A crystalline end product produced by the hydrolytic cleavage of an RNA-like fragment by an organometallointercalator: 1,10-phenanthroline-platinum(II)-ethylenediamine-cytidine 3'' monophosphate

1,10-Phenanthroline-platinum(II)-ethylenediamine ( PEPt ) forms a crystalline complex with cytidine-3'-phosphate (3'-CMP) and its structure has been determined by X-ray crystallography. 3'-CMP molecules are hemiprotonated and form hydrogen-bonded pairs that stack above and below the phenanthroline-platinum(II) drug molecule. Sugar residues are in the C2' endo conformation, with glycosidic torsional angles intermediate between the high and low anti forms. The structure is of particular interest since it forms as an end product of the hydrolytic cleavage of the dinucleoside monophosphate, CpG, by the platinum organometallointercalator ( PEPt ). This hydrolytic activity appears to be specific for the RNA dinucleoside monophosphate fragment, since deoxycytidylyl (3'-5')deoxyguanosine (d-CpG) and other deoxyribooligonucleotides are not cleaved under similar conditions.     

Functional properties of factor Va subunits after proteolytic alterations by activated protein C

The two-subunit structure of the factor Va molecule is essential to its function in the prothrombinase complex. In the presence of phospholipids, the cleavage of the light chain of bovine factor Va by activated protein C proceeded at the same rate as the cleavage of the heavy chain. The limited proteolysis of factor Va is accompanied by a parallel loss of factor Va activity. Evidence that loss of activity was solely the result of the cleavage of the heavy chain, was obtained from reconstitution experiments utilizing cleaved and intact chains. The pseudo first-order rate constant of factor Va inactivation by activated protein C was found to be dependent on the amount of phospholipid-bound activated protein C and not on the amount of phospholipid-bound factor Va. However, phospholipids enhance the rate of proteolysis of the phospholipid-binding subunit, i.e. the light chain, and not the cleavage of the heavy chain. Cleavage of the heavy chain and as a consequence the inactivation of factor Va by activated protein C is mediated by phospholipid-bound light chain. After cleavage of the light chain, the 'two-subunit' structure, as well as the phospholipid-binding properties of factor Va were found to be conserved.     

Properties of the hatching enzyme from Xenopus laevis.

Using an anti-(glutathione S-transferase-UVS.2 cDNA) Ig and uterine egg vitelline envelope (UEVE) protein of Xenopus laevis as probes, the hatching enzyme (HE) from Xenopus was solubilized in hatching medium and purified by gel-filtration and ion-exchange chromatography, and characterized in terms of its molecular mass and enzymatic properties. The hatching medium solubilized the UEVE and contained molecules reactive to the anti-(GST UVS.2) Ig against Xenopus HE. It was found that the HE had a molecular mass of 60 kDa, and often preparations also contained a 40-kDa form. The 60-kDa HE had a high hydrolytic and UEVE-solubilizing activity, and its activities against Boc-Leu-Gly-Arg-7-amino-4-methylcoumarin (-NH-Mec) and UEVE were inhibited by anti-(GST UVS.2) Ig in a dose-dependent manner. The 60-kDa form was easily autodigested into a 40-kDa form. The 40-kDa molecule alone had no detectable UEVE-solubilizing activity, even it still had high hydrolytic activity. It probably represents the main protease domain of the 60-kDa form after loss of two CUB repeats during autodigestion or digestion. The autodigestion of the 60-kDa molecule into 40-kDa molecule is probably a congenital behavior for successfully dissolving the embryo envelope during the hatching process. The two molecules may play different roles at different stages of the hatching process, during which they co-ordinate with each other to achieve complete solubilization of the embryo envelope, similar to the high and low choriolytic enzymes in medaka (Oryzias latipes). Their hydrolytic activity against Boc-Leu-Gly-Arg-NH-Mec was optimal at pH of 7.4, and with an apparent Km value of 200 micromol.L-1 at 30 degrees C. The HE is very sensitive to trypsin-specific inhibitors such as leupeptin, (4-amidino-phenyl)methane sulfonyl fluoride, diisopropyl fluorophosphate (DFP) and N-alpha-tosyl-L-lysylchloromethane (Tos-Lys-CH2Cl), indicates that it is a trypsin-type protease. The results on EDTA and some metal ions, combined with the occurrence of a astacin family metalloprotease-specific 'HExHxxGFxHE' sequence in the deduced HE amino-acid sequence, indicates that this HE is a Zn2+ metalloprotease.     

