Hormone processing and membrane-bound proteinases in yeast.

A search for maturating peptidases of the precursor protein of the mating hormone (pheromone) alpha-factor of Saccharomyces cerevisiae was performed using short model peptides representing those sequences of the precursor protein, where cleavage is thought to occur in vivo. This search was done in a mutant lacking several of the unspecific vacuolar peptidases. The chromogenic peptide Cbz-Tyr-Lys-Arg-4-nitroanilide led to the detection of a membrane-bound enzyme called proteinase yscF. Cleavage of the synthetic peptide derivative occurs after the basic amino acid pair, a proposed signal for hormone processing. Optimum pH for the reaction is 7.2. The enzyme does not cleave after single basic amino acid residues indicating that it is distinct from trypsin-like proteinases. Proteolytic activity is enhanced by Triton X-100. The enzyme is strongly inhibited by EGTA, EDTA and mercurials but insensitive to phenylmethylsulfonyl fluoride. The enzyme activity is strongly dependent on Ca2+ ions. In a mutant (kex2), which accumulates an over-glycosylated alpha-factor precursor, no proteinase yscF activity can be found. Membrane-bound peptidase activity possibly involved in removal of the arginyl and lysyl residues remaining at the carboxy terminus of the alpha-factor pheromone peptide after the initial cut of the precursor molecule could be identified by using the model peptides Cbz-Tyr-Lys-Arg and Cbz-Tyr-Lys.     

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.     

Specific cleavage of glucosephosphate isomerases at cysteinyl residues using 2-nitro-5-thiocyanobenzoic acid: analyses of peptides eluted from polyacrylamide gels and localization of active site histidyl and lysyl residues

Specific chemical cleavage of human placental and porcine muscle glucosephosphate isomerases at three amino peptide bonds of cysteinyl residues with 2-nitro-5-thiocyanobenzoic acid was achieved. Four primary peptides were generated from the cyanylated human glucosephosphate isomerase, indicating the quantitative cleavage of this enzyme. Four primary plus six overlap peptides were obtained from the cleavage of the swine muscle enzyme. The peptides were separated by SDS-polyacrylamide gel electrophoresis and eluted from the gels. Amino acid and carboxyl terminal analyses of the eluted peptides have permitted the alignment of these peptides with respect to the native polypeptide chain. The analysis of the enzyme which had been specifically covalently labeled at the essential lysine and histidine residues of the active center revealed that the active-site histidine and lysine residues are located on two distinct peptides with molecular weights of 27,500 and 14,000, respectively.     

Some Characteristics of Hydrolysis of Synthetic Substrates and Proteins by the Alkaline Proteinase from Aspergillus sojae

The specificity of the alkaline proteinase from Aspergillus sojae was investigated. In the specificity studies with synthetic substrates, the enzyme hydrolyzed the peptide linkages involving the carboxyl group of leucine, tyrosine, phenylalanine, arginine and lysine. In the hydrolysis of natural proteins, the enzyme liberated relatively large peptides and traces of free amino acids, suggesting that the enzyme is of a typical endo-type.

N- and C-Terminal amino acid residues appearing during time course digestion of various proteins were determined. Considering the influence of amino acid composition of substrates on the frequencies of appearance of the terminal amino acids, it was estimated that the susceptibility of peptide bonds of substrate to the enzyme depends mainly on the carboxyl side residues, and, to far less extent, on the amino side residues of the peptide bonds. The enzyme showed relatively high specificity for lysine, tyrosine, histidine, arginine and phenylalanine residues at the carboxyl side of the susceptible linkages.     

