Preparations of Ψ-peptide bond and peptide-aldehyde inhibitors of atrial granule serine proteinase, a candidate processing enzyme of pro-atrial natriuretic factor

Pseudo-peptide bond inhibitors (-bond inhibitors) and peptide-aldehyde inhibitors of atrial granule serine proteinase, the candidate processing enzyme of pro-atrial natrieuretic factor, are prepared in high yield and purity by novel synthetic routes. The -bond compounds retain essential residues for enzyme binding, but place the enzyme inhibition site in the midst of the peptide sequence. Thus, Bz-APR--LR and Bz-APR--SLRR can be considered readthrough inhibitors of atrial granule serine proteinase. The most potent -peptide, Bz-APR--SLRR (IC₅₀=250 M), is about fivefold less potent than the best peptide-aldehyde inhibitor (EACA-APR-CHO), and both the -bond and peptide-aldehyde compounds are competitive, reversible inhibitors of the enzyme. The -bond peptides containing two C-terminal Arg residues are three-to tenfold more potent than the analogous compounds containing only one C-terminal Arg residue, confirming the importance of both Arg residues in the enzyme processing recognition site. As expected, because of their moderate potencies, the -peptides are not useful affinity ligands for purification of atrial granule serine proteinase, but both peptide aldehydes are effective affinity ligands [Damodaran and Harris (1995),J. Protein Chem., this issue].Abbreviations AGSP atrial granule serine proteinase - ANF atrial natriuretic factor - Bz benzoyl - DIEA diisopropylethylamine - DIPCDI diisopropylcarbodiimide - DMF dimethylformamide - DMSO dimethylsulfoxide - EACA 6(e)-aminocaproic acid - EtOAc ethyl acetate - HEPES N-2-hydroxyethylpiperazine-N-propanesulfonic acid - HOBt N-hydroxybenzotriazole - HPLC high-performance liquid chrornatography - NMR nuclear magnetic resonance - PEG polyethylene glycol-3350 - PyBOP benzotriazole-1-yl-oxy-trispyrrolidino-phosphonium-hexafluorophospate - TEA triethylamine - TFA trifluoroacetic acid - THF tetrahydrofuran - TLC thin-layer chromatography - UV ultraviolet - pseudo-peptide bond -CH₂-NH-. Single-letter abbreviations are used to denote amino acids     

Cleavage specificity of a myofibril-bound serine proteinase from carp (Cyprinus carpio) muscle

Previously, we reported the purification and characterization of a myofibril-bound serine proteinase (MBP) from carp muscle (Osatomi K, Sasai H, Cao M-J, Hara K, Ishihara T. Comp Biochem Physiol 1997;116B:159–66). In the present study, the N-terminal amino acid sequence of the enzyme was determined, which showed high identity with those of other trypsin-like serine proteases. The cleavage specificity of MBP for dibasic and monobasic residues was investigated using various fluorogenic substrates and peptides. Analyses of the cleaved peptide products showed that the enzyme hydrolyzed peptides both at monobasic and dibasic amino acid residues. Monobasic amino acid residues were hydrolyzed at the carboxyl side; dibasic residues were cleaved either at the carboxyl side of the pair or between the two basic residues and the enzyme showed a cleavage preference for the Arg-Arg pair. Unexpectedly, MBP hydrolyzed lysyl-bradykinin and methionyl–lysyl–bradykinin at the carboxyl side of Gly fairly specifically and efficiently displaying a unique cleavage. Because MBP also degraded protein substrates such as casein and myofibrillar proteins, the substrate specificity of MBP appeared not to be strictly specific.     

Characterization of homogeneous atrial granule serine proteinase, a candidate processing enzyme of pro-atrial natriuretic factor.

We previously reported the discovery and partial characterization of bovine atrial granule serine proteinase, a candidate processing enzyme of pro-atrial natriuretic factor, which is associated with atrial granule membranes. We now report the physicochemical properties of electrophoretically homogeneous enzyme purified by a series of chromatography steps from a subcellular fraction enriched for atrial granules. The enzyme tends to associate during purification to higher molecular weight species, but SDS-PAGE analysis reveals a single polypeptide chain of molecular weight 70,000. The enzyme is activated 2-3 fold by Ca+2 and 1.5-fold by Mg+2 and is nearly 100% inhibited by Zn+2 or Co+2. Thus, the enzyme can be considered a calcium activated, neutral pH, serine proteinase. Based on the hydrolysis of numerous synthetic peptide substrates, the recognition sequence for the enzyme within the pro-hormone has been mapped to A96PRSLRR102; cleavage occurs at the Arg98-Ser99 bond yielding bioactive atrial natriuretic peptide directly from the pro-hormone. The doublet of basic amino acids is part of the recognition sequence but is not the primary cleavage site. It is our hypothesis that the processing site sequence acts as a recognition element for the endoproteinase and resides at the surface of the pro-hormone and thus contributes to the molecular basis for limited proteolysis.     