Role of phosphorolytic cleavage in cellobiose and cellodextrin metabolism by the ruminal bacterium Prevotella ruminicola.

In bacteria, cellobiose and cellodextrins are usually degraded by either hydrolytic or phosphorolytic cleavage. Prevotella ruminicola B(1)4 is a noncellulolytic ruminal bacterium which has the ability to utilize the products of cellulose degradation. In this organism, cellobiose hydrolytic cleavage activity was threefold greater than phosphorolytic cleavage activity (113 versus 34 nmol/min/mg of protein), as measured by an enzymatic assay. Cellobiose phosphorylase activity (measured as the release of P(i)) was found in cellobiose-, mannose-, xylose-, lactose-, and cellodextrin-grown cells (> 92 nmol of P(i)/min/mg of protein), but the activity was reduced by more than 74% for cells grown on fructose, L-arabinose, sucrose, maltose, or glucose. A small amount of cellodextrin phosphorylase activity (19 nmol/min/mg of protein) was also detected, and both phosphorylase activities were located in the cytoplasm. Degradation involving phosphorolytic cleavage conserves more metabolic energy than simple hydrolysis, and such degradation is consistent with substrate-limiting conditions such as those often found in the rumen.     

Ontogenetic variations of hydrolytic enzymes in the Pacific oyster Crassostrea gigas

Occurrence and level of hydrolytic enzymatic activity (proteases, glycosidases, phosphatases, lipases, and esterases) were studied in oocytes, larvae, juveniles, and adult haemolymph of the Pacific oyster Crassostrea gigas. Samples were obtained as oocyte lysate supernatant, larval homogenate supernatant, juvenile homogenate supernatant, haemocyte lysate supernatant, and plasma. The presence of enzymes was demonstrated by colorimetric and lysoplate assay techniques. Between stages, significant differences in enzymatic activity determined by the colorimetric technique were found. Higher levels of enzymatic activity were found in the adult stage. Lysozyme-like activity was not found in oocytes, but was present in larvae, juveniles, and adults. In larvae, the highest lysozyme-like activity was in 3-d larvae. Juveniles had a 48-fold higher level of lysozyme-like activity, compared with 20-h larvae and was six-fold higher compared with 3-d larvae. In adults, lysozyme-like activity had a five-fold higher level in haemocyte lysate supernatant compared with plasma and was 98-fold higher compared with 20-h larvae. As determined with the API ZYM kit, 19 hydrolytic enzymatic activities were present, in oocytes, larvae, juveniles, and adult haemolymph of C. gigas. The presence of important lysozyme-like activity was confirmed from trochophora larvae (20 h) to adult stages.     

Methene bridge carbon atom elimination in oxidative heme degradation catalyzed by heme oxygenase and NADPH-cytochrome P-450 reductase

