Phylogenetic and Morphological Analyses of Androctonus crassicuda from Khuzestan Province, Iran (Scorpiones: Buthidae).

The Androctonus crassicuda is the most diverse scorpion species in the family of Buthidae, which is endemic to Khuzestan province, Iran. Investigation of the relationship of species by means of a molecular study of specimens is one of the new approaches due to the limitations of the morphological approaches. In the current study, the analysis was based on 32 morphological characteristics of A. crassicuda native to southwest Iran. Moreover, the DNA sequencing of two mitochondrial markers, namely cytochrome oxidase subunit I and 12sRNA loci was performed, and the phylogenetic tree was constructed using maximum likelihood method with 1000 replications using MEGA software (version 7). Based on the results of the phylogenetic tree, A. crassicuda was classified into a monophyletic group. However, the genetic diversity of this species populations was not significant (0.001). The highest and lowest genetic distance of A. crassicuda was compared with the reports obtained in Urmia and west Azerbaijan, Iran. There was a clear divergence between the A. crassicuda isolated from northern and southern areas of Iran. This study showed the importance of geographical and climate features of the region and genetic distance among the populations. The phylogenetic analysis of Androctonus species from other regions showed the highest and lowest genetic distance with A. gonneti (Morocco) and A. amoreuxi (Portugal), respectively. The comparison of the morphological characteristics and morphometric results revealed that metasoma characteristics are important in the identification of A. crassicuda. The results of the analysis of the morphometric values of A. crassicuda were mainly compatible with the phylogenetic trees and supported the traditional morphological classification, thereby presenting a clearly definition of the genera of Androctonus species.

Antigenic and Substrate Preference Differences between Scorpion and Spider Dermonecrotic Toxins, a Comparative Investigation.

The Hemiscorpius lepturus scorpion and brown spider Loxosceles intermedia represent a public health problem in Asia and America, respectively. Although distinct, these organisms contain similar toxins responsible for the principal clinical signs of envenomation. To better understand the properties of these toxins, we designed a study to compare recombinant Heminecrolysin (rHNC) and rLiD1, the major phospholipase D toxins of scorpion and spider venom, respectively. Using a competitive ELISA and a hemolytic inhibition test, we come to spot a cross reaction between scorpion and spider venoms along with an epitopic similarity between rHNC and rLiD1 associated with neutralizing antibodies. Results show that the ability of the rHNC to hydrolyze lysophosphatidylcholine (LPC) is equivalent to that of rLiD1 to hydrolyze sphingomyelin and vice-versa. rHNC exclusively catalyze transphosphatidylation of LPC producing cyclic phosphatidic acid (cPA). The in-silico analysis of hydrogen bonds between LPC and toxins provides a possible explanation for the higher transphosphatidylase activity of rHNC. Interestingly, for the first time, we reveal that lysophosphatidic acid (LPA) can be a substrate for both enzymes using cellular and enzymatic assays. The finding of the usage of LPA as a substrate as well as the formation of cPA as an end product could shed more light on the molecular basis of Hemiscorpius lepturus envenomation as well as on loxoscelism.

Novel Mutant Phospholipase D from Hemiscorpius lepturus Acts as A Highly Immunogen in BALB/c Mice Against the Lethality of Scorpion Venom.

Hemiscorpius lepturus (H. lepturus) which belongs to the Scorpionidae family, is the deadliest scorpion in Iran. It causes pathological manifestations like dermonecrosis, hemolysis, renal failure, necrotic ulcers, and in some cases, even death. The venom of this scorpion is well-known for its cytotoxic effects in comparison with the other venomous scorpions which show significant neurotoxic effects. Due to the painless nature of the sting of this scorpion, the clinical symptoms occur in victims 24 to 72 h post-sting. In our previous studies during the last decade, we demonstrated that the medical complications are attributable to the presence of phospholipase D (PLD) as a major toxin in the venom. With the purpose of designing and constructing a vaccine against H. lepturus for humans, animal model experiments were performed. To achieve this goal, non-toxic PLD was developed by mutation of two critical catalytic residues-His12 and His48-into alanines and the product was then denominated mut-rPLD1. The in-vivo tests showed that the mice immunized with interval doses of 10 µg of mut-rPLD1, were completely protected against 10× the LD100 of the venom. In conclusion, this mutant may be an effective vaccine candidate against scorpion envenomation by H. lepturus in future clinical studies.