Purification and Characterization of Serine Proteinase 2 from Bacillus intermedius 3-19

A proteinase secreted in the late stationary phase was isolated from the culture fluid of Bacillus intermedius 3-19 by ion-exchange chromatography on CM-cellulose followed by FPLC on a Mono S column. The enzyme was completely inhibited by the serine proteinase inhibitors diisopropyl fluorophosphate and phenylmethylsulfonyl fluoride. The maximum proteolytic activity against the synthetic chromogenic substrate Z-Ala-Ala-Leu-pNA was observed at pH 9.0. The molecular weight of the enzyme is 28 kD and its isoelectric point is 9.2. We have also determined pH- and thermostability and Km and kcat of this proteinase. The enzyme has been classified as a thiol-dependent serine proteinase. N-Terminal amino acid sequence (10 residues) and amino acid composition of the protein were also determined. By the mode of hydrolysis of peptide bonds in the oxidized B-chain of insulin, this enzyme is similar to the thiol-dependent serine proteinase 1 from B. intermedius 3-19 secreted during vegetative growth.     

Characteristics of the Chromaffin Granule Aspartic Proteinase Involved in Proenkephalin Processing

Abstract: Proteolytic processing of neuropeptide precursors is required for production of active neurotransmitters and hormones. In this study, a chromaffin granule (CG) aspartic proteinase of 70 kDa was found to contribute to enkephalin precursor cleaving activity, as assayed with recombinant ([³⁵S]Met)preproenkephalin. The 70-kDa CG aspartic proteinase was purified by concanavalin A-Sepharose, Sephacryl S-200, and pepstatin A agarose affinity chromatography. The proteinase showed optimal activity at pH 5.5. It was potently inhibited by pepstatin A, a selective aspartic proteinase inhibitor, but not by inhibitors of serine, cysteine, or metalloproteinases. Lack of inhibition by Val-d -Leu-Pro-Phe-Val-d -Leu—an inhibitor of pepsin, cathepsin D, and cathepsin E—distinguishes the CG aspartic proteinase from classical members of the aspartic proteinase family. The CG aspartic proteinase cleaved recombinant proenkephalin between the Lys¹⁷²-Arg¹⁷³ pair located at the COOH-terminus of (Met)enkephalin-Arg⁶-Gly⁷-Leu⁸, as assessed by peptide microsequencing. The importance of full-length prohormone as substrate was demonstrated by the enzyme's ability to hydrolyze ³⁵S-labeled proenkephalin and proopiomelanocortin and its inability to cleave tri- and tetrapeptide substrates containing dibasic or monobasic cleavage sites. In this study, results provide evidence for the role of an aspartic proteinase in proenkephalin and prohormone processing.     

Atrial granules contain an amino-terminal processing enzyme of atrial natriuretic factor

At least three enzymes have been identified in atrial tissue homogenates that are capable of processing pro-atrial natriuretic factor to active atrial peptides. The atrial peptides possess potent natriuretic, diuretic, vasorelaxant, and hemodynamic properties, and their existence has implicated the mammalian heart as an endocrine organ. We have purified and characterized a serine proteinase (Mr approximately equal to 70,000) associated with atrial granules that preferentially hydrolyzes the Arg-Ser bond in the synthetic substrates Gly-Pro-Arg-Ser-Leu-Arg, benzoyl-Gly-Pro-Arg-Ser-Leu-Arg, and benzoyl-Gly-Pro-Arg-Ser-Leu-Arg-Arg-2-naphthylamide, the Arg-2-naphthylamide bond in the substrate benzoyl-Gly-Pro-Arg-2-naphthylamide, and the Arg-Ser bond in a 31-residue substrate (Gly96-Tyr126 peptide) corresponding to residues Arg98-Ser99 in pro-atrial natriuretic factor. The Gly96-Tyr126 peptide contains the putative processing site in pro-atrial natriuretic factor and the sequence for the bioactive peptides. Our results indicate that the minimum processing site sequence is -Gly-Pro-Arg-Ser-Leu-Arg-Arg- and that the Ser99-Tyr126 natriuretic peptide is the predominant hydrolytic product. After prolonged incubation or at high enzyme concentrations, the Ser103-Tyr126 natriuretic peptide may also be formed. The Ser103-Arg125 natriuretic peptide was only a very minor product. The doublet of basic amino acids is not the primary processing site in pro-atrial natriuretic factor, but their presence may influence cleavage at the single Arg residue "upstream." Our findings are consistent with the idea that the pro-protein and the processing enzymes are packaged into the secretory granule and in response to the proper stimulus, the pro-protein is processed to the active peptides, probably during the process of secretion. The processing pathway of pro-atrial natriuretic factor is discussed.     

