Purification and characterization of protease So, a cytoplasmic serine protease in Escherichia coli

A new cytoplasmic endoprotease, named protease So, was purified to homogeneity from Escherichia coli by conventional procedures with casein as the substrate. Its molecular weight was 140,000 when determined by gel filtration on Sephadex G-200 and 77,000 when estimated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Thus, it appears to be composed of two identical subunits. Protease So had an isoelectric point of 6.4 and a K(m) of 1.4 muM for casein. In addition to casein, it hydrolyzed globin, glucagon, and denatured bovine serum albumin to acid-soluble peptides but did not degrade insulin, native bovine serum albumin, or the "auto alpha" fragment of beta-galactosidase. A variety of commonly used peptide substrates for endoproteases were not hydrolyzed by protease So. It had a broad pH optimum of 6.5 to 8.0. This enzyme is a serine protease, since it was inhibited by diisopropyl fluorophosphate and phenylmethylsulfonyl fluoride. Although it was not inhibited by chelating agents, divalent cations (e.g., Mg(2+)) stabilized its activity. Protease So was sensitive to inhibition by N-tosyl-l-phenylalanine chloromethyl ketone but not by N-tosyl-l-lysine chloromethyl ketone. Neither ATP nor 5'-diphosphate-guanosine-3'-diphosphate affected the rate of casein hydrolysis. Protease So was distinct from the other soluble endoproteases in E. coli (including proteases Do, Re, Mi, Fa, La, Ci, and Pi) in its physical and chemical properties and also differed from the membrane-associated proteases, protease IV and V, and from two amino acid esterases, originally named protease I and II. The physiological function of protease So is presently unknown.     

Isolation of a neutral histone-hydrolyzing protease from bovine spleen]

Neutral histone-hydrolyzing protease has been isolated by fractionation of bovine spleen extract. The low level of the protease activity in the extract may be due to the presence of an inhibitor. The enzyme activity was increased 100--1200-fold during ammonium sulfate fractionations, gel filtration on Sephadex G-100 and G-75, chromatography on CM- and DEAE-celluloses. The protease was detected in the fraction with a molecular weight lower than 25000. The enzyme was markedly activated by dithiothreitol and EDTA and inhibited by p-chloromercuribenzoate and iodoacetic acid. It was also inhibited by N-tosyl-L-lysyl chloromethyl ketone, N-tosyl-L-phenylalanyl chloromethylketone, bovine blood serum and partially by soybean trypsin inhibitor DFP, trasylol and epsilon-amino caproic acid had no effect. Beside histone, the neutral protease hydrolyzed casein and gamma-globulin and fibrinogen in a low extent. The enzyme had no activity toward N-benzoyl-D,L-arginine p-nitroanilide, N-benzoyl-L-arginine ethyl ester and N-acetyl-L-tyrosine ethyl ester, collagen, elastin and fibrin. Some properties of the enzyme were similar to those of neutral SH-dependent proteases described by Hayashi and Lo Spalluto et al.     

Purification and characterization of myocardial fructose-6-phosphate,2-kinase and fructose-2,6-bisphosphatase

Fructose-6-P,2-kinase:fructose-2,6-bisphosphatase has been purified to homogeneity from beef heart. The enzyme was bifunctional and the specific activities of the kinase and the phosphatase of the pure enzyme were 60 and 30 milliunits/mg, respectively. The molecular weight of the enzyme was 118,000, consisting of two subunits of 58,000. In some preparations of the enzyme a minor protein with a subunit Mr of 54,000 was present. This minor protein (54,000) was also bifunctional and showed the same immunoreactivity as the major protein. The specific activity of fructose-6-P,2-kinase of the minor component was three times higher than that of the major enzyme (58,000), but fructose-2,6-bisphosphatase activity was the same. These two forms have been separated by phosphocellulose chromatography. The tryptic peptide maps of these enzymes were very similar. The 58,000 enzyme was phosphorylated by cAMP-dependent protein kinase but the 54,000 enzyme was not. These results indicated that the minor 54,000 protein might be a proteolytically digested form of the 58,000 enzyme. The Km of the kinase for fructose-6-P and ATP was 70 microM and 260 microM, respectively for both the 58,000 and the 54,000 enzymes. Km for fructose-2,6-P2 and Ki for fructose-6-P of the phosphatase was approximately 40 and 11 microM, respectively. The enzyme was phosphorylated by fructose-2,6-P2 but the stoichiometry of the phosphate incorporation was 0.05 mol/mol subunit, while 0.4 mol/mol was incorporated in rat liver enzyme under the same conditions.     

