Cleavage of Rabbit Myelin Basic Protein by Thrombin

Rabbit myelin basic protein (BP) contains several Arg-X bonds with differing susceptibilities to thrombic cleavage as measured by the yields of the various cleavage products obtained under three different conditions. Under conditions where the thrombin-to-substrate ratio was very low (1 NIH unit/mg BP), the concentration of substrate was relatively low (4 mg BP/ml), and the incubation time was short (2 h), the rabbit BP was cleaved essentially completely and specifically at a single site, the Arg(95)-Thr(96) bond. The BPs of other species (beef, pig, guinea pig, rat) were similarly cleaved, no doubt because all have the same amino acid sequence in this region of the protein. Under conditions in which the enzyme-to-substrate ratio and the substrate concentration were higher (2 NIH units/mg BP, 8 mg BP/ml) and the incubation time was long (24 h), additional, partial cleavages occurred, principally at the Arg(43)-Phe(44) and Arg(128)-Ala(129) bonds, but with some cleavage at the Arg(31)-His(32) and Arg(63)-Thr(64) bonds as well. Under conditions in which all three variables were elevated (5 NIH units/mg peptide, 20 mg peptide/ml, 24 h), more extensive cleavage occurred at the above sites. In peptide (96-168), which we examined in detail, nearly complete cleavage of the Arg(128)-Ala(129) bond occurred, with partial cleavage at the unmethylated Arg(105)-Gly(106), Arg(111)-Phe(112), Arg(150)-Leu(151), and Arg(160)-Ser(161) bonds. The susceptibilities to cleavage of the Arg-X bonds in the BP can be explained with varying degrees of success in terms of the known specificity of thrombin. Cleavage of two of the bonds, Arg(128)-Ala(129) and Arg(160)-Ser(161), suggests the occurrence of a chain reversal or beta-turn in the sequence preceding the scissile bonds. Most cleavages of the BP with thrombin do not occur in the more hydrophobic regions; in particular, the hydrophobic region in the center of the molecule that includes the Phe-Phe(87-88) sequence is left intact.     

Enzymatic inactivation of bradykinin by rat brain neuronal perikarya

1. Bradykinin (Bk; Arg1-Pro2-Pro3-Gly4-Phe5-Ser6-Pro7-Phe8-Arg8) inactivation by bulk isolated neurons from rat brain is described. 2. Bk is rapidly inactivated by neuronal perikarya (4.2 +/- 0.6 fmol/min/cell body). 3. Sites of inactivating cleavages, determined by a kininase bioassay combined with a time-course Bk-product analysis, were the Phe5-Ser6, Pro7-Phe8, Gly4-Phe5, and Pro3-Gly4 peptide bonds. The cleavage of the Phe5-Ser6 bond inactivated Bk at least five fold faster than the other observed cleavages. 4. Inactivating peptidases were identified by the effect of inhibitors on Bk-product formation. The Phe5-Ser6 bond cleavage is attributed mainly to a calcium-activated thiol-endopeptidase, a predominantly soluble enzyme which did not behave as a metalloenzyme upon dialysis and was strongly inhibited by N-[1(R,S)-carboxy-2-phenylethyl]-Ala-Ala-Phe-p-aminobenzoate and endo-oligopeptidase A antiserum. Thus, neuronal perikarya thiol-endopeptidase seems to differ from endo-oligopeptidase A and endopeptidase 24.15. 5. Endopeptidase 24.11 cleaves Bk at the Gly4-Phe5 and, to a larger extent, at the Pro7-Phe8 bond. The latter bond is also cleaved by angiotensin-converting enzyme (ACE) and prolyl endopeptidase (PE). PE also hydrolyzes Bk at the Pro3-Gly4 bond. 6. Secondary processing of Bk inactivation products occurs by (1) a rapid cleavage of Ser6-Pro7-Phe8-Arg8 at the Pro7-Phe8 bond by endopeptidase 24.11, 3820ACE, and PE; (2) a bestatin-sensitive breakdown of Phe8-Arg9; and (3) conversion of Arg1-Pro7 to Arg1-Phe5, of Gly4-Arg9 to both Gly4-Pro7 and Ser6-Arg9, and of Phe5-Arg9 to Ser6-Arg9, Phe8-Arg9, and Ser6-Pro7, by unidentified peptidases. 7. A model for the enzymatic inactivation of bradykinin by rat brain neuronal perikarya is proposed.     