Consensus sequence for processing of peptide precursors at monobasic sites

Many regulatory peptide precursors undergo post-translational processing at mono- and/or dibasic residues. Comparison of amino acids around the monobasic cleavage sites suggests that these cleavages follow certain sequence motifs and can be described as the rules that govern monobasic cleavages: (i) a basic amino acid it present at either 3, 5, or 7 amino acids N-terminal to the cleavage site, (ii) hydrophobic aliphatic amino acids (leucine, isoleucine, valine, or methionine) are never present in the position C-terminal to the monobasic amino acid at the cleavage site, (iii) a cysteine is never present in the vicinity of the cleavage site, and (iv) an aromatic amino acid is never present at the position N-terminal to the monobasic amino acid at the cleavage site. In addition to these rules, the monobasic cleavages follow certain tendencies: (i) the amino acid at the cleavage site tends to be predominantly arginine, (ii) the amino acid at the position C-terminal to the cleavage site tends to be serine, alanine or glycine in more than 60% of the cases, (iii) the amino acid at either 3, 5, or 7 position N-terminal to the cleavage site tends to be arginine, (iv) aromatic amino acids are rare at the position C-terminal to the monobasic amino acid at the cleavage site, and (v) aliphatic amino acids tend to be in the two positions N-terminal to and the two positions C-terminal to the cleavage site, except as noted above. When compared with a large number of sequence containing single basic amino acids, these rules and tendencies are capable of not only correctly predicting the processing sites, but also are capable of excluding most of the single basic sequences that are known to be uncleaved. Many or these rules can also be applied to correctly predict the dibasic and multibasic cleavage sites suggesting that the rules and tendencies could govern endoproteolytic processing at the monobasic, dibasic and multibasic sites.     

Purification and Characterization of Alkaline Serine Proteinase from Photosynthetic Bacterium,Rubrivivax gelatinosus KDDS1

In order to reduce the protein content of wastewater, photosynthetic bacteria producing proteinases were screened from wastewater of various sources and stocked in culture. An isolated strain, KDDS1, was identified as Rubrivivax gelatinosus, a purple nonsulfur bacterium that secretes proteinase under micro-aerobic conditions under light at 35°C. Molecular weight of the purified enzyme was estimated to be 32.5 kDa. The enzyme showed the highest activity at 45°C and pH 9.6, and the activity was completely inhibited by phenylmethyl sulfonyl fluoride (PMSF), but not by EDTA. The amino-terminal 24 amino acid sequence of the enzyme showed about 50% identity to those of serine proteinases from Pseudoalteromonas piscicida strain O-7 and Burkholderia pseudomallei. Thus, the enzyme from Rvi. gelatinosus KDDS1 was thought to be a serine-type proteinase. This was the first serine proteinase characterized from photosynthetic bacteria.     

Cloning and Characterization of the cDNA Encoding the Masquerade-like Serine Proteinase Homologue Gene of the Silkworm, Bombyx mori

ABSTRACT From Bombyx mori larvae, RT-PCR and cDNA library screening isolated masquerade-like serine proteinase homologue cDNA gene, proposed to be related to insect immunity and its characteristics were examined. The isolated gene is composed of 1.3 kb of nucleotide and 420 amino acid residues were encoded. According to the results of database search, the isolated gene showed high sequence homology with Holotrichia and Tenebrio's 45 kDa protein, Drosophila CG5390 gene. Moreover, it is composed of regulatory domain and catalytic domain, which is characteristic of serine proteinase that can be found in the insect immune reaction and embryonic development processes. Enzyme activation site by proteolytic cleavage and the sequence of three amino acids participate in the catalytic triad of enzyme and 14 cystein residues used in disulfide bridges are well conserved with the compared genes. The mRNA expression was increased following E. coli injection and constitutive expression was also observed before injection by Northern blot analysis.     