Subunit 4 of the 26 S protease is a member of a novel eukaryotic ATPase family.

Ubiquitinated proteins are degraded by a 26 S ATP-dependent protease. SDS-polyacrylamide gel electrophoresis analysis of the purified 26 S enzyme reveals more than 20 polypeptides ranging in apparent molecular masses from 20 to 110 kDa. Although many of the subunits smaller than 30 kDa are members of the multicatalytic protease family, the identity and function of the larger polypeptides have remained unknown. We report here the cDNA sequence for subunit 4, a 51-kDa chain of the 26 S protease. Subunit 4 belongs to a recently identified eukaryotic ATPase family, which includes proteins involved in peroxisome formation, secretion, and human immunodeficiency virus gene expression. Subunit 4 also shows weak similarity to ClpA, the ATP-binding subunit of the Escherichia coli protease, Clp.     

The energy utilized in protein breakdown by the ATP-dependent protease (La) from Escherichia coli

A crucial enzyme in the pathway for protein degradation in Escherichia coli is protease La, an ATP-hydrolyzing protease encoded by the lon gene. This enzyme degrades various proteins to small polypeptides containing 10-20 amino acid residues. To learn more about its energy requirement, we determined the number of ATP molecules hydrolyzed by the purified protease for each peptide bond cleaved. The enzyme hydrolyzed about 2 molecules of ATP for each new amino group generated with casein, bovine serum albumin, glucagon, or guanidinated casein as substrates, even though these proteins differ up to 20-fold in size and 3-4 fold in rates of hydrolysis of peptide bonds. Similar values for the stoichiometry (from 1.9 to 2.4) were obtained using fluorescamine or 2,4,6-trinitrobenzene sulfonic acid to estimate the appearance of new amino groups. These values appeared lower at 1 mM than at 10 mM Mg2+. The coupling between ATP and peptide bond hydrolysis appeared very tight. However, when the protease was assayed under suboptimal conditions (e.g. at lower pH or with ADP present), many more ATP molecules (from 3.5 to 12) were consumed per peptide bond cleaved. Our data would indicate that the early steps in protein degradation consume almost as much energy (2 ATPs for each cleavage) as does the formation of peptide bonds during protein synthesis.     

Purification and characterization of peptidylglycine alpha-amidating monooxygenase from bovine neurointermediate pituitary

Extracts of bovine neurointermediate pituitary secretory granules and frozen bovine neurointermediate pituitary contain multiple forms of peptidylglycine alpha-amidating monooxygenase (PAM) activity differing in apparent molecular weight and in charge. Metal chelate affinity chromatography, substrate affinity chromatography, and gel filtration resulted in the purification of two forms of amidation activity from frozen bovine neurointermediate pituitary: PAM-A, apparent molecular weight 54,000, was purified 7,000-fold and PAM-B, apparent molecular weight 38,000, was purified 21,000-fold. Enzyme activity of similar molecular weights was observed in the starting material. Purified PAM-A and PAM-B correspond to two of the three charge forms present in crude extracts, and both exhibited optimal activity at alkaline pH. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of PAM-B revealed the presence of two bands with apparent molecular weights of 42,000 and 37,000; autoradiography of 125I-labeled PAM-B revealed only the same two bands, and 125I-labeled PAM-B co-eluted with enzyme activity during gel filtration. PAM-A was still heterogeneous based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The properties of purified PAM-A and PAM-B were very similar to those of amidation activity in crude extracts: activity was reduced upon removal of molecular oxygen; activity was stimulated by the addition of CuSO4 and eliminated by the addition of diethyldithiocarbamate; activity was stimulated by the addition of ascorbate, with optimal levels of ascorbate increasing as the concentration of peptide substrate was increased. In the presence of 1.25 mM ascorbate, PAM-B exhibited a Km of 7.0 microM for D-Tyr-Val-Gly and a Vmax of 84 nmol/micrograms/h.     