Structural and functional characterization of mutants of recombinant single-chain urokinase-type plasminogen activator obtained by site-specific mutagenesis of Lys158, Ile159 and Ile160

Single-chain urokinase-type plasminogen activator (scu-PA) is converted to urokinase by hydrolysis of the Lys158-Ile159 peptide bond. Site-directed mutagenesis of Lys158 to Gly or Glu yields plasmin-resistant mutants with a 10-20-fold reduced catalytic efficiency for the activation of plasminogen [Nelles et al. (1987) J. Biol. Chem. 262, 5682-5689]. In the present study, we have further evaluated the enzymatic properties of derivatives of recombinant scu-PA (rscu-PA), produced by site-directed mutagenesis of Lys158, Ile159 or Ile160, in order to obtain additional information on the structure/function relations underlying the enzymatic properties of the single- and two-chain u-PA moieties. [Arg158]rscu-PA (rscu-PA with Lys158 substituted with Arg) appeared to be indistinguishable from wild-type rscu-PA with respect to plasminogen-activating potential (catalytic efficiency k2/Km = 0.21 mM-1 s-1 versus 0.64 mM-1 s-1), conversion to active two-chain urokinase by plasmin (k2/Km = 0.13 microM-1 s-1 versus 0.28 microM-1 s-1), as well as its specific activity (48,000 IU/mg as compared to 60,000 IU/mg) and its fibrinolytic potential in a plasma medium (50% lysis in 2 h with 2.8 micrograms/ml versus 2.1 micrograms/ml). [Pro159]rscu-PA (Ile159 substituted with Pro) and [Gly159]rscu-PA (Ile159 converted to Gly) are virtually inactive towards plasminogen (k2/Km less than 0.004 mM-1 s-1). They are however converted to inactive two-chain derivatives by plasmin following cleavage of the Arg156-Phe157 peptide bond in [Pro159]rscu-PA and of the Lys158-Gly159 peptide bond in [Gly159]rscu-PA. [Gly158,Lys160]rscu-PA (with Lys158 converted to Gly and Ile160 to Lys) has a low catalytic efficiency towards plasminogen both as a single-chain form (k2/Km = 0.012 mM-1 s-1) and as the two-chain derivative (k2/Km = 0.13 mM-1 s-1) generated by cleavage of both the Arg156-Phe157 and/or the Lys160-Gly161 peptide bonds by plasmin. These findings suggest that the enzymatic properties of rscu-PA are critically dependent on the amino acids in position 158 (requirement for Arg or Lys) and position 159 (requirement for Ile). Conversion of the basic amino acid in position 158 results in a 10-20-fold reduction of the catalytic efficiency of the single-chain molecule but yields a fully active two-chain derivative. The presence of Ile in position 159 is not only a primary determinant for the activity of the two-chain derivative, but also of the single-chain precursor. Cleavage of the Arg156-Phe157 or the Lys160-Gly161 peptide bonds by plasmin yields inactive two-chain derivatives.     

A Proteinase from Germinated Barley : II. Hydrolytic Specificity of a 30 Kilodalton Cysteine Proteinase From Green Malt

The hydrolytic specificity of a 30 kilodalton cysteine proteinase purified from germinated barley (Hordeum vulgare L. cv Morex) was investigated using high performance liquid chromatography to characterize its hydrolysis of two small barley seed proteins, the α- and β-hordothionins. The reduced and pyridylethylated thionins were rapidly cleaved, resulting in the production of a limited number of peptides. Peptide bonds Gly9-Arg10, Cys 16-Arg17, Cys25-Ala26, and Thr34-Ser35 were most susceptible to hydrolysis, the peptide bonds Arg5-Ser6, Arg19-Gly20 in both thionins and Lys38-Cys39 in β-hordothionin and Cys29-Arg30 of α-hordothionin being broken at much slower rates. The hydrolysis patterns were highly reproducible from assay to assay and with various enzyme preparations. The specificity was apparently defined by the amino acids in the P₂ position, not those immediately adjacent to the susceptible bonds. The P₂ amino acid residues of the released peptides were always either leucine, valine, tyrosine, or pyridylethylcysteine. From these observations and from the rates of release of the various peptides, it appears that the barley 30 kilodalton endoproteinase has an S2 subsite that preferentially binds the leucine side chain: i.e. for hydrolyzing the peptide bond P₁-P₁′ in the general sequence NH₂—P₂-P₁-P₁′—COOH, the enzyme is selective for leucine and, to a lesser extent, valine and tyrosine at position P₂. The barley proteinase thus resembles two other cysteine proteinases, papain and Streptococcal proteinase, in its specificity.     