Mapping of human plasma kallikrein active site by design of peptides based on modifications of a Kazal-type inhibitor reactive site

Human plasma kallikrein (huPK) is a proteinase that participates in several biological processes. Although various inhibitors control its activity, members of the Kazal family have not been identified as huPK inhibitors. In order to map the enzyme active site, we synthesized peptides based on the reactive site (PRILSPV) of a natural Kazal-type inhibitor found in Cayman plasma, which is not an huPK inhibitor. As expected, the leader peptide (Abz-SAPRILSPVQ-EDDnp) was not cleaved by huPK. Modifications to the leader peptide at P'1, P'3 and P'4 positions were made according to the sequence of a phage display-generated recombinant Kazal inhibitor (PYTLKWV) that presented huPK-binding ability. Novel peptides were identified as substrates for huPK and related enzymes. Both porcine pancreatic and human plasma kallikreins cleaved peptides at Arg or Lys bonds, whereas human pancreatic kallikrein cleaved bonds involving Arg or a pair of hydrophobic amino acid residues. Peptide hydrolysis by pancreatic kallikrein was not significantly altered by amino acid replacements. The peptide Abz-SAPRILSWVQ-EDDnp was the best substrate and a competitive inhibitor for huPK, indicating that Trp residue at the P'4 position is important for enzyme action.     

Bitter Peptides in Cow Milk Casein Digests with Bacterial Proteinase

Isolation and determination of amino acid sequence of the bitter peptides formed in the digestion of cow milk casein with alkaline proteinase of Bacillus subtilis were investigated. The casein digest with the enzyme was extracted with butanol and the extracted bitter peptides were fractionally purified by treating with several other organic solvents followed by subjecting to chromatography and gel-filtration. The amino acid sequence of one of the bitter peptides was determined as follows: Arg-Gly-Pro-Pro-Phe-Ileu-Val. Liberation of N-terminal Arg with trypsin or bacterial aminopeptidase did not affect the bitterness. Also, splitting off of Val and Ileu or Ileu-Val at the C-terminus by carboxypeptidase, or a bacterial neutral proteinase gave no influence on the bitterness. However, liberation of Arg and Gly from the peptide with bacterial aminopeptidase gave rise to a non bitter peptide.     

Proteinase Enzyme System of Lactic Streptococci III. Substrate Specificity of Streptococcus lactis Intracellular Proteinase

The substrate specificity of an intracellular proteinase from Streptococcus lactis was investigated in an effort to understand the role of the enzyme in the cell. Peptides in which the N-terminal residue was glycine were not hydrolyzed by the enzyme (exceptions were glycyl-alanine, glycyl-aspartic acid, and glycyl-asparagine), but the peptide was hydrolyzed if the N-terminal residue was alanine. The enzyme also showed activity toward peptides containing aspartic acid or asparagine. Hydrolysis of only the peptide bonds of alanyl, aspartyl, or asparaginyl residues was confirmed by the action of the enzyme on oxidized bovine ribonuclease A- and B- chain insulin. The N-terminal residues of the peptide fragments liberated were identified. The enzyme attacked both substrates only at alanyl, aspartyl, and asparaginyl residues, releasing these as free amino acids. In addition to alanine, aspartic acid, and asparagine, certain other amino acids were liberated from ribonuclease A, but these were accounted for by the relation of their position to alanine, aspartic acid, and asparagine residues.     

The primary structure of Aspergillus niger acid proteinase A

The complete amino acid sequence of the acid proteinase A, a non-pepsin type acid proteinase from the fungus Aspergillus niger var. macrosporus, was determined by protein sequencing. The enzyme was first dissociated at pH 8.5 into a light (L) chain and a heavy (H) chain, and the L chain was sequenced completely. Further sequencing was performed with the reduced and pyridylethylated or aminoethylated derivative of the whole protein, using peptides obtained by digestions with Staphylococcus aureus V8 protease, trypsin, chymotrypsin, and lysylendopeptidase. The location of the two disulfide bonds was determined by analysis of cystine-containing peptides obtained from a chymotryptic digest of the unmodified protein. These results established that the protein consists of a 39-residue L chain and a 173-residue H chain that associate noncovalently to form the native enzyme of 212 residues (Mr 22,265). This is, to our knowledge, the first time that such a protein with a rather short peptide chain associated noncovalently has been found. No sequence homology is found with other acid or aspartic proteinases, except for Scytalidium lignicolum acid proteinase B, an enzyme unrelated to pepsin by sequence, which has about 50% identity with the present enzyme. These two enzymes, however, are remarkably different from each other in some structural features.     