Inhibition of Serine Proteinases by P-Carbethoxyphenyl Esters of ϵ-Guanidino- And ϵ-Amino Caproic Acid: Thermodynamic and Molecular Modeling Study

Abstract

The inhibitory effect of the clinically used p-carbethoxyphenyl ester of ?-guanidino-caproic acid metha-nesulphonate (?-GCA-CEP) on the catalytic properties of human LYS⁷⁷-plasmin (EC 3.4.21.7), bovine factor Xa (EC 3.4.21.6), bovine α-thrombin (EC 3.4.21.5), ancrod (EC 3.4.21.28), crotalase (EC 3.4.21.30), bovine β-trypsin (EC 3.4.21.4), porcine pancreatic β-kallikrein-B (EC 3.4.21.39, human urinary kallikrein (EC 3.4.21.35) and the M_r 54,000 species of human urokinase (EC 3.4.21.31) was investigated (between pH 2.0 and 8.5, I = 0.1 M;T = 21 ? 0.5?C), and analyzed in parallel with that of the homologous derivative p-carbethoxyphenyl ?-amino-caproate hydro chloride (?-ACA-CEP). On lowering the pH from 5.5 to 3.0, values of the apparent dissociation inhibition constant (K_i) for ?-GCA. CEP and ?-ACA-CEP interaction with the serine proteinases considered increase, reflecting the acidic pK-shift upon inhibitor binding of a single ionizing group. Over the whole pH range explored, (i) ?-GCA-CEP interacts with bovine factor Xa and bovine α-thrombin with an higher affinity than that observed for ?-ACA-CEP binding; (ii) both inhibitors associate to bovine β-trypsin with the same affinity; and (iii) ?-ACA-CEP inhibits human Lys⁷⁷-plasmin and the M_r 54,000 species of human urokinase with an higher affinity than that reported for ?-GCA-CEP association, thus reflecting the known enzyme primary specificity properties. However, the affinity of ?-ACA-CEP for ancrod, crotalase, porcine pancreatic β-kallikrein-B and human urinary kallikrein, all of which preferably bind arginyl rather than lysyl side chains at the primary position of substrates and/or inhibitors, is paradoxically higher than that displayed by ?-GCA-CEP. By considering the amino acid sequences, the X-ray three-dimensional structures and/or the computer-generated molecular models of serine proteinase: inhibitor adducts, the observed binding behaviour of ?-GCA-CEP and ?-ACA-CEP to the enzymes considered has been related to the inferred stereochemistry of proteinase: inhibitor contact region(s).     

Effects of eel atrial natriuretic peptide on NaCl and water transport across the intestine of the seawater eel

Summary Eel atrial natriuretic peptide inhibited the serosa-negative transepithelial potential difference and short-circuit current, accompanied by a decrease in NaCl and water absorption across the seawater eel intestine. Similar effects were obtained after treatment with N-terminally truncated eel atrial natriuretic peptide (5–27), indicating that N-terminal amino acids are not essential for the action of eel atrial natriuretic peptide. Although mammalian atrial natriuretic peptides also inhibited the short-circuit current, a 100-fold higher concentration was reuired to obtain the same effect as with eel atrial natriuretic peptide, indicating that eel atrial natriuretic peptide is 100 times as potent in eel intestine as the mammalian atrial natriuretic peptides. Similarly, in mammalian atrial natriuretic peptide, the four N-terminal amino acids had no significant effects. However, when the C-terminal tyrosine was removed, the potency of rat atrial natriuretic peptide was lowered. Compared with the effects of acetylcholine, serotonin and histamine, eel atrial natriuretic peptide was the most potent inhibitor, with 100% inhibition at 10^-7 M; 50% inhibition was obtained at 10^-2 M in acetylcholine, and 30% inhibition in serotonin (10^-5 M) and histamine (10^-3 M). These inhibitory effects of eel atrial natriuretic peptide were not diminished even in the presence of tetradoxin, and were mimicked by 8-bromoguanosine 3,5-cyclic monophosphate. Based on these results, structure-activity relationships of eel atrial natriuretic peptide and a possible mechanism of action of eel atrial natriuretic peptide are discussed.Abbreviations 8BrcGMP 8-bromoguanosine 3,5-cyclic monophosphate - eANP eel atrial natriuretic peptide - hANP human atrial natriuretic peptide - 5-HT 5-hydroxytryptamine creatine sulphate - I _sc short-circuit current - PD transepithelial potential difference - rANP rat atrial natriuretic peptide - R _t tissue resistance - TTX tetrodotoxin     