Molecular analysis of dibasic endoproteolytic cleavage signals.

Biologically active peptide hormones are synthesized from larger precursor proteins by a variety of post-translational processing reactions. To characterize these processing reactions further we have expressed preprogastrin in two endocrine cell lines and examined the molecular determinants involved in endoproteolysis at dibasic cleavage sites. The Gly93-Arg94-Arg95 carboxyl-terminal processing site of progastrin must be processed sequentially by an endoprotease, a carboxypeptidase, and an amidating enzyme to produce bioactive gastrin. For these studies the dibasic Arg94-Arg95 residues that serve as signals for the initiation of this processing cascade were mutated to Lys94-Arg95, Arg94-Lys95, and Lys94-Lys95. In the GH3 cells the Lys94-Arg95 mutation slightly diminished synthesis of carboxyl-terminally amidated gastrin, whereas in the MTC 6-23 cells this mutation had no effect on amidated gastrin synthesis. In contrast, both Arg94-Lys95 and Lys94-Lys95 mutations resulted in significantly diminished production of amidated gastrin in both cell lines. A specific hierarchy of preferred cleavage signals at this progastrin processing site was demonstrated in both cell lines, indicating that cellular dibasic endoproteases have stringent substrate specificities. Progastrins with the Lys94-Arg95 mutation in GH3 cells also demonstrated diminished processing at the Lys74-Lys75 dibasic site, thus single amino acid changes at one processing site may alter cleavage at distant sites. These studies provide insight into the post-translational processing and biological activation of not only gastrin but other peptide hormones as well.     

Tailoring structure-function properties of L-asparaginase: engineering resistance to trypsin cleavage

Bacterial L-ASNases (L-asparaginases) catalyse the conversion of L-asparagine into L-aspartate and ammonia, and are widely used for the treatment of ALL (acute lymphoblastic leukaemia). In the present paper, we describe an efficient approach, based on protein chemistry and protein engineering studies, for the construction of trypsin-resistant PEGylated L-ASNase from Erwinia carotovora (EcaL-ASNase). Limited proteolysis of EcaL-ASNase with trypsin was found to be associated with a first cleavage of the peptide bond between Lys53 and Gly54, and then a second cleavage at Arg206-Ser207 of the C-terminal fragment, peptide 54-327, showing that the initial recognition sites for trypsin are Lys53 and Arg206. Site-directed mutagenesis of Arg206 to histidine followed by covalent coupling of mPEG-SNHS [methoxypoly(ethylene glycol) succinate N-hydroxysuccinimide ester] to the mutant enzyme resulted in an improved modified form of EcaL-ASNase that retains 82% of the original catalytic activity, exhibits enhanced resistance to trypsin degradation, and has higher thermal stability compared with the wild-type enzyme.     

Inhibition mechanism of a peanut trypsin-chymotrypsin inhibitor, B-III: determination of the reactive sites for trypsin and chymotrypsin

Peanut inhibitor B-III was found to form two types of complexes with trypsin, T2I and TI, by gel filtration HPLC. Two cleaved peptide bonds, Arg(10)-Arg(11) and Arg(38)-Ser(39), in the trypsin modified inhibitor (TM-B-III*R*S) (J. Biochem. 93, 479-485 (1983] were resynthesized by the complex formation with 2 mol of trypsin. These results suggest that the two peptide bonds may be the reactive sites for trypsin. TM-B-III*R*S inhibited bovine trypsin as well as native B-III but had little chymotrypsin inhibitory activity. The two peptide bonds, Arg(10)-Arg(11) and Arg(38)-Ser(39), in B-III were cleaved partly by prolonged incubation with a catalytic amount of chymotrypsin. But gel filtration HPLC of the chymotrypsin-inhibitor complex showed the formation of only CI complex. Incubation of TM-B-III*R*S with an equimolar amount of chymotrypsin resulted in the resynthesis of only the Arg(10)-Arg(11) bond. These findings suggest that Arg(10)-Arg(11) may be a true reactive site for chymotrypsin. An inhibition mechanism of B-III against trypsin and chymotrypsin was proposed from the results obtained by the present studies.     