Purification and properties of a bovine brain thyrotropin-releasing-factor deamidase. A post-proline cleaving enzyme of limited specificity

A bovine brain thyrotropin-releasing-factor (thyroliberin) deamidase has been purified 1100-fold to apparent homogeneity. Molecular weight estimates by gel filtration and sodium dodecylsulfate gel electrophoresis indicate that the enzyme consists of a single polypeptide chain of molecular weight of about 62 000-65 000. The enzyme is inactivated by sulfhydryl blocking agents. Serine proteinase inhibitors, phenylmethanesulfonyl fluoride and benzamidine, have no effect. Besides thyroliberin, the enzyme hydrolyzes peptide bonds involving the carboxyl group of proline residues in luliberin, tuftsin, angiotensin II, melanotropin, and neurotensin. Oxytocin, vasopressin, and bradykinin are not cleaved; they are, however, strong competitive inhibitors of thyroliberin deamidation. The specificity studies indicate that the enzyme is a "post-proline cleaving enzyme" which hydrolyzes peptides of the general structure, Yaa-Pro-Xaa, in which Xaa = amino acid, peptide, or amide (not Pro), and Yaa = N-blocked basic amino acid or a peptide sequence in which the C-terminal residue (i.e. the residue prior to Pro) is a basic amino acid such as His, Lys, or Arg. The enzyme is compared to other post-proline cleaving enzymes.     

Identification of calmodulin isoform-specific binding peptides from a phage-displayed random 22-mer peptide library

Plants express numerous calmodulin (CaM) isoforms that exhibit differential activation or inhibition of CaM-dependent enzymes in vitro; however, their specificities toward target enzyme/protein binding are uncertain. A random peptide library displaying a 22-mer peptide on a bacteriophage surface was constructed to screen peptides that specifically bind to plant CaM isoforms (soybean calmodulin (ScaM)-1 and SCaM-4 were used in this study) in a Ca2+-dependent manner. The deduced amino acid sequence analyses of the respective 80 phage clones that were independently isolated via affinity panning revealed that SCaM isoforms require distinct amino acid sequences for optimal binding. SCaM-1-binding peptides conform to a 1-5-10 ((FILVW)XXX(FILV) XXXX(FILVW)) motif (where X denotes any amino acid), whereas SCaM-4-binding peptide sequences conform to a 1-8-14 ((FILVW)XXXXXX(FAILVW)XXXXX(FILVW)) motif. These motifs are classified based on the positions of conserved hydrophobic residues. To examine their binding properties further, two representative peptides from each of the SCaM isoform-binding sequences were synthesized and analyzed via gel mobility shift assays, Trp fluorescent spectra analyses, and phosphodiesterase competitive inhibition experiments. The results of these studies suggest that SCaM isoforms possess different binding sequences for optimal target interaction, which therefore may provide a molecular basis for CaM isoform-specific function in plants. Furthermore, the isolated peptide sequences may serve not only as useful CaM-binding sequence references but also as potential reagents for studying CaM isoform-specific function in vivo.     

Schistosoma mansoni: purification and characterization of the major acidic proteinase from adult worms

We report purification of the major digestive proteinase from adult worms of Schistosoma mansoni. This enzyme is a thiol proteinase with a pH optimum of 5 and is activated by thiol reagents. It was purified 300-fold using a combination of gel chromatography and chromatofocusing. It readily hydrolyzed hemoglobin with an apparent Km of 0.29 microM and a specific activity of 27 micrograms degraded/min/mg enzyme at 37 C. Peptides with positively charged amino acids were preferentially cleaved. The enzyme degraded Boc-Arg-Arg-7-amino-4-methyl coumarin with a kcat/Km of 9083 M-1 sec-1. Lengthening the peptide chain to 3 amino acids or substituting glycine for the amino terminal arginine resulted in decreased activity. The enzyme was inhibited by chloromethylketone-derivatized peptides of similar sequence and by leupeptin. The purified proteinase exhibits microheterogeneity in different preparations with forms ranging in molecular weight from 30,000 to 35,000, and pI 5.7-6.0.     

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.     