The Dual α-Amidation System in Scorpion Venom Glands.

Many peptides in scorpion venoms are amidated at their C-termini. This post-translational modification is paramount for the correct biological function of ion channel toxins and antimicrobial peptides, among others. The discovery of canonical amidation sequences in transcriptome-derived scorpion proproteins suggests that a conserved enzymatic α-amidation system must be responsible for this modification of scorpion peptides. A transcriptomic approach was employed to identify sequences putatively encoding enzymes of the α-amidation pathway. A dual enzymatic α-amidation system was found, consisting of the membrane-anchored, bifunctional, peptidylglycine α-amidating monooxygenase (PAM) and its paralogs, soluble monofunctional peptidylglycine α-hydroxylating monooxygenase (PHMm) and peptidyl-α-hydroxyglycine α-amidating lyase (PALm). Independent genes encode these three enzymes. Amino acid residues responsible for ion coordination and enzymatic activity are conserved in these sequences, suggesting that the enzymes are functional. Potential endoproteolytic recognition sites for proprotein convertases in the PAM sequence indicate that PAM-derived soluble isoforms may also be expressed. Sequences potentially encoding proprotein convertases (PC1 and PC2), carboxypeptidase E (CPE), and other enzymes of the α-amidation pathway, were also found, confirming the presence of this pathway in scorpions.

Functional characterization and FTIR-based 3D modeling of full length and truncated forms of Scorpio maurus venom phospholipase A2.

BACKGROUND: Heterodimeric phospholipase A2 from venom glands of Tunisian scorpion Scorpio maurus (Sm-PLGV) had been purified. It contains long and short chains linked by a disulfide bridge. Sm-PLGV exhibits hemolytic activity towards human erythrocytes and interacts with phospholipid monolayers at high surface pressure. The investigation of structure-function relationships should provide new clues to understand its activity. METHODS: Molecular cloning of Sm-PLGV and heterologous expression in Escherichia coli of three recombinant forms was used to determine the role of the short chain on enzymatic activity. Infrared spectroscopy assisted 3D model building of the three recombinant constructs (phospholipases with and without the penta-peptide and Long chain only) allowed us to propose an explanation of the differences in specific activities and their interaction with various phospholipids. RESULTS: Nucleotide sequence of Sm-PLGV encodes 129 residues corresponding to the Long chain, the penta-peptide and the short chain. Although recombinant phospholipases without and with the penta-peptide have different specific activities, they display a similar substrate specificity on various phospholipid monolayers and similar bell-shaped activity profiles with maxima at high surface pressure. The absence of the short chain reduces significantly enzymatic and hemolytic activities. The 3D models pointed to an interaction of the short chain with the catalytic residues, what might explain the difference in activities of our constructs. CONCLUSION: Infrared spectroscopy data and 3D modeling confirm the experimental findings that highlight the importance of the short chain for the Sm-PLGV activity. GENERAL SIGNIFICANCE: New informations are given to further establish the structure-function relationships of the Sm-PLGV.

[Identification of Scolopendra subspinipes mutilans and its adulterants using DNA barcode].

In this study, the COI barcode was used to identify the Scolopendra medicinal materials and its adulterants in order to provide a new method for the identification of Scolopendra. Genomic DNA was extracted from the experimental samples. The COI sequences were amplified and sequenced bi-directionally. Sequence alignment and NJ tree construction was carried out by MEGA6.0 software. The results showed that the COI sequences can be obtained from all experimental samples. The average inter-specific K2P distance of Scolopendra was 0.222 and the minimum inter-specific distance was 0.190. All the Scolopendra subspinipes mutilans medicinal samples clustered into a clade in the NJ tree and can be distinguished from its adulterants. In a conclusion, COI can be used to correctly identify Scolopendra medicinal materials, and it will be a potential DNA barcode for identifying other animal medicinal materials.

Cloning and molecular characterization of scorpion Buthus martensi venom hyaluronidases: a novel full-length and diversiform noncoding isoforms.