Purification and characterization of an extracellular acid protease from Neurospora crassa

An extracellular acid protease was purified 1420-fold from sulfur-starved protein-induced cultures of Neurospora crassa. The enzyme was homogeneous as determined by polyacrylamide electrophoresis. The purification procedure consisted of an ultrafiltration step, cation-exchange chromatography, and affinity chromatography on Sepharose-linked pepstatin. The enzyme is homologous to aspartyl proteases that are characterized by pepstatin inhibition and trypsinogen activation. It is extremely autolytic, especially under denaturing conditions. The protease is stable between pH 3 and 7, showing optimal activity near pH 4.0 for both trypsinogen activation and hydrolysis of bovine serum albumin. The molecular weight of the enzyme was 34,500 by gel electrophoresis and gel filtration, and 34,975 by amino acid analysis.     

Intracellular serine protease of Bacillus subtilis: sequence homology with extracellular subtilisins.

Intracellular serine protease was isolated from stationary-grown Bacillus subtilis A-50 cells and purified to homogeneity. The molecular weight of the enzyme is 31,000 +/- 1,000, with an isoelectric point of 4.3. Its amino acid composition is characteristically enriched in glutamic acid content, differing from that of extra-cellular subtilisins. The enzyme is completely inhibited with phenylmethylsulfonyl fluoride and ethylenediaminetetraacetic acid. Intracellular protease possesses negligible activity towards bovine serum albumin and hemoglobin, but has 5- to 20-fold higher specific activity against p-nitroanilides of benzyloxycarbonyl tripeptides than subtilisin BPN'. Esterolytic activity of the enzyme is also higher than that of subtilisin BPN'. The enzyme is sequence homologous with secretory subtilisins throughout 50 determined NH2-terminal residues, indicating the presence of duplicated structural genes for serine proteases in the B. subtilis genome. The occurrence of two homologous genes in the cell might accelerate the evolution of serine protease not only by the loosening of selective constrainst, but also by creation of sequence variants by means of intragenic recombination. Three molecular forms of intracellular protease were found, two of them with NH2-terminal glutamic acid and one minor form, three residues longer, with asparagine as NH2 terminus. These data indicate the possible presence of an enzyme precursor proteolytically modified during cell growth.     

Leishmania (Leishmania) amazonensis: purification and characterization of a promastigote serine protease

Pathogenic protozoan proteases play crucial roles in the host-parasite interaction, and its characterization contributes to the understanding of protozoan disease mechanisms. A Leishmania amazonensis promastigote protease was purified 36-fold, using aprotinin-agarose affinity chromatography and gel filtration high performance liquid chromatography, yielding a total recovery of 49%. The molecular mass of active enzyme obtained from native gel filtration HPLC and SDS-PAGE under conditions of reduction and non-reduction was 68 kDa, suggesting that the enzyme may exist as a monomer. The protease isoelectric point (pI) was around 4.45 and, as demonstrated by deglycosylation assay, it did not have any carbohydrate content. The optimal pH and temperature of the enzyme were 8.0 and 28 degrees C, respectively, determined using alpha-N-rho-tosyl-L-arginyl-methyl ester (L-TAME) as substrate. Assays of thermal stability indicated that 50% of the enzymatic activity was preserved after 4 min of pre-treatment at 42 degrees C and after 24 h of pre-treatment at 37 degrees C, both in the absence of substrate. Hemoglobin, bovine serum albumin (BSA), ovalbumin, and both gelatin and peptide substrates containing arginine in ester bound were hydrolyzed by 68 kDa protease. The insulin beta-chain was also hydrolyzed by the protease, and four peptidic bonds (L11-V12, E13-A14, L15-Y16, and Y16-L17) were susceptible to the 68-kDa protease action. Inhibition studies suggested that the enzyme belonged to a serine protease class inhibited by calcium ions and activated by manganese ions. These findings demonstrate that the L. amazonensis 68-kDa serine protease differs from those of other protozoan parasites.     