Identification of a calcium-dependent microsomal proteinase responsible for monobasic cleavage of chicken proalbumin

The location and nature of the endoproteolytic activity involved in processing of proproteins has been studied in chicken liver microsomes. A membrane-bound, calcium-dependent proteinase was found to cleave chicken proalbumin with a monobasic cleavage site approx. 10-times faster than human proalbumin, which has a dibasic cleavage site. The mutant (human) proalbumin Christchurch (Arg(-1)----Gln), with a potential monobasic site, was not processed. The enzyme, which had a pH optimum of between 5.0 and 7.0, was not inhibited by serine or aspartyl proteinase inhibitors but was affected by some inhibitors of cysteine proteinases. The convertase was specifically inhibited by the reactive centre variant alpha 1-antitrypsin Pittsburgh, but not by normal alpha 1-antitrypsin.     

Purification, Properties, and N-Terminal Amino Acid Sequence of a Kallikrein-like Enzyme from the Venom of Lachesis muta rhombeata (Bushmaster)

Pit viper venoms contain multiple proteinases which cause considerable damage in tissues and systemic effects after envenomation. A proteinase, kallikrein-like enzyme, belonging to the serine group must play a very important role on systemic effects. The corresponding enzyme from Lachesis muta rhombeata venom was purified to homogeneity by a combination of isoelectrofocusing fractionation followed by one step of gel filtration HPLC. The enzyme focused with pI 5.0–6.5, it had a molecular mass of 32 kDa by gel filtration HPLC, had edematogenic activity, and induced a hypotensic effect in anesthetized rats. It exhibited strong N--tosyl-L-Arg methyl esterase (955.38 units/mg) and N-BZ-DL-Arg-pNA amidolytic (233.02 units/mg) activities, hydrolyzed tripeptide nitroanilide derivatives weakly or not at all, and cleaved selectively the A- and B- chains of fibrinogen, apparently leaving the Y-chain unaffected. The 30 N-terminal amino acid sequence of the L. m. rhombeata protein showed greatest identity (74% in 26 amino acids) with Crotalus viridis kallikrein-like protein, but significant similarities in sequence were observed with enzymes from other snake venoms and pig pancreatic kallikrein.     

Purification and characterisation of a novel 34,000-Mr cell-associated proteinase from the dermatophyte Trichophyton rubrum

Abstract A novel cell-associated proteinase was purified to homogeneity from cytoplasmic antigen preparations of Trichophyton rubrum by sequential isoelectric focusing and gel filtration chromatography. The enzyme exhibited relative molecular masses of 34,000- M _r (non-reduced sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE)), 15,000- M _r (reduced SDS-PAGE) and 37,000- M _r (substrate SDS-PAGE). It had a pH optimum of 7.5 and a p I of 4.5. The proteinase exhibited broad substrate specificity and it was strongly inhibited by the serine proteinase inhibitors phenylmethylsulfonyl fluoride and chymostatin. The N-terminal amino acid sequence of the 34,000- M _r proteinase shared 50% homology with the deduced amino acid sequence of a Coccidioides immitis wall-associated chymotrypsin-type serine proteinase. This is the first cell-associated proteinase to be purified and characterised from T. rubrum and it would appear to be related to the chymotrypsin-type serine proteinases, a class of enzymes that have rarely been isolated from fungi. The function of the proteinase remains speculative although it may play a role in the development and subsequent proliferation of the fungus in vivo.     

A new tyrosine-specific chymotrypsin-like and angiotensin-degrading serine proteinase from Vipera lebetina snake venom