Hyaluronidase is a common component of scorpion venom and has been considered as "spreading factor" that promotes a fast penetration of the venom in the anaphylactic reaction. In the current study, a novel full-length of hyaluronidase BmHYI and three noncoding isoforms of BmHYII, BmHYIII and BmHYIV were cloned by using a combined strategy based on peptide sequencing and Rapid Amplification of cDNA Ends (RACE). BmHYI has 410 amino acid residues containing the catalytic, positional and five potential N-glycosylation sites. The deduced protein sequence of BmHYI shares significant identity with venom hyaluronidases from bees and snakes. The phylogenetic analysis showed early divergence and independent evolution of BmHYI from other hyaluronidases. An extraordinarily high level of sequence similarity was detected among four sequences. But, BmHYII, BmHYIII and BmHYIV were short of stop-codon in the open reading frame and poly(A) signal in the 3' end.

Fibrin(ogen)olytic enzymes in scorpion (Tityus discrepans) venom.

Several fibrin(ogen)olytic enzymes from Tityus discrepans (Buthidae, Buthoidea) venom (TdV) were partially purified on a Sephadex G-50 column, by affinity and molecular exclusion high-performance chromatography. Fractions SB1-I and SB1-II had fibrinolytic, fibrinogenolytic (Aα-chains degradation) and tissue plasminogen activator (t-PA)-like activities. SB1-III was only fibrinogenolytic (fast degradation of Aα-chains and slower degradation of fibrinogen Bß-chains). These results showed the presence of α-fibrinogenases in TdV. The fibrino(geno)lytic activity in these fractions was abolished by metalloprotease inhibitors (MPI). Fractions SB3-I and SB3-II contain fibrinogenolytic (Aα-chains degradation) and fibronectinolytic activities. Also fraction SB3-I had a t-PA-like activity. Activities in SB3-I and SB3-II were abolished by serine protease inhibitors (SPI). None of the fractions degraded fibrinogen γ-chains. Fibrinogen degradation by active fractions is associated with an anticoagulant effect supported by a reduced coagulant activity. The overall outcome suggests that metalloproteases and serine proteases in TdV are responsible for fibrin(ogen)olytic activity because MPI and SPI inhibited these activities.

Cloning and molecular characterization of BumaMPs1, a novel metalloproteinases from the venom of scorpion Buthus martensi Karsch.

Scorpion venoms metalloproteinase is involved in a number of important biological, physiological and pathophysiological processes. In this work, a complete sequence of metalloproteinase was first obtained from venom of scorpion Buthus martensi and named as BumaMPs1. BumaMPs1 has 393 amino acid residues containing with a molecular mass of 44.53 kDa, showing an isoelectric point of 5.66. The primary sequence analysis indicated that the BumaMPs1 contains a zinc-binding motif (HELGHNLGISH), methionine-turn motif (YIM), disintegrin-like domain (ETCD) and N-glycosylation site. The multiple alignment of its deduced amino acid sequence and those of other metalloproteinase showed a high structural similarly, mainly among class reprolysin proteases. The phylogenetic analysis showed early divergence and independent evolution of BumaMPs1 from other metalloproteinase.

A new chymotrypsin-like serine protease involved in dietary protein digestion in a primitive animal, Scorpio maurus: purification and biochemical characterization.

BACKGROUND: Most recent works on chymotrypsins have been focused on marine animals and insects. However, no study was reported in chelicerate. RESULTS: Scorpion chymotrypsin-like protease (SCP) was purified to homogeneity from delipidated hepatopancreases. The protease NH2-terminal sequence exhibited more than 60% monoacids identity with those of insect putative peptidases. The protease displayed no sequence homology with classical proteases. From this point of view, the protease recalls the case of the scorpion lipase which displayed no sequence homology with known lipases. The scorpion amylase purified and characterized by our time, has an amino-acids sequence similar to those of mammalian amylases. The enzyme was characterized with respect its biochemical properties: it was active on a chymotrypsin substrate and had an apparent molecular mass of 25 kDa, like the classically known chymotrypsins. The dependence of the SCP activity and stability on pH and temperature was similar to that of mammalian chymotrypsin proteases. However, the SCP displayed a lower specific activity and a boarder pH activity range (from 6 to 9). CONCLUSION: Lower animal have a less evaluated digestive organ: a hepatopancreas, whereas, higher ones possess individualized pancreas and liver. A new chymotrypsin-like protease was purified for the first time from the scorpion hepatopancreas. Its biochemical characterization showed new features as compared to classical chymotrypsin-higher-animals proteases.