Purification and characterization of an acidic amino acid specific endopeptidase of Streptomyces griseus obtained from a commercial preparation (Pronase)

A protease was purified 163-fold from Pronase, a commercial product from culture filtrate of Streptomyces griseus, by a series of column chromatographies on CM-Toyopearl (Fractogel), Sephadex G-50, hydroxyapatite, and Z-Gly-D-Phe-AH-Sepharose 4B using Boc-Ala-Ala-Pro-Glu-pNA as a substrate. The final preparation was homogeneous by polyacrylamide gel electrophoresis (PAGE), sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and gel isoelectric focusing. Studies on the substrate specificity with peptide p-nitroanilides revealed that this protease preferentially hydrolyzed peptide bonds on the carbonyl-terminal side of either glutamic acid or aspartic acid. It was most active at pH 8.8 for the hydrolysis of Boc-Ala-Ala-Pro-Glu-pNA. The molecular weight of the protease was estimated to be 20,000 by gel filtration on Sepharose 6B using 6 M guanidine hydrochloride as an eluent, and 22,000 by SDS-PAGE in the presence of 2-mercaptoethanol. The isoelectric point of the enzyme was 8.4. The enzyme was inactivated by diisopropyl phosphofluoridate (DFP) but not by p-chloromercuribenzoate (PCMB) or EDTA.     

Characterization and purification of a protease in serum that cleaves proatrial natriuretic factor (ProANF) to its circulating forms

Atrial natriuretic factor (ANF) is synthesized and stored in atrial cardiocytes as a 17-kilodalton (kDa), 126 amino acid polypeptide, proANF, but circulates as smaller, 24 and 28 amino acid peptide fragments of the carboxy terminus of proANF. It has previously been shown that proANF is secreted intact from cultured atrial cardiocytes and can be cleaved by a serum protease to smaller, 3-kDa peptides believed to be the circulating forms. This report describes the purification and characterization of this proANF-cleaving protease from rat serum. The cleavages both of 35S-labeled proANF derived from rat atrial cell cultures, as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)/autoradiography, and of a synthetic p-nitroanilide-containing substrate were used as assays for the detection of enzyme activity. ProANF-cleaving activity was found in rat serum, with no such activity detectable in rat plasma. Cleavage in serum was not dependent on the presence of platelets or other cellular elements. Complete inhibition of proANF cleavage was obtained with the protease inhibitors benzamidine, leupeptin, phenylmethanesulfonyl fluoride, and diisopropyl fluorophosphate (DFP) but not with aprotinin, soybean trypsin inhibitor, pepstatin, or hirudin. Unlike the vitamin K dependent plasma proteins, the proANF-cleaving protease did not adsorb to barium sulfate. With the sequential application of ion-exchange, hydroxylapatite, lectin affinity, and gel filtration chromatography, a 5000-6000-fold purification of the enzyme from rat serum was achieved. Fractionation of either whole serum or the purified enzyme by gel filtration chromatography revealed a single peak of activity corresponding to a protein with a Stokes radius of 45 A.(ABSTRACT TRUNCATED AT 250 WORDS)     

Autoproteolysis of the small subunit of calcium-dependent protease II activates and regulates protease activity

