the heavy chain. The fragments were aligned by comparison with known sequences of cathepsins H and L from other species. Cathepsins H and L exhibit a high degree of sequence homology to cathepsin B (EC 3.4.22.1) and other cysteine proteinases of the papain superfamily.     

A 34-amino acid peptide of the third component of complement mediates properdin binding

In this study, a peptide of 34 amino acids from the Mr 40,000 C terminus alpha-chain fragment of C3 was found to mediate properdin (P) binding. Treatment of the Mr 40,000 fragment with CNBr generated one major Mr 17,000 fragment that was capable of binding P. Amino acid sequence data placed the Mr 17,000 fragment within residues 1385 to 1540 of the C3 sequence. After analyzing this sequence for highly conserved segments within the C3 from other species (which bind P) and segments of low similarity within human C4, mouse C5, and alpha 2-macroglobulin (which do not bind P), a 34-amino acid (1402 to 1435) peptide was synthesized. This synthetic peptide bound to P and inhibited its binding to C3b. In addition, it exhibited negative regulatory activity on the alternative pathway as it inhibited the lysis of rabbit erythrocytes by normal human serum. These results show that the P-binding site is located within the residues 1402 to 1435 of the C3 sequence.     

Localization of the human complement component C3 binding site on the IgG heavy chain

The location of the covalent binding site of the third component of complement (C3) on the IgG heavy chain was determined by sequence analysis of peptides generated by cyanogen bromide digestion of C3-IgG adducts. Activation of the alternative pathway by incubation of heat-aggregated human IgG1 with fresh normal human plasma formed covalent adducts of C3b-IgG. CNBr peptides of these adducts were transferred to a polyvinylidene difluoride membrane, and amino-terminal sequences were determined. A 40-kDa dipeptide containing the covalent bond was identified by labeling the free thiol group (generated during activation of the internal thioester of C3b) with iodo[1-14C]acetamide and analyzed by amino acid sequencing. The resulting double sequence suggested an adduct with NH2 termini at residue 938 (pro-C3 numbering) of C3 (75 residues NH2-terminal to the thioester) and residue 84 in the variable region of the IgG heavy chain. These results combined with results from hydroxylamine treatment (splits ester linkage between C3b and IgG) imply that this adduct peptide consists of a 22-kDa C3 fragment and an 18-kDa IgG fragment. Therefore, C3 binds covalently within the region extending from the last 20 residues of the variable region through the first 20 residues of CH2.     

Synthesis of Penicillins and Cephalosporins by Penicillin Acylase Entrapped in Fibres

Tryptic peptides from two cyanogen bromide (CNBr) fragments CB II and CB III of the Ala chain of ricin D were sequenced by manual Edman degradation. Chymotryptic or peptic peptides from the two fragments were isolated by Dowex 1 x 2 column chromatography to obtain overlaps for the tryptic peptides, and the complete amino acid sequences of fragments CB II and III were established. The amino acid residues in fragments CB II and CB III accounted for 75 and 45 residues, respectively, of 260 residues in the Ala chain.

These sequences together with the sequence of fragment CBI described in the preceding paper established the complete sequence of the 260 amino acid residues in the Ala chain. Some structural characteristics of the protein are also discussed.     

Amino acid sequence of the Bb fragment from complement Factor B. Sequence of the major cyanogen bromide-cleavage peptide (CB-II) and completion of the sequence of the Bb fragment.

The amino acid sequence of peptide CB-II, the major product (mol.wt. 30 000) of CNBr cleavage of fragment Bb from human complement Factor B, is given. The sequence was obtained from peptides derived by trypsin cleavage of peptide CB-II and clostripain digestion of fragment Bb. Cleavage of two Asn-Gly bonds in peptide CB-II was also found useful. These results, along with those presented in the preceding paper [Gagnon & Christie (1983) Biochem. J. 209, 51-60], yield the complete sequence of the 505 amino acid residues of fragment Bb. The C-terminal half of the molecule shows strong homology of sequence with serine proteinases. Factor B has a catalytic chain (fragment Bb) with a molecular weight twice that of proteinases previously described, suggesting that it is a novel type of serine proteinase, probably with a different activation mechanism.     

Amino acid sequence of the 203-residue fragment of the heavy chain of chicken gizzard myosin containing the SH1-type cysteine residue

A fluorescent fragment of Mr = 23,800 was obtained by the papain digestion of N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylene diamine (abbreviated as IAEDANS)-modified chicken gizzard myosin. The fragment was isolated by gel filtration on a Sephadex G-100 column in the presence of 5 M guanidine-HCl followed by anion exchange chromatography on a QAE Sephadex A-50 column. This fragment contained 203 amino acid residues which could be assigned as a COOH-terminal part of the S-1 heavy chain based on the homology with the known sequence of rabbit skeletal myosin fragment. The amino acid sequence was K-G-M-F-R-T-V- G-Q-L-Y-K-E-Q-L-T-K-L-M-T-T-L-R-N-T-N-P-N-F-V-R-C-I-I-P-N-H-E-K-R-A- G-K-L-D-A-H-L-V-L-E-Q-L-R-C-N-G-V-L-E-G-I-R-I-C-R-Q-G-F-P-N-R-I-V-F-Q- E-F-R-Q-R-Y-E-I-L-A-A-N-A-I-P-K-G-F-M-D-G-K-Q-A-C-I-L-M -I-K-A-L-E-L- D-P-N-L-Y-R-I-G-Q-S-K-I-F-F-R-T-G-V-L-A-H-L-E-E-E-R-D-L-K- I-T-D-V-I-I-A- F-Q-A-Q-C-R-G-Y-L-A-R-K-A-F-A-K-R-Q-Q-Q-L-T-A-M-K-V-I-Q-R-N-C-A -A-Y-L-K-L-R-N-W-Q-W-W-R-L-F-T-K-V-K-P-L-L-Q-V-T-R. The cysteine residue which was modified with IAEDANS was of the SH1 type (Cys-65). Pro-197 was suggested to be the NH2-terminal boundary of the alpha-helical coiled-coil rod sequence of gizzard myosin, based on the homology with the nematode sequence reported by MacLachlan and Karn (Proc. Natl. Acad. Sci. U.S. 80, 4253-4257 (1983)). Three different COOH-terminal peptides (Val-Lys-Pro-Leu-Leu-Gln-Val-Thr-Arg, Val-Lys-Pro-Leu-Leu-Gln, and Val-Lys-Pro-Leu-Leu) were isolated from the tryptic digest of this fragment.(ABSTRACT TRUNCATED AT 400 WORDS)