Structural analysis of a group III Glu62-phospholipase A2 from the scorpion, Mesobuthus tamulus: Targeting and reversible inhibition by native peptides.

Group III phospholipase A(2) enzyme transcript from the Mesobuthus tamulus (Indian red scorpion) codes for three distinct products that include a large enzymatic subunit, a pentameric peptide and a small non-enzymatic subunit. The structures of these two subunits were modeled based on their sequence identity to bee venom PLA(2) and the partial sequence of MU2 adaptin subunit of AP2 clathrin adaptor, respectively. The enzymatic subunit comprises of three helices, the calcium binding loop and a substrate binding hydrophobic channel where the structure is stabilized by four disulfide bonds. The active site of the enzyme shows a catalytic histidine residue. Interestingly, there is a conservative mutation of the conserved aspartic acid, a classical participant of catalysis in this enzyme family, to glutamic acid. However, the side chain oxygen atoms of this glutamate are oriented away from the catalytic histidine implicating the non-participation of this residue in stabilizing the tautomeric conformation of the histidine. The acidic non-enzymatic subunit comprises of extensive hydrophobic residues with a conformation of an anti-parallel ß-sheets making it ideal for tissue specific targeting. The native pentapeptide with the sequence Alanine-Arginine-Serine-Alanine-Arginine was docked to the enzymatic subunit. The peptide ligand occupies the hydrophobic cavity and makes a plethora of interactions with the residues in the channel, including a hydrogen bond with the crucial catalytic histidine and coordinate bond with the calcium ion. This ligand has a binding constant (K(D)) of 1.5µM. This makes the ligand a potential reversible inhibitor, ideal to prevent the enzyme from interacting with non-specific molecules enroute to the target. The enzyme-ligand complex also provides a model to understand the stereochemistry required for the design of more potent drug molecules against such enzyme drug targets.

Enzymatic analysis of Hemiscorpius lepturus scorpion venom using zymography and venom-specific antivenin.

Hemiscorpius lepturus envenomation exhibits various pathological changes in the affected tissues, including skin, blood cells, cardiovascular and central nervous systems. The enzymatic activity and protein component of the venom have not been described previously. In the present study, the electrophoretic profile of H. lepturus venom was determined by SDS-PAGE (12 and 15%), resulting in major protein bands at 3.5-5, 30-35 and 50-60 kDa. The enzymatic activities of the venom was, for the first time, investigated using various zymography techniques, which showed the gelatinolytic, caseinolytic, and hyaluronidase activities mainly at around 50-60 kDa, 30-40 kDa, and 40-50 kDa, respectively. Among these, the proteolytic activities was almost completely disappeared in the presence of a matrix metalloproteinase inhibitor, 1, 10-phenanthroline. Antigen-antibody interactions between the venom and its Iranian antivenin was observed by Western blotting, and it showed several antigenic proteins in the range of 30-160 kDa. This strong antigen-antibody reaction was also demonstrated through an enzyme-linked immunosorbent assay (ELISA). The gelatinase activity of the venom was suppressed by Razi institute polyvalent antivenin, suggesting the inhibitory effect of the antivenin against H. lepturus venom protease activities. Prudently, more extensive clinical studies are necessary for validation of its use in envenomed patients.

Digestive amylase of a primitive animal, the scorpion: purification and biochemical characterization.

Scorpion, one of the most ancient invertebrates was chosen, as a model of a primitive animal, to purify and characterize an amylase located in the hepatopancreas. The scorpion digestive amylase (SDA) was purified. Pure SDA was obtained after heat treatment followed by ammonium sulfate fractionation and three steps of chromatography. The pure amylase is not glycosylated and has a molecular mass of 59,101 Da determined by MALDI-TOF MS analysis. The maximal amylase activity was measured at pH 7.0 and 50 degrees C, in the presence of Ca2+ and using potato starch as substrate. The enzyme was able to hydrolyze also, glycogen and amylose. The 23 NH2-terminal amino acid SDA residues were sequenced. The sequence obtained is similar to those of mammalian and avian pancreatic amylases. Nevertheless, polyclonal antibodies directed against SDA failed to recognize classical digestive amylases like the porcine pancreatic one.

Vesicle-associated membrane protein (VAMP) cleavage by a new metalloprotease from the Brazilian scorpion Tityus serrulatus.