Physiological heme degradation is mediated by the heme oxygenase system consisting of heme oxygenase and NADPH-cytochrome P-450 reductase. Biliverdin IX alpha is formed by elimination of one methene bridge carbon atom as CO. Purified NADPH-cytochrome P-450 reductase alone will also degrade heme but biliverdin is a minor product (15%). The enzymatic mechanisms of heme degradation in the presence and absence of heme oxygenase were compared by analyzing the recovery of 14CO from the degradation of [14C]heme. 14CO recovery from purified NADPH-cytochrome P-450 reductase-catalyzed degradation of [14C]methemalbumin was 15% of the predicted value for one molecule of CO liberated per mole of heme degraded. 14CO2 and [14C]formic acid were formed in amounts (18 and 98%, respectively), suggesting oxidative cleavage of more than one methene bridge per heme degraded, similar to heme degradation by hydrogen peroxide. The reaction was strongly inhibited by catalase, but superoxide dismutase had no effect. [14C]Heme degradation by the reconstituted heme oxygenase system yielded 33% 14CO. Near-stoichiometric recovery of 14CO was achieved after addition of catalase to eliminate side reactions. Near-quantitative recovery of 14CO was also achieved using spleen microsomal preparations. Heme degradation by purified NADPH-cytochrome P-450 reductase appeared to be mediated by hydrogen peroxide. The major products were not bile pigments, and only small amounts of CO were formed. The presence of heme oxygenase, and possibly an intact membrane structure, were essential for efficient heme degradation to bile pigments, possibly by protecting the heme from indiscriminate attack by active oxygen species.     

Effects of human polymorphonuclear leukocyte collagenase on sub-component C1q of the first component of human complement

The similarities in the structure and properties of C1q and collagen prompted us to examine the susceptibility of C1q to human polymorphonuclear leukocyte collagenase. Incubation of C1q with a collagenase preparation resulted in no change in (1) the binding of C1q to immunoglobulin aggregates, (2) the hemolytic function of C1q as measured by reconstitution of C1q-depleted serum in immune hemolysis, or (3) the structural properties of C1q as revealed by gel electrophorettic patterns of the whole molecule or its polypeptide chains. In contrast, rapid inactivation and degradation of C1q was caused by leukocyte elastase.The collagenase preparation was, however, capable of cleaving reduced and carboxamidomethylated C1q into discrete fragments. This activity was attributed to a gelatinase present in the enzyme preparation since (1) the cleavage reaction was inhibited by denatured collagen but not by native collagen and (2) a collagenase fraction free of gelatinolytic activity could not degrade reduced and carboxamidomethylated C1q, while a gelatinase fraction devoid of collagenase activity retained the capacity to effect reduced and carboxamidomethylated C1q. Both collagenase and gelatinase activities were activated from the latent form by trypsin, and inhibited by EDTA.Therefore, it appears that native C1q lacks the structural features present in collagen which are recognized by leukocyte collagenase for hydrolytic action even though the denatured molecule still contains that region capable of being cleaved by gelatinase.     

Ribonuclease III cleavage of a bacteriophage T7 processing signal. Divalent cation specificity, and specific anion effects.

Escherichia coli ribonuclease III, purified to homogeneity from an overexpressing bacterial strain, exhibits a high catalytic efficiency and thermostable processing activity in vitro. The RNase III-catalyzed cleavage of a 47 nucleotide substrate (R1.1 RNA), based on the bacteriophage T7 R1.1 processing signal, follows substrate saturation kinetics, with a Km of 0.26 microM, and kcat of 7.7 min.-1 (37 degrees C, in buffer containing 250 mM potassium glutamate and 10 mM MgCl2). Mn2+ and Co2+ can support the enzymatic cleavage of the R1.1 RNA canonical site, and both metal ions exhibit concentration dependences similar to that of Mg2+. Mn2+ and Co2+ in addition promote enzymatic cleavage of a secondary site in R1.1 RNA, which is proposed to result from the altered hydrolytic activity of the metalloenzyme (RNase III 'star' activity), exhibiting a broadened cleavage specificity. Neither Ca2+ nor Zn2+ support RNase III processing, and Zn2+ moreover inhibits the Mg(2+)-dependent enzymatic reaction without blocking substrate binding. RNase III does not require monovalent salt for processing activity; however, the in vitro reactivity pattern is influenced by the monovalent salt concentration, as well as type of anion. First, R1.1 RNA secondary site cleavage increases as the salt concentration is lowered, perhaps reflecting enhanced enzyme binding to substrate. Second, the substitution of glutamate anion for chloride anion extends the salt concentration range within which efficient processing occurs. Third, fluoride anion inhibits RNase III-catalyzed cleavage, by a mechanism which does not involve inhibition of substrate binding.     