The importance of the N-terminal beta-turn in bradykinin antagonists

Three peptides, B-10148 (Lys-1-Lys0-Arg1-Pro2-Hyp3-Gly4-Igl5-Ser6- DF5F7-Oic8; where Hyp is trans-4-hydroxyproline, Igl is alpha-(2-indanyl)glycine, F5F is 2,3,4,5,6-pentafluorophenylalanine and Oic is (3aS,7aS)-octahydroindole-2-carboxylic acid), B-10206 (DArg0-Arg1-Pro2-Hyp3-Gly4-Igl5-Ser6-DF 5F7-Nc7G8-Arg9; where Nc7G is N-cycloheptylglycine) and B- 10284 (Arg1-Pro2-Pro3-Gly4-Phe5-Thr6-DTic7-Oic8- NH2; where Tic is 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid), were studied in detail by NMR spectroscopy in 60% CD3OH /40% H2O and modeled by a simulated annealing protocol to determine their solution structure. B-10148, an extremely potent BK B1 receptor antagonist with very high BK B2 receptor antagonist activity, despite lacking a C-terminal Arg, displayed an ideal type II beta-turn from Pro2 to Igl5, as well as a salt bridge between the guanidino group of Arg1 and the carboXylate group of Oic8. B-10206, the most potent B2 antagonist, also displayed an ideal type II beta-turn from Pro2 to Igl5 but secondary structure was not observed at the C-terminal end. The third peptide, B-10284, a des-Arg9 analog with a C-terminal amide and a very potent B2 antagonist, had no definite solution structure. The high activity of these peptides emphasizes the importance of the N-terminal beta-turn and the hydrophobic character at the C-terminus in determining the activity of bradykinin antagonists.     

Isolation of brain endopeptidases: influence of size and sequence of substrates structurally related to bradykinin.

Two thiol-activated endopeptidases with pH optima near pH 7.5 were isolated from the supernatant fraction of rabbit brain homogenates by DEAE-cellulose chromatography, gel filtration and isoelectrofocusing. Peptide bond hydrolysis was measured quantitatively by ion-exchange chromatography with an amino acid analyzer. Brain kininase A hydrolyzes the Phe5-Ser6 peptide bond in bradykinin (Bk), Arg1-Pro2-Pro3-Gly4-Phe5-Ser6-Pro7-Phe8-Arg9. It is isoelectric near pH 5.2 and has a molecular weight of approximately 71 000. The enzyme also hydrolyzes the Phe-Ser peptide bond in Lys-Bk, Met-Lys-Bk, des-Arg1-Bk, Lys9-Bk, Pro-Gly-Phe-Ser-Pro-Phe-Arg, and Gly-Pro-Phe-Ser-Pro-Phe-Arg, but does not hydrolyze (0.1%) this bond in des-Phe8-Arg9-Bk. Brain kininase B hydrolyzes the Pro7-Phe8 peptide bond in Bk. It is isoelectric at pH 4.9 and has a molecular weight of approximately 68 000. Brain kininase B also hydrolyzes the Pro-Phe bond in Lys-Bk, Met-Lys-Bk, Lys9-Bk, Ser-Pro-Phe-Arg, and Phe-Ser-Pro-Arg. Pretreatment of denatured kininogen with brain kininase A or B did not reduce the amount of trypsin-releasable Bk from this precursor protein, indicating that the Bk sequence, when part of a large protein, is not a substrate for either enzyme. However, kininase A and B hydrolyze the octadecapeptide Gly-Leu-Met-Lys-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg-Ser-Val-Gin-Val. The data show that a large part of the C-terminal portion of bradykinin is important for the brain kininase A activity and, for both enzymes, the size of the peptide and presumably the residues adjacent to the scissle bond are important in determining the rate of peptide bond hydrolysis by these endopeptidases.     