Vipera lebetina venom contains different metallo- and serine proteinases that affect coagulation and fibrin(ogen)olysis. A novel serine proteinase from V. Lebetina venom having ChymoTrypsin Like Proteolytic activity (VLCTLP) was purified to homogeneity from the venom using Sephadex G-100sf, DEAE-cellulose, heparin-agarose and FPLC on Superdex 75 chromatographies. VLCTLP is a glycosylated serine proteinase with a molecular mass of 41926 Da. It reacts with N-acetyl-l-tyrosine ethyl ester (ATEE) but not with Suc-Ala-Ala-Pro-Phe-pNA or Suc-Ala-Ala-Pro-Leu-pNA. The complete amino acid sequence of the VLCTLP is deduced from the nucleotide sequence of the cDNA encoding this protein. The full-length cDNA sequence of the VLCTLP encodes open reading frame of 257 amino acid residues that includes a putative signal peptide of 18 amino acids, a proposed activation peptide of six amino acid residues and serine proteinase of 233 amino acid residues. VLCTLP belongs to the S1 (chymotrypsin) subfamily of proteases. The multiple alignment of its deduced amino acid sequence showed structural similarity with other serine proteases from snake venoms. The protease weakly hydrolyses azocasein, Aα-chain and more slowly Bβ-chain of fibrinogen. VLCTLP does not cleave fibrin and has no gelatinolytic activity. Specificity studies against peptide substrates (angiotensin I and II, oxidized insulin B-chain, glucagon, fibrinogen fragments etc.) showed that VLCTLP catalysed the cleavage of peptide bonds after tyrosine residues. VLCTLP is the only purified and characterized serine proteinase from snake venoms that catalyses ATEE hydrolysis. We detected ATEE-hydrolysing activities also in 9 different Viperidae and Crotalidae venoms.     

A new alkaline proteinase with pI 2.8 from alkalophilicBacillus sp.

A new serine alkaline proteinase (ALPase II) was purified from the culture broth of an alkalophilicBacillus sp. NKS-21. The molecular weight of ALPase II was estimated to be 32,000 by SDS-polyacrylamide gel electrophoresis. The enzyme had a very low isoelectric point (pI), which was determined to be 2.8. An optimum pH of this enzyme was 10.2. The specific activity was 0.28 katal/kg of protein for milk casein, 0.34 katal/kg for succinyl-l-alanyl-l-alanyl-l-prolyl-l-phenylalanyl-4-methyl-coumaryl-7-amide (Suc-Ala-Ala-Pro-Phe-MCA) and 8.5 katal/kg for succinyl-l-alanyl-l-alanyl-l-prolyl-l-phenylalanyl-p-nitroanilide (Suc-Ala-Ala-Pro-Phe-pNA).The substrate specificity of the alkaline proteinase was studied with the synthetic fluorogenic and chromogenic substrates. It was most favorable for the enzyme that the P₁ site of the substrate might be hydrophobic and bulky amino residue (Phe or Tyr). When the substrate contained four amino residues, the proteinase efficiently expressed its activity. The alkaline proteinase had higher specificity than those of the bacterial serine proteinases, subtilisins Carlsberg and BPN, and lower specificity than that of serine alkaline proteinase with pI 8.2 (ALPase I) obtained from the same bacteria NKS-21. ALPase II did not react with the anti-ALPase I antiserum.     

Extracellular serine proteinase from Bacillus licheniformis

The serine proteinase from B. licheniformis was purified by affinity chromatography on the sorbent obtained by attachment of p-(omega-aminomethyl)-phenylboronic acid via an amino group to CH-Sepharose. The use of this sorbent specific to the serine proteinases active sites resulted in a 35-fold purification of the enzyme with an apparent activity yield of 288%. Such a high activity yield is due to a removal of the enzyme inhibitors. The N-terminal sequence of B. licheniformis extracellular serine proteinase traced for 35 amino acid residues coincides with that of subtilisin Carlberg, a serine proteinase presumed to be secreted by a B. subtilis strain. Since the amino acid composition as well as the functional properties of these two enzymes did not reveal any noticeable differences, it was assumed that both proteinases are very similar, if not identical. This conclusion leads to reconsideration of the existing concept on an extremely fast rate of subtilisin evolution. Three multiple forms of B. licheniformis extracellular serine proteinase were found to differ only in their net charges, presumably as a result of partial deamidation of Asn or Gln residues within their structure.     

Identification of a peptidase which processes atrial natriuretic factor precursor to its active form with 28 amino acid residues in particulate fractions of rat atrial homogenate

With the objective of identifying specific peptidase responsible for the processing of atrial natriuretic factor precursor pro-ANF to the circulating active form ANF (99-126), a fluorometric assay method was devised using synthetic fluorogenic substrate Boc-Ala-Gly-Pro-Arg-MCA(methylcoumarinamide) which contains the amino acid sequence immediately adjacent to the arginyl peptide bond which is cleaved in the natural processing of pro-ANF. A protease which selectively cleaves this bond and produces the natural circulating peptide was identified in the particulate fraction of rat atrial homogenate and was solubilized by 1.6 M KCl. It was partially purified by affinity chromatography heparin-agarose column and was shown to be a serine protease. Its reaction product with natural pro-ANF was identified as ANF (99-126) containing 28 amino acid residues.