Variations in the enzymatic properties of human complement subcomponent C1s by treatment with human plasma kallikrein

We have investigated the effect of plasma kallikrein digestion upon hydrolytic activities of human C1s. Incubation of C1s (85 kDa) with plasma kallikrein led to progressive cleavages on the heavy chain to yield C1s-K1 (70 kDa) then C1s-K2 (53 kDa). Although these cleavages caused little change in the C2 hydrolytic and esterase activities of C1s, a marked loss in the C4 hydrolytic activity was observed. C1s-K1 and C1s-K2 were purified by DE-52 chromatography and it was found that the proteolysis of C1s into C1s-K1 was accompanied with a decrease in the C4 hydrolytic activity. Although the turnover numbers for the hydrolysis of C4 by C1s-K1 and C1s-K2 were almost the same as that of intact C1s, the Kms for C4 of C1s-K1 and C1s-K2 were found to be increased to 10 times that of intact C1s. This result suggests that the apparent decrease in the C4 hydrolytic activity upon plasma kallikrein digestion of C1s is not due to disruption in the active site but is due to decrease in the affinity between C4 and the C1s derivatives. In support of this assumption, C1s-K1 was found to be devoid of the ability to bind C4b-Sepharose. C1s is capable of forming a dimer through the C1s-binding domain in the N-terminal side of the heavy chain. Although C1s-K1 is still capable of forming a dimer, C1s-K2 fails to form a dimer, suggesting that the N-terminal C1s-binding site is released during cleavage of C1s-K1 into C1s-K2.(ABSTRACT TRUNCATED AT 250 WORDS)     

The caspase-derived C-terminal fragment of betaAPP induces caspase-independent toxicity and triggers selective increase of Abeta42 in mammalian cells

During its physiopathological maturation, the beta-amyloid precursor protein undergoes several distinct proteolytic events by activities called secretases. In Alzheimer's disease, the main histological hallmark called senile plaque is clearly linked to the overproduction of the amyloid peptides Abeta40 and Abeta42, two highly aggregable betaAPP-derived fragments generated by combined cleavages by beta- and gamma-secretases. Recently, an alternative hydrolytic pathway was described, involving another category of proteolytic activities called caspases, responsible for the production of a 31 amino acids betaAPP C-terminal fragment called C31. C31 was reported to lower the viability of N2a cells but the exact mechanisms mediating C31-toxicity remained to be established. Here we show that the transient transfection of pSV2 vector encoding C31 lowers by about 80% TSM1 neuronal cells viability. Arguing against a C31-stimulated apoptotic response, we demonstrate by combined enzymatic and immunological approaches that C31 expression did not modulate basal or staurosporine-induced caspase 3-like activity and pro-caspase-3 activation. Furthermore, C31 did not modify Bax and p53 expressions, poly-(ADP-ribose)-polymerase cleavage and cytochrome c translocation into the cytosol. However, we established that C31 overexpression triggers selective increase of Abeta42 but not Abeta40 production by HEK293 cells expressing wild-type betaAPP751. Altogether, our data demonstrate that C31 induces a caspase-independent toxicity in TSM1 neurons and potentiates the pathogenic betaAPP maturation pathway by increasing selectively Abeta42 species in wild type-betaAPP-expressing human cells.     