Proteolysis of non-phosphorylated and phosphorylated tau by thrombin

The microtubule-associated protein tau aggregates intracellularly by unknown mechanisms in Alzheimer's disease and other tauopathies. A contributing factor may be a failure to break down free cytosolic tau, thus creating a surplus for aggregation, although the proteases that degrade tau in brain remain unknown. To address this issue, we prepared cytosolic fractions from five normal human brains and from perfused rat brains and incubated them with or without protease inhibitors. D-Phenylalanyl-L-prolylarginyl chloromethyl ketone, a thrombin-specific inhibitor, prevented tau breakdown in these fractions, suggesting that thrombin is a brain protease that processes tau. We next exposed human recombinant tau to purified human thrombin and analyzed the fragments by N-terminal sequencing. We found that thrombin proteolyzed tau at multiple arginine and lysine sites. These include Arg(155)-Gly(156), Arg(209)-Ser(210), Arg(230)-Thr(231), Lys(257)-Ser(258), and Lys(340)-Ser(341) (numbering according to the longest human tau isoform). Temporally, the initial cleavage occurred at the Arg(155)-Gly(156) bond. Proteolysis of the resultant C-terminal tau fragment then proceeded bidirectionally. When tau was phosphorylated by glycogen synthase kinase-3beta, most of these proteolytic processes were inhibited, except for the first cleavage at the Arg(155)-Gly(156) bond. Furthermore, paired helical filament tau prepared from Alzheimer's disease brain was more resistant to thrombin proteolysis than following dephosphorylation by alkaline phosphatase. The results suggest a possible role for thrombin in proteolysis of tau under physiological and/or pathological conditions in human brains. They are consistent with the hypothesis that phosphorylation of tau inhibits proteolysis by thrombin or other endogenous proteases, leading to aggregation of tau into insoluble fibrils.     

Nonpeptide antagonists for kinin receptors

Kinins are a family of small peptides acting as mediators of inflammation and pain in the peripheral and central nervous system. The two main 'kinins' in mammals are the nonapeptide bradykinin (BK, Arg1-Pro2-Pro3-Gly4-Phe5-Ser6-Pro7-Phe8-Arg9) and the decapeptide kallidin (KD, [Lys0]-BK, Lys1-Arg2-Pro3-Pro4-Gly5-Phe6-Ser7-Pro8-Phe9- Arg10). Their biological actions are mediated by two distinct receptors, termed B1 and B2. Kinin B and B2 receptor antagonists may be useful drugs endowed with analgesic and anti-inflammatory properties, with potential use in asthma, allergic rhinitis and other diseases. The first nonpeptide kinin B2 receptor antagonist, WIN 64338, was reported in 1993. Despite its low selectivity, the compound provided a reference for pharmacological and modeling studies. Several quinoline and imidazo[1,2-a]pyridine derivatives have been shown by Fujisawa to possess high affinity and selectivity for kinin B2 receptors. Among them, FR 173657 displayed excellent in vitro and in vivo antagonistic activity, while FR 190997 emerged as the first nonpeptide agonist for B2 receptor. Two structurally related Fournier compounds were recently published. Other kinin B2 receptor ligands were obtained by rational design, through library screening or from natural sources. The only example of a nonpeptide kinin B1 receptor ligand has been reported in a patent by Sanofi.     

High-resolution NMR studies of fibrinogen-like peptides in solution: interaction of thrombin with residues 1-23 of the A alpha chain of human fibrinogen