We present evidence that venom from the Brazilian scorpion Tityus serrulatus and a purified fraction selectively cleave essential SNARE proteins within exocrine pancreatic tissue. Western blotting for vesicle-associated membrane protein type v-SNARE proteins (or synaptobrevins) reveals characteristic alterations to venom-treated excised pancreatic lobules in vitro. Immunocytochemistry by electron microscopy confirms both the SNARE identity as VAMP2 and the proteolysis of VAMP2 as a marked decrease in secondary antibody-conjugated colloidal gold particles that are predominantly associated with mature zymogen granules. Studies with recombinant SNARE proteins were used to determine the specific cleavage site in VAMP2 and the susceptibility of VAMP8 (endobrevin). The VAMP2 cleavage site is between the transmembrane anchor and the SNARE motif that assembles into the ternary SNARE complex. Inclusion of divalent chelating agents (EDTA) with fraction nu, an otherwise active purified component from venom, eliminates SNARE proteolysis, suggesting the active protein is a metalloprotease. The unique cleavages of VAMP2 and VAMP8 may be linked to pancreatitis that develops following scorpion envenomation as both of these v-SNARE proteins are associated with zymogen granule membranes in pancreatic acinar cells. We have isolated antarease, a metalloprotease from fraction nu that cleaves VAMP2, and report its amino acid sequence.

Switch between tyrosinase and catecholoxidase activity of scorpion hemocyanin by allosteric effectors.

Phenoloxidases and hemocyanins have similar type 3 copper centers although they perform different functions. Hemocyanins are oxygen carriers, while phenoloxidases (tyrosinase/catecholoxidase) catalyze the initial step in melanin synthesis. Tyrosinases catalyze two subsequent reactions, whereas catecholoxidases catalyze only the second one. Recent results indicate that hemocyanins can also function as phenoloxidases and here we show for the first time that hemocyanin can be converted to phenoloxidase. Furthermore, its substrate specificity can be switched between catecholoxidase and tyrosinase activity depending on effectors such as hydroxymethyl-aminomethan (Tris) and Mg(2+)-ions. This demonstrates that substrate specificity is not caused by a chemical modification of the active site.

Sequence analysis and phylogenetic relationship of genes encoding heterodimeric phospholipases A2 from the venom of the scorpion Anuroctonus phaiodactylus.

Some scorpion venom contain heterodimeric phospholipases A2. They were shown to be toxic to insects and to cause edema and/or hemolysis of mammalian erythrocytes. This manuscript describes the results of cDNA cloning of five different heterodimeric phospholipases from the venomous glands of the Mexican scorpion Anuroctonus phaiodactylus. The amino acid sequence deduced from the heterodimeric phospholipases open reading frames corresponds in each case to a different isoform. The nucleotide sequences corresponding to two of these genes were also obtained by directly sequencing genomic DNA. The cDNA isoforms show high similarity with the heterodimeric phospholipase Phaiodactylipin purified from the same scorpion. However, similar phospholipases were also found in scorpions from other species and the sequences available were used to construct a phylogenetic tree. In order to understand better the gene structure and phylogeny of these enzymes we analyzed their sequences and compared them with secretory phospholipases of other sources from groups I, II and III. The genomic DNA sequence of a similar phospholipase from bee venomous glands was also cloned. The information available on a Drosophila phospholipase was included in this analysis. The phospholipases of groups I and II contain a conserved exon-intron structure (four or five exons of the mature segment of the enzyme are separated by three or four introns). Also, the gene structure of the phospholipases from A. phaiodactylus and that of the bee venom, belonging to group III phospholipases, are interrupted by three introns. The mature peptide of the bee enzyme is a single polypeptide chain, coded by four exons, whereas those from the scorpion studied here although having four exons, showed the presence of two different polypeptides in its native state. The mature protein is processed after synthesis, producing the heterodimeric structure: a long and a short-peptide chain, linked by a disulfide bridge. The small subunit is the one coded by the fourth exon. The human phospholipase A2 and that of Drosophila, also classified into the group III phospholipases, have a considerably different exon-intron organization.

Cloning, sequence analysis and homology modeling of a novel phospholipase A2 from Heterometrus fulvipes (Indian black scorpion).