A diagonal electrophoretic method for selective purification of methionine peptides

1. A method is described that selectively purifies methionine peptides from enzymic digests of a protein. The peptides, after paper electrophoresis, are treated on paper with iodoacetamide at acid pH. This specifically converts methionine residues into their sulphonium salts. When the paper is submitted to electrophoresis at right angles to the original direction, the carbamoylmethylmethionine peptides emerge from an undifferentiated diagonal. 2. Heating at neutral pH converts carbamoylmethylmethionine into homoserine and thereby specifically cleaves the peptides. 3. The effect of the modifications on amino acid composition and sequence analyses of the peptides was studied. 4. When the method was applied to a tryptic digest of S-aminoethyl-chymotrypsinogen A, two peptides were selectively purified that had the expected amino acid sequence.     

Purification, characterization and gene cloning of a novel glutamic acid-specific endopeptidase from Staphylococcus aureus ATCC 12600.

Twenty strains of Staphylococcus aureus from ATCC type cultures and strains found in clinical studies were cultivated, and their endopeptidase activity specific for glutamic acid was surveyed using benzyloxycarbonyl-Phe-Leu-Glu-p-nitroanilide (Z-Phe-Leu-Glu-pNA) as a substrate. The activity was found in two of the strains, ATCC 12600 and ATCC 25923. A glutamic acid-specific proteinase, which we propose to call SPase, was purified from the culture filtrate of S. aureus strain ATCC 12600 by a series of column chromatographies on DEAE-Sepharose twice and on Sephacryl S-200. A single band was observed on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of the purified SPase. The molecular weight of the proteinase was estimated to be 34000 by SDS-PAGE. When synthetic peptides and oxidized insulin B-chain were used as substrates, SPase showed the same substrate specificity as V8 proteinase, EC 3.4.21.9, which specifically cleaves peptide bonds on the C-terminal side of glutamic acid and aspartic acid. Examination with p-nitroanilides of glutamic acid and aspartic acid as substrates, however, revealed that both proteinases are highly specific for a glutamyl bond in comparison with an aspartyl bond. To elucidate the complete primary structure of SPase, its gene was cloned from genomic DNA of S. aureus ATCC 12600, and the nucleotide sequence was determined. Taking the amino acid sequence of SPase from the NH2-terminus to the 27th residue into consideration, the clones encode a mature peptide of 289 amino acids, which follows a prepropeptide of 68 residues. SPase was confirmed to be a novel endopeptidase specific for glutamic acid, being different from V8 proteinase which consists of 268 amino acids.     

Amino acid sequence of an active fragment of potato proteinase inhibitor IIa.

The complete amino acid sequence of an active fragment of potato proteinase inhibitor IIa has been established by the Edman degradation procedure and the carboxypeptidase technique. Sequence analyses were carried out on the reduced and carboxymethylated active fragment and its tryptic peptides. To aid in the alignment of some tryptic peptides, the partial sequences of two fragments obtained by selective tryptic cleavage of the reactive site peptide bond of inhibitor IIa at acidic pH, with subsequent reduction and carboxymethylation, were also analyzed. The active fragment consisted of 45 amino acid residues including 6 half-cystine residues. Degradation of the intact active fragment by subtilisin [EC 3.4.21.14.] at pH 6.5. yielded 3 cystine-containing peptides. Sequence analyses of these peptides revealed that the 3 disulfide linkages were located between Cys(10) and Cys(24), Cys(14) and Cys(35), and Cys(20) and Cys(43). The reactive site peptide bond of inhibitor IIa, a Lys-Ser bond, was located between positions 32 and 33 of the active fragment. The overall sequence of the active fragment was quite different from those of potato chymotrypsin inhibitor I (subunit A) and potato carboxypeptidase inhibitor.     

Purification and characterization of a cysteine endopeptidase in cotyledons of germinated mung bean seeds

A cysteine endopeptidase (EC 3.4.22.-) present in cotyledons of mung bean (Vigna radiata) seedlings was purified to homogeneity, as judged by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). This proteinase has an apparent molecular mass of 33 kilodaltons as estimated by SDS-PAGE and belongs to the class of cysteine proteinases as judged by the effects of various proteinase inhibitors on the activity of the enzyme. When proangiotensin is used as a substrate, the enzyme preferentially hydrolyzes the peptide bonds formed by the amino group of Leu or lle in this oligopeptide chain; for the enzyme to cleave those bonds, peptide sequences consisting of at least three amino acid residues on the amino side of Leu or lle must be present. The proteinase readily digests globulin present in mung bean cotyledons to smaller polypeptides.     