The interaction of the following human fibrinogen-like peptides with bovine thrombin was studied by use of one- and two-dimensional NMR techniques in aqueous solution: Ala(1)-Asp-Ser-Gly-Glu-Gly-Asp-Phe(8)-Leu-Ala-Glu-Gly-Gly-Gly-Val-Arg(16 )- Gly(17)-Pro-Arg(19)-Val(20)-Val-Glu-Arg (F10), residues 1-16 of F10 (fibrinopeptide A), residues 17-23 of F10 (F12), residues 1-20 of F10 (F13), residues 6-20 of F10 with Arg(16) replaced by a Gly residue (F14), and residues 6-19 of F10 with Arg(16) replaced by a Leu residue (F15). At pH 5.3 and 25 degrees C, the Arg(16)-Gly(17) peptide bonds of both peptides F10 and F13 were cleaved instantaneously in the presence of 0.6 mM thrombin, whereas the cleavage of the Arg(19)-Val(20) peptide bonds in peptides F12, F13, and F14 took over 1 h for completion. On the basis of observations of line broadening, fibrinopeptide A was found to bind to thrombin. While resonances from residues Ala(1)-Glu(5) were little affected, binding of fibrinopeptide A to thrombin caused significant line broadening of NH and side-chain proton resonances within residues Asp(7)-Arg(16). There is a chain reversal within residues Asp(7)-Arg(16) such that Phe(8) is brought close to the Arg(16)-Gly(17) peptide bond in the thrombin-peptide complex, as indicated by transferred NOEs between the aromatic ring protons of Phe(8) and the C alpha H protons of Gly(14) and the C gamma H protons of Val(15). A similar chain reversal was obtained in the isolated peptide F10 at a subzero temperature of -8 degrees C. The titration behavior of Asp(7) in peptide F13 does not deviate from that of the reference peptide, N-acetyl-Asp-NHMe at both 25 and -8 degrees C, indicating that no strong interaction exists between Asp(7) and Arg(16) or Arg(19). Peptides with Arg(16) replaced by Gly and Leu, respectively, i.e., F14 and F15, were also found to bind to thrombin but with a different conformation, as indicated by the absence of the long-range NOEs observed with fibrinopeptide A. Residues Asp(7)-Arg(16) constitute an essential structural element in the interaction of thrombin with fibrinogen.     

Relaxation of smooth muscle by cardiodilatin/atrial natriuretic peptide is inhibited by cAMP-dependent phosphorylation

Cardiodilatins/atrial natriuretic peptides (CDD/ANP) exhibit a common amino acid sequence: Arg101-Arg102-Ser103-Ser104. Cyclic AMP-dependent phosphorylation of Ser104 of atrial peptides with [gamma-32P]ATP enables rapid identification of cardiac hormones. The biological activity of in vitro phosphorylated cardiodilatin (CDD-28/alpha-hANP) is dramatically altered compared to the unphosphorylated peptide: the vaso-relaxant effect of cardiodilatin 28 is inhibited upon phosphorylation.     

Neuropeptide-Metabolizing Peptidases in Neuro-2a Neuroblastoma and C6 Glioma Cells

Mouse Neuro-2a neuroblastoma and rat C6 glioma cloned cells were screened for neuropeptide-metabolizing peptidases using a kininase bioassay combined with a time-course bradykinin-product analysis, and a fluorimetric assay for prolyl endopeptidase. The complementary peptide products Arg1----Phe5/Ser6----Arg9 and Arg1----Pro7/Phe8-Arg9 were released during bradykinin (Arg1-Pro2-Pro3-Gly4-Phe5-Ser6-Pro7-Phe8-Arg9) inactivation by homogenates of Neuro-2a and C6 cells. The 1:1 stoichiometry of the complementary fragments and their high yields, at 10% bradykinin inactivation, demonstrated the sites of hydrolysis. The initial rate of Phe5-Ser6 bond cleavage was six-fold higher than that of the Pro7-Phe8 bond. These sites of cleavage can be attributed to enzymes similar to endopeptidase A (Phe5-Ser6) and prolyl endopeptidase (Pro7-Phe8) on the basis of the specificity and sensitivity to inhibitors of the kininase activity in Neuro-2a and C6 cell homogenates. Kininase and prolyl endopeptidase specific activities (fmol/min/cell) were 10.5 and 12.4 for Neuro-2a, and 1.5 and 2 for C6 homogenate, respectively. The recovery of kininase activity was 2.2-fold higher in the particulate than in the soluble (105,000 g for 1 h) neuronal fraction, whereas the amount of prolyl endopeptidase activity was about the same in both fractions. Kininase and prolyl endopeptidase activities in C6 cells were recovered mostly in the soluble fraction. Prolyl endopeptidase specific activity decreased 10-fold in serum-starved Neuro-2a cultured cells, with no change in activity in similarly treated C6 cells. In contrast, kininase specific activity in both cell types was essentially unaffected on serum-deprivation-induced differentiation.     