Calcium-dependent protease II (CDP-II) from bovine heart is a heterodimer with subunit molecular weights of 80,000 and 26,000. Previous studies have demonstrated that the protease requires 350 microM Ca2+ for half-maximal activity and that the large subunit contains both the catalytic and Ca2+ binding functions of the enzyme. The function of the small subunit has been unclear. We have examined the effect of Ca2+ on structural and catalytic properties of CDP-II in the presence and absence of substrate proteins. When incubated with Ca2+ in the absence of substrate, CDP-II undergoes a series of autoproteolytic cleavages that sequentially reduce the small subunit's molecular weight from 26,000 to 24,000 to 22,000 to 17,000. During this time there is no detectable change in the 80-kDa subunit, which remains associated with the autolyzed small subunit. The rate of autoproteolysis is dependent on temperature and on the concentration of Ca2+ (half-maximal rate at approximately 600 microM Ca2+). The first cleavage appears to be unimolecular because its rate is unaffected by CDP-II concentration or by the presence of exogenous protein substrates. Subsequent cleavages result in the formation of the 80-kDa/17-kDa heterodimer and appear to occur by bimolecular reactions; rates of these reactions were slowed by decreasing CDP-II concentrations and by the presence of protein substrates. Autoproteolysis of the small subunit has two distinct functional consequences, each of which is associated with different forms of the autolyzed protease. Our results indicate that the 80-kDa/26-kDa form of CDP-II represents an inactive proenzyme and that the initial Ca2+-dependent cleavage of the 26-kDa subunit results in activation of the protease. The activated enzyme hydrolyzes protein substrates with a Ca2+ concentration requirement of 350 microM for half-maximal rates. The further autoproteolysis, which results in the formation of the 80-kDa/17-kDa heterodimer, serves to reduce the Ca2+ concentration requirement for protease activity by 25-fold. Thus, these results provide evidence for specific roles of the small subunit in the regulation of CDP-II activity.     

Primary structure of the catalytic subunit of calf thymus DNA polymerase delta: sequence similarities with other DNA polymerases.

The 125- and 48-kDa subunits of bovine DNA polymerase delta have been isolated by SDS-polyacrylamide gel electrophoresis and demonstrated to be unrelated by partial peptide mapping with N-chlorosuccinimide. A 116-kDa polypeptide, usually present in DNA polymerase delta preparations, was shown to be a degraded form of the 125-kDa catalytic subunit. Amino acid sequence data from Staphylococcus aureus V8 protease, cyanogen bromide, and trypsin digestion of the 125- and 116-kDa polypeptides were used to design primers for the polymerase chain reaction to determine the nucleotide sequence of a full-length cDNA encoding the catalytic subunit of bovine DNA polymerase delta. The predicted polypeptide is 1106 amino acids in length with a calculated molecular weight of 123,707. This is in agreement with the molecular weight of 125,000 estimated from SDS-polyacrylamide gel electrophoresis. Comparison of the deduced amino acid sequence of the catalytic subunit of bovine DNA polymerase delta with that of its counterpart from Saccharomyces cerevisiae showed that the proteins are 44% identical. The catalytic subunit of bovine DNA polymerase delta contains the seven conserved regions found in a number of bacterial, viral, and eukaryotic DNA polymerases. It also contains five additional regions that are highly conserved between bovine and yeast DNA polymerase delta, but these regions share little or no homology with the alpha polymerases. Four of these additional regions are also highly homologous to the herpes virus family of DNA polymerases, whereas one region is not homologous to any other DNA polymerase that has been sequenced thus far.(ABSTRACT TRUNCATED AT 250 WORDS)     

The protease problem in Neurospora. Structural modification of the arom multienzyme system during its extraction and isolation.