We report the cloning and sequencing of group III phospholipaseA(2) from Heterometrus fulvipes (HfPLA(2)), Indian black scorpion. The cDNA sequence codes for the mature portion of the group PLA(2) of 103 amino acids. The sequence has 85% identity with Mesobuthus tamulus (Indian red scorpion) PLA(2) and a 40% identity with bee venom PLA(2) and human group III PLA(2). Most of the essential features of group III PLA(2) like Ca(2+) binding loop and catalytic residues are conserved. Homology modeling was done with the known structure of group III bee venom PLA(2). All the secondary structural motifs and the disulfide bridges are as predicted. The variation like the replacement of aspartic acid residue with glutamic acid in the well known histidine-aspartic acid dyad is a rare feature. This is the first structural model report of an Indian black scorpion PLA(2).

Scorpion digestive lipase: a member of a new invertebrate's lipase group presenting novel characteristics.

Unlike classical digestive lipases, the scorpion digestive lipase (SDL) has a strong basic character. The SDL activity's optimal pH, when using tributyrin or olive oil as substrate, was 9.0. Added to that, the estimated isoelectric point of the native SDL using the electrofocusing technique, was found to be higher than 9.6. To our knowledge, this is the first report of an animal digestive lipase having such a basic character. When olive oil was used as substrate, SDL was shown to be insensitive to the presence of amphiphilic proteins such as bovine serum albumin (BSA). Furthermore, the hydrolysis was found to be specifically dependent on the presence of Ca(2+) ions, since no significant SDL activity was detected in the presence of ions chelator such as EDTA. Nevertheless, the SDL does not require Ca(2+) to trigger the hydrolysis of tributyrin emulsion. Interestingly Zn(2+) and Cu(2+) ions act as strong inhibitors of SDL activity when using tributyrin as substrate. An internal chymotryptic cleavage of SDL generated two fragments of 28 and 25 kDa having the same N-terminal sequence. This sequence of 19 residues does not share any homology with known animal and microbial lipases. Polyclonal antibodies directed against SDL (pAbs anti-SDL) failed to recognise ostrich pancreatic and dog gastric lipases (OPL and rDGL). Moreover, both pAbs anti-OPL and anti-rDGL failed to immunoreact with SDL. These immunological as well as distinct biochemical properties strengthen the idea that SDL appears to belong to a new invertebrate's lipase group.

Immunocytochemical localization of scorpion digestive lipase.

The scorpion hepatopancreas consists of digestive diverticula and interstitial tissue. A digestive diverticulum is composed of two differentiated cell types: the secretory zymogene-like cells and the digestive cells which are the most abundant. The scorpion digestive lipase (SDL) has been previously purified from scorpion hepatopancreas, but its cellular localization has not yet been established. Polyclonal antibodies specific to SDL were prepared and used in immunofluorescence and immunogold techniques to determine the cellular location of SDL. Our results clearly established that SDL was detected intracellularly in specific vesicles tentatively named (SDL+) granules of the digestive cells. No immunolabelling was observed in secretory zymogene-like cells. This immunocytolocalization indicates that lipid digestion might occur in specific granules inside the digestive cells, as suggested by previous studies on the scorpion digestive process.

Purification and properties of hyaluronidase from Palamneus gravimanus (Indian black scorpion) venom.

Scorpion venoms are a rich source of enzymes. Some of the enzymes such as phospholipase A2, proteolytic enzymes and phosphodiesterase are well characterized. However, hyaluronidase has not been studied extensively. In this paper we describe the purification and characterization of hyaluronidase (Hyaluronate lyase, E.C.3.2.1.35) from the Palamneus gravimanus scorpion venom by a combination of gel filtration on Sephadex G-75 and ion-exchange chromatography on DEAE-cellulose. The optimal pH and temperature for its maximum activity of the isolated enzyme were 4.5 and 37 degrees C, respectively, and its K(m) was 47.61 microg/ml at 37 degrees C and its specific activity was 6411.7 +/- 117TRU/min per mg against 250 +/- 4.0 TRU/min per mg for the whole desiccated venom suggesting 25-fold purification. The molecular weight of the isolated enzyme was 52 +/- 1 kDa as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration chromatography on Sephadex G-75. The enzyme was stable for 30 days in the presence of NaCl; no loss of activity was observed up to 37 degrees degrees C and showed a sharp decrease in its activity at 40 degrees C. Heparin inhibited the enzyme activity.