Structural characterization of peptides derived from prosomatostatins I and II isolated from the pancreatic islets of two species of teleostean fish: the daddy sculpin and the flounder

The primary structures of three peptides from extracts from the pancreatic islets of the daddy sculpin (Cottus scorpius) and three analogous peptides from the islets of the flounder (Platichthys flesus), two species of teleostean fish, have been determined by automated Edman degradation. The structures of the flounder peptides were confirmed by fast-atom bombardment mass spectrometry. The peptides show strong homology to residues (49-60), (63-96) and (98-125) of the predicted sequence of preprosomatostatin II from the anglerfish (Lophius americanus). The amino acid sequences of the peptides suggest that, in the sculpin, prosomatostatin II is cleaved at a dibasic amino acid residue processing site (corresponding to Lys61-Arg62 in anglerfish preprosomatostatin II). The resulting fragments are further cleaved at monobasic residue processing sites (corresponding to Arg48 and Arg97 in anglerfish preprosomatostatin II). In the flounder the same dibasic residue processing site is utilised but cleavage at different monobasic sites takes place (corresponding to Arg50 and Arg97 in anglerfish preprosomatostatin II). A peptide identical to mammalian somatostatin-14 was also isolated from the islets of both species and is presumed to represent a cleavage product of prosomatostatin I.     

Lysine can replace arginine 67 in the mediation of covalent attachment of FAD to histidine 71 of 6-hydroxy-D-nicotine oxidase

The requirements for FAD-attachment to His71 of 6-hydroxy-D-nicotine oxidase (6-HDNO) were investigated by site-directed mutagenesis. The following amino acid replacements were introduced into the sequence Arg67-Ser68-Gly69-Gly70-His71 of the 6-HDNO-polypeptide: 1) Arg67 was replaced with Ala (A1 mutant); 2) Ser68 was replaced with Ala (A2 mutant); and 3) Arg67 was replaced with Lys (K mutant). The substitution in mutant A2 had no effect on flavinylation, measured as [14C]FAD incorporation into apo-6-HDNO. Replacement of Arg67 with Ala prevented, but replacement with Lys permitted the flavinylation of His71. Mutant A1 showed no 6-HDNO activity, whereas the replacement of Ser with Ala in mutant A2 had only a slight effect on 6-HDNO activity. The substitution of Lys for Arg67, however, reduced the specific 6-HDNO activity in extracts of Escherichia coli cells expressing the mutant polypeptide from 50.3 to 17.5 milliunits/mg protein. It is concluded that a basic amino acid residue (Arg67 or Lys67) is required to mediate the attachment of FAD to His71, and while Lys can substitute for Arg67 in this function, it can only partially replace Arg67 in the enzyme reaction mechanism of 6-HDNO.     

Treatment of an encephalitogenic peptide from guinea pig myelin basic protein with alpha-protease and thermolysin. Isolation of fragments and determination of cleavage sites.

Two peptic fragments (residues 37-88 and 43-88) of guinea pig myelin basic protein which are capable of inducing experimental allergic encephalomyelitis in Lewis rats were cleaved to shorter fragments with alpha-protease (Crotalus atrox proteinase, EC 3.4.24.1) and thermolysin (EC 3.4.24.4). The fragments were isolated, purified, and identified by amino acid composition and NH2- and COOH-terminal residues. The time courses of the reactions, monitored by thin layer electrophoresis of the digests, showed that alpha-protease cleaves peptide (43-88) initially at the Pro(71)-Gln(72) bond, and that the product peptides are subsequently attacked at the Arg(63) -Thr(64), Ser(74)-Gln(75), Arg(78)-Ser(79), and Ser(76)-Gln(80) bonds. No significant cleavages occurred at the -Leu, -Val, and -Ala bonds. These results are in striking contrast to those obtained previously by others workers with other peptide substrates, where selective cleavage at hydrophobic residues occurred. Thermolysin was found to attack peptide (37-88) at the Phe(42)-Phe(43) bond very rapidly; the product peptides were subsequently attacked at the His(60)-Ala(61), Ser(38)-Ile(39)-Tyr(67)-Gly(68), and Pro(84)-Val(85) bonds. These cleavages are compatible with the known specificity of this enzyme. Several of the fragments prepared with these two enzymes, peptides (43-71), (61-88), (75-88), and (72-84) have been used in other studies to locate the encephalitogenic site in the parent peptic peptide.     