The “aromatic complex” or “arom aggregate” of Neurospora crassa catalyzes five consecutive reactions in the central pathway leading to the biosynthesis of the aromatic amino acids. Previously, this multienzyme system was shown variously to have a molecular weight of 230,000 to 300,000 and to contain up to four subunits. Recently, a protease and a corresponding specific inhibitor have been isolated from N. crassa and, as described in this report, a new method for isolating the multienzyme system has been developed. We have made the following observations: (a) Detergent (sodium dodecyl sulfate) gel electrophorograms of the “complex” isolated by two different methods are not comparable. In an earlier method, which involved more manipulations and time, the detergent gel banding patterns showed four polypeptides with molecular weights totaling about 300,000. With the new purification procedure, there are two major bands: the first with an apparent molecular weight of about 150,000 and the second with a molecular weight of 50,000. (b) When the freshly purified multienzyme system is incubated at 25 °C, four new bands appear within 30 h and a fifth is visible after 40 h. (c) The formation of these new bands is prevented for up to 40 h by the addition of phenylmethanesulfonylfluoride or a purified preparation of the specific N. crassa protease inhibitor, (d) The multienzyme system appears to remain intact, as shown by standard polyacrylamide gel electrophoresis, even after it has suffered several proteolytic clips. These results demonstrate that the purified complex is contaminated with a small but influential quantity of the inhibitable N. crassa protease and show that this protease is capable of creating an artificial subunit structure in the multienzyme system. Based on these observations, we hypothesize that the arom enzyme system is a five-component multifunctional enzyme.     

A study of extracellular alkaline protease from Bacillus subtilis NCIM 2713

An alkaline protease was isolated from culture filtrate of B. subtilis NCIM 2713 by ammonium sulphate precipitation and was purified by gel filtration. With casein as a substrate, the proteolytic activity of the purified protease was found to be optimal at pH 8.0 and temperature 70 degrees C. The purified protease had molecular weight 20 kDa, Isoelectric point 5.2 and km 2.5 mg ml(-1). The enzyme was stable over the pH range 6.5-9.0 at 37 degrees C for 3 hr. During chromatographic separation this protease was found to be susceptible to autolytic degradation in the absence of Ca2+. Ca2+ was not only required for the enzyme activity but also for the stability of the enzyme above 50 degrees C. About 62% activity was retained after 60 min at pH 8.0 and 55 degrees C. DFP and PMSF completely inhibited the activity of this enzyme, while in the presence of EDTA only 33% activity remained. However, it was not affected either by sulfhydryl reagent, or by divalent metal cations, except SDS and Hg2+. The results indicated that this is a serine protease.     

Partial characterization of a 29 kDa cysteine protease purified from Taenia solium metacestodes

A 29 kDa cysteine protease of Taenia solium metacestodes was purified by Mono Q anion-exchanger and Superose 6 HR gel filtration chromatography. The enzyme was effectively inhibited by cysteine protease inhibitors, such as iodoacetic acid (IAA) and trans-epoxy-succinyl-L-leucyl-amido (4-guanidino) butane (E-64) while inhibitors acting on serine- or metallo-proteases did not affect the enzyme activity. The purified enzyme degraded human immunoglobulin G (IgG), collagen and bovine serum albumin (BSA), but human IgG was more susceptible for proteolysis by the enzyme. To define the precise biological roles of the enzyme, more detailed biochemical and functional studies would be required.     

Characteristics of an Acid protease from maize endosperm

An assay has been developed to measure protease activity in endosperm extracts of maize seeds. With hemoglobin as substrate, the enzyme(s) has a pH optimum of 3.8 and a temperature optimum of 46 C. It also degrades gliadin, edestin, bovine serum albumin, and partially hydrolyzed zein and glutelin under standard assay conditions. The enzyme(s) has endopeptidase activity with all substrates tested. When undenatured zein and glutelin are suspended in an agar gel, both are efficiently degraded. Using this assay, the protease activity increases from day 3 to day 8 after inhibition and then declines.     

Multiple forms of the 20 S multicatalytic and the 26 S ubiquitin/ATP-dependent proteases from rabbit reticulocyte lysate.