Specificity of Proteinase K from Tritirachium album Limber for Synthetic Peptides

The specificity of proteinase K from Tritirachium album Limber was determined using various synthetic peptide substrates. The esterase activity against N-acylated amino acid esters indicated that the enzyme is primarily specific against aromatic or hydrophobic amino acid residues at the carboxyl side of the splitting point. Secondary interaction for hydrolysis was also studied using peptide esters or others, which showed that the enzyme activity is markedly promoted by elongating the peptide chain to the N-terminal from the splitting point. Thus, peptide chloromethyl ketone derivatives such as Cbz-Ala-Gly-PheCH₂Cl inactivated the enzyme activity markedly.     

Putative N-terminal splitting enzyme of amyloid A4 peptides is the multicatalytic proteinase, ingensin, which is widely distributed in mammalian cells

The main characteristic changes observed in Alzheimer's disease (AD) are the presence of neurofibrillary tangles and the deposition of amyloid A4 peptides. The most abundant amyloid A4 peptide species in AD (which we tentatively named A4') is composed of 39 amino acids, which is devoid of the 3 N-terminal amino acids, Asp-Ala-Glu, of the originally reported A4 peptide. We synthesized a model peptide substrate, Suc-Ala-Glu-methylcoumarinamide (MCA), to identify the proteinase that splits the A4' peptide. DEAE-cellulose column chromatography of rat liver and porcine brain extracts showed that only one peak material digested the synthetic substrate at pH 8. The results for the final preparation indicate that the Suc-Ala-Glu-MCA-degrading enzyme is a high-molecular-mass proteinase, with a molecular mass of above 500,000, and is composed of several low-molecular-mass subunits. These results suggest that a non-lysosomal multicatalytic proteinase (we named this enzyme ingensin (ingens = large in Latin). Ishiura, S. et al. (1985) FEBS Lett. 189, 119-123) catalyzes the above reaction. Antiserum against the purified multicatalytic proteinase, ingensin, crossreacted with the purified Suc-Ala-Glu-MCA-degrading proteinase. It is likely that ingensin shows a similar action toward amyloid precursor protein (APP) in vivo.     

Identification of endothelin converting enzyme in bovine lung membranes using a new fluorogenic substrate.

An enzyme partially purified from bovine lung membranes appears to be endothelin converting enzyme (ECE). This enzyme specifically cleaves big endothelin-1 (big ET-1) at the proper site, between Trp21 and Val22, with maximum activity at pH 7.5 and with a Km of roughly 3 microM, to produce endothelin-1 (ET-1) and C-terminal peptide (CTP). This same enzyme hydrolyzes the fluorogenic substrate succinyl-Ile-Ile-Trp-methylcoumarinamide to release the highly fluorescent 7-amino-4-methylcoumarin. The peptide derivative has the same amino acid sequence as big ET-1 and is a good substrate with a Km of about 27 microM. This enzyme is a metalloproteinase. It is not inhibited by five common proteinase inhibitors (pepstatin A, PMSF, NEM, E-64 and thiorphan) but it is inhibited by phosphoramidon and chelating compounds. The apoenzyme is restored to nearly full activity by a zinc-EDTA buffer with pZn = 13.     

Structural Investigations and Identification of the Extracellular Bacteriolytic Endopeptidase L1 from Lysobacter sp. XL1

The N-terminal amino acid sequence (23 amino acid residues) and the amino acid composition of the extracellular bacteriolytic enzyme L1 of 21 kD from the bacterium Lysobacter sp. XL1 have been determined. The enzyme was hydrolyzed by trypsin, the resulting peptides were isolated, and their primary structures were determined. A high extent of homology (92%) of the N-terminal amino acid sequence and the primary structure of isolated peptides of the enzyme L1 (62 amino acid residues or 31% of protein sequence) to the corresponding sites of alpha-lytic proteinases (EC 3.4.21.12) of Lysobacter enzymogenes and Achromobacter lyticus was found. These data allowed identification of the endopeptidase L1 of Lysobacter sp. XL1 as alpha-lytic proteinase EC 3.4.21.12.