Structure-based rational design of streptavidin mutants with pseudo-catalytic activity

Introduction of enzymatic activity into proteins or other types of polymers by rational design is a major objective in the life sciences. To date, relatively low levels of enzymatic activity could be introduced into antibodies by using transition-state analogues of haptens. In the present study, we identify the structural elements that contribute to the observed hydrolytic activity in egg white avidin, which promote the cleavage of active biotin esters (notably biotinyl p-nitrophenyl ester). The latter elements were then incorporated into bacterial streptavidin via genetic engineering. The streptavidin molecule was thus converted from a protector to an enhancer of hydrolysis of biotin esters. The conversion was accomplished by the combined replacement of a "lid-like loop" (L3,4) and a leucine-to-arginine point mutation in streptavidin. Interestingly, neither of these elements play a direct role in the hydrolytic reaction. The latter features were thus shown to be responsible for enhanced substrate hydrolysis. This work indicates that structural and non-catalytic elements of a protein can be modified to promote the induced fit of a substrate for subsequent interaction with either a catalytic residue or water molecules. This approach complements the conventional design of active sites that involves direct modifications of catalytic residues.     

Generation of DNA double-strand breaks by two independent enzymatic activities in nuclear extracts

We have reported the existence in rat nuclear extracts of a specific cleavage activity on a DNA fragment containing the human minisatellite MsH42 region (minisatellite plus its flanking sequences). Here, we have developed a system to analyse the nature of the cleavage products from the MsH42 region generated by the nuclear extracts. Our results demonstrated the formation of DNA double-strand breaks (DSB) in the MsH42 region by two different enzymatic activities, and that their distribution along this fragment changes depending on the presence of Mg2+. In the assays with Mg2+, the DSB were located in the minisatellite and its 3'-flanking region, showing preference for G-rich stretches. Oligonucleotide mutagenesis analysis confirmed that this enzymatic activity has a strong preference for G-tracts and that the recognition site is polarized towards the 3' end. Moreover, this activity cuts GC palindromes efficiently. In contrast, in the experiments without Mg2+, most DSB were mapped within the minisatellite flanking sequences. The analysis with oligonucleotides showed that G-tracts are recognized by this endonuclease activity, but with differences in the cleavage behaviour with respect to the reactions observed with Mg2+. The existence of two separate activities (Mg2+-dependent and Mg2+-independent) for the production of DSB was confirmed by analysing the effect of EGTA, N-ethyl maleimide, ionic strength, and by preincubations of the nuclear extracts at different temperatures. The tissue distribution of both DSB-producing activities was also different. The in vitro system used in the present work may be a useful tool for studying the formation of DSB and for investigation of the mechanisms of DNA repair.     

Osmolytes protect mitochondrial F(0)F(1)-ATPase complex against pressure inactivation

We have previously reported that carbohydrates and polyols protect different enzymes against thermal inactivation and deleterious effects promoted by guanidinium chloride and urea. Here, we show that these osmolytes (carbohydrates, polyols and methylamines) protect mitochondrial F(0)F(1)-ATPase against pressure inactivation. Pressure stability of mitochondrial F(0)F(1)-ATPase complex by osmolytes was studied using preparations of membrane-bound submitochondrial particles depleted or containing inhibitor protein (IP). Hydrostatic pressure in the range from 0.5 to 2.0 kbar causes inactivation of submitochondrial particles depleted of IP (AS particles). However, the osmolytes prevent pressure inactivation of the complex in a dose-dependent manner, remaining up to 80% of hydrolytic activity at the highest osmolyte concentration. Submitochondrial particles containing IP (MgATP-SMP) exhibit low ATPase activity and dissociation of IP increases the hydrolytic activity of the enzyme. MgATP-SMP subjected to pressure (2.2 kbar, for 1 h) and then preincubated at 42 degrees C to undergo activation did not have an increase in activity. However, particles pressurized in the presence of 1.5 M of sucrose or 3.0 M of glucose were protected and after preincubation at 42 degrees C, showed an activation very similarly to those kept at 1 bar. In accordance with the preferential hydration theory, we believe that osmolytes reduce to a minimum the surface of the macromolecule to be hydrated and oppose pressure-induced alterations of the native fold that are driven by hydration forces.     

Activation of C1r by proteolytic cleavage.

C1r was unable to cleave and activate proenzyme C1s unless first incubated at 37 degrees C in the absence of calcium before the addition of C1s. The acquisition of ability to activate C1s was associated with, and paralleled by, cleavage of each of the two noncovalently bonded 95,000 dalton chains of the molecule into disulfide linked subunits of 60,000 and 35,000 daltons, respectively. Thus, C1r is converted from an inactive form into an enzyme, C1r, able to cleave and activate C1s by proteolytic cleavage in marked analogy to the activation of several other complement enzymes. Trypsin was also found to cleave C1r but at a different site, and its action did not lead to C1r activation. C1r activation was inhibited by calcium, polyanethol sulfonate, C1 inactivator, and DFP but not by a battery of other protease inhibitors. C1 inactivator inhibited C1r by forming a complex with C1r via sites located on the light chain of the molecule. In other studies, cleavage of C1r was not accelerated by the addition of C1r ot C1s. C1r and C1r were found to have the same m.w., sedimentation coefficient, and diffusion coefficients. They differed, however, in charge with C1r migrating as a Beta-globulin and C1r as a gammaglobulin on electrophoresis in agarose. The amino acid composition of C1r and of each of the two polypeptide chains of Clr was determined. Both chains contained carbohydrate. Proteolytic cleavage of the C1r molecule was found to occur on addition of aggregated IgG to a mixture of C1q, C1r, and C1s in the presence of calcium. Neither C1q, C1s nor aggregated IgG alone, not C1r nor C1s induced C1r cleavage. Liquoid, an inhibitor of C1 activation, inhibited C1r cleavage. Thus, proteolytic cleavage of C1r appears to be a biologically meaningful event occurring during the activation of C1.     

Natural substrates and inhibitors of mannan-binding lectin-associated serine protease-1 and -2: a study on recombinant catalytic fragments

Mannan-binding lectin-associated serine protease (SP) (MASP)-1 and MASP-2 are modular SP and form complexes with mannan-binding lectin, the recognition molecule of the lectin pathway of the complement system. To characterize the enzymatic properties of these proteases we expressed their catalytic region, the C-terminal three domains, in Escherichia coli. Both enzymes autoactivated and cleaved synthetic oligopeptide substrates. In a competing oligopeptide substrate library assay, MASP-1 showed extreme Arg selectivity, whereas MASP-2 exhibited a less restricted, trypsin-like specificity. The enzymatic assays with complement components showed that cleavage of intact C3 by MASP-1 and MASP-2 was detectable, but was only approximately 0.1% of the previously reported efficiency of C3bBb, the alternative pathway C3-convertase. Both enzymes cleaved C3i 10- to 20-fold faster, but still at only approximately 1% of the efficiency of MASP-2 cleavage of C2. We believe that C3 is not the natural substrate of either enzyme. MASP-2 cleaved C2 and C4 at high rates. To determine the role of the individual domains in the catalytic region of MASP-2, the second complement control protein module together with the SP module and the SP module were also expressed and characterized. We demonstrated that the SP domain alone can autoactivate and cleave C2 as efficiently as the entire catalytic region, while the second complement control protein module is necessary for efficient C4 cleavage. This behavior strongly resembles C1s. Each MASP-1 and MASP-2 fragment reacted with C1-inhibitor, which completely blocked the enzymatic action of the enzymes. Nevertheless, relative rates of reaction with alpha-2-macroglobulin and C1-inhibitor suggest that alpha-2-macroglobulin may be a significant physiological inhibitor of MASP-1.     

Evidence for processing enzymes in the abdominal gland of the newt, Cynops pyrrhogaster, that generate sodefrin from its biosynthetic precursor

Sodefrin (Ser-Ile-Pro-Ser-Lys-Asp-Ala-Leu-Leu-Lys) is a female-attracting peptide pheromone secreted by the