We have used native gel electrophoresis followed by fluorogenic peptide overlay to identify multiple forms of rabbit reticulocyte multicatalytic protease (MCP) or 20 S protease, and two forms of rabbit 26 S ubiquitin/ATP-dependent protease. An abundant, fast-migrating 20 S complex (20 SF) possesses modest ability to hydrolyze the fluorogenic peptide succinyl-Leu-Leu-Val-Tyr-4-methyl-coumaryl-7-amide. In contrast, two minor, slower migrating species cleave the peptide at high rates. A unique 30-kDa polypeptide is associated with one of the active MCPs, and a 160-kDa subunit is associated with the other. Two electrophoretically distinct 26 S proteases can also be isolated from rabbit reticulocyte lysate. The faster migrating form, 26 SF, is more resistant to inactivation by ATP depletion. Despite the differential response to nucleotides and the distinctive electrophoretic mobilities of 26 SF and 26 SS, we have not identified any subunit differences between the two enzymes. In addition to active 26 S proteases, we have discovered and purified a proteolytically inactive particle that contains subunits characteristic of the 26 S protease (e.g. molecular masses between 30 and 110 kDa). Incubation of this protein complex with purified MCP and ATP results in the formation of the 26 S proteases.     

A serine protease from a detergent-soluble extract of Leishmania (Leishmania) amazonensis

Proteases mediate important crucial functions in parasitic diseases, and their characterization contributes to the understanding of host-parasite interaction. A serine protease was purified about 43-fold with a total recovery of 60% from a detergent-soluble extract of promastigotes of Leishmania amazonensis. The purification procedures included aprotinin-agarose affinity chromatography and gel filtration high performance liquid chromatography. The molecular mass of active enzyme was 110 kDa by native gel filtration HPLC and by SDS-PAGE gelatin under non-reducing conditions. Under conditions of reduction using SDS-PAGE gelatin analyses the activity of enzyme was observed in two proteins of 60 and 45 kDa, suggesting that the enzyme may be considered as a dimer. The Leishmania protease was not glycosylated, and its isoelectric point (pI) was around 4.8. The maximal protease activity was at pH 7.0 and 28 degrees C, using a-N-o-tosyl-L-arginyl-methyl ester (L-TAME) as substrate. Assays of thermal stability indicated that this enzyme was totally denatured after pre-treatment at 42 degrees C for 12 min and preserved only 20% of its activity after pre-treatment at 37 degrees C for 24 h, in the absence of substrate. Hemoglobin, bovine serum albumin (BSA), ovalbumin and gelatin were hydrolyzed by Leishmania protease. Inhibition studies indicated that the enzyme belonged to a serine protease class because of a significant impediment by serine protease inhibitors such as benzamidine, aprotinin, and antipain. The activity of the present serine protease is negatively modulated by calcium and zinc and positively modulated by manganese ions. This is the first study that reports the purification of a protease from a detergent-soluble extract of Leishmania species.     

Structural polypeptides of rabbit, bovine, and human papillomaviruses.

The number and apparent molecular weight of the structural polypeptides of Shope rabbit papilloma virus (RPV), bovine papilloma virus (BPV), and human papilloma virus (HPV) were estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Up to 10 polypeptides were detected in highly purified BPV and HPV full particles; a close homology was found between the polypeptide composition of both viruses. Purified RPV virions gave a similar polypeptide pattern. The main components of the three papillomaviruses are the major polypeptide (VP1) with a mol wt of approximately 54,000 and the three smaller polypeptides (VP8, 9, 10) with mol wt of about 16,500, 15,500 and 12,500, respectively. VP8, VP9, and VP10 are never detected in empty capsids. When BPV virions were disrupted with alkaline buffer, the six lower-molecular-weight polypeptides (VP5 to 10) remained associated with viral DNA. This suggests that they are internal components of the virions and that the four higher-molecular-weight polypeptides (VP1 to 4) may represent external components. The polypeptide compositions of BPV and polyoma virus, another papovavirus, have been compared. The number of BPV and polyoma virus components (10 and 6, respectively) and the molecular weight of their major polypeptide (54,000 and 44,500, respectively) are different; however, the three main DNA-associated polypeptides of BPV (VP8, 9, 10) and the three histone-like components of polyoma virus (VP4, 5, 6) were shown to have identical apparent molecular weights. The possibility that some of the minor components of papillomaviruses may be proteolytic degradation products or cell protein contaiminants is discussed.