Limited Digestion of Guinea Pig Myelin Basic Protein and Its Carboxy-Terminal Fragment (Residues 89–169) with Staphylococcus aureus V8 Protease

Staphylococcus aureus V8 protease has been reported to have a strict specificity for cleavage of the Glu-X bond in ammonium bicarbonate (pH 7.9). With myelin basic protein and one of its major peptic fragments (residues 89-169) as substrates, selective cleavage of Asp(32)-Thr(33), Asp(37)-Ser(38), and Glu(118-Gly(119) bonds was observed, as well as the unusual cleavage of the Gly(127)-Gly(128) bond. The Asp-Glu and Glu-Asn bonds in the sequence of Gln-Asp-Glu-Asn-Pro(81-84) were resistant to V8 protease attack. The following peptides were identified as products of limited cleavage of basic protein by V8 protease: (1-32), (1-37), (33-169), (38-169), (33-118), (38-118), (33-127), (38-127), (119-169), and (128-169). Cleavage of the peptic peptide (89-169) yielded fragments (89-118), (89-127), (119-169), and (128-169). All peptides were identified by amino acid analysis, as well as NH2- and COOH-terminal analyses. Time course studies with basic protein showed that V8 protease initially attacked the bonds between Asp(32) and Thr(33) and Asp(37) and Ser(38). With peptide (89-169) the initial cleavage was between Glu(118) and Gly(119). Peptides (89-118) and (89-127) were encephalitogenic in the Lewis rat. The activity of these peptides in the rat confirms the presence of a minor encephalitogenic site in guinea pig basic protein. Peptide (89-127) was encephalitogenic in the guinea pig, as expected, because it contains the intact encephalitogenic site. V8 protease digestion of basic protein yields some interesting new fragments, not previously available for biologic studies.     

Domain-domain interactions of HtpG, an Escherichia coli homologue of eukaryotic HSP90 molecular chaperone.

In the present study, we investigated the domain structure and domain-domain interactions of HtpG, an Escherichia coli homologue of eukaryotic HSP90. Limited proteolysis of recombinant HtpG, revealed three major tryptic sites, i.e. Arg7-Gly8, Arg336-Glu337 and Lys552-Leu553, of which the latter two were located at the positions equivalent to the major cleavage sites of human HSP90alpha. A similar pattern was obtained by papain treatment under nondenaturing conditions but not under denaturing conditions. Thus, HtpG consists of three domains, i.e. Domain A, Met1-Arg336; domain B, Glu337-Lys552; and domain C, Leu553-Ser624, as does HSP90. The domains of HtpG were expressed and their interactions were estimated on polyacrylamide gel electrophoresis under nondenaturing conditions. As a result, two kinds of domain-domain interactions were revealed: domain B interaction with domain A of the same polypeptide and domain C of one partner with domain B of the other in the dimer. Domain B could be structurally and functionally divided into two subdomains, the N-terminal two-thirds (subdomain BI) that interacted with domain A and the C-terminal one-third (subdomain BII) that interacted with domain C. The C-terminal two-thirds of domain A, i.e. Asp116-Arg336, were sufficient for the binding to domain B. We finally propose the domain organization of an HtpG dimer.     

Generation of interleukin-8 by plasmin from AVLPR-interleukin-8, the human fibroblast-derived neutrophil chemotactic factor

Plasmin mainly cleaved the Arg5-Ser6 bond of Arg-Val-Leu-Pro-Arg-interleukin-8 (AVLPR-IL-8) produced by human dermal fibroblasts, which resulted in the conversion of AVLPR-IL-8 to IL-8 and the inactive pentapeptide, though a minor cleavage of AVLPR-IL-8 by plasmin at Lys8-Glu9 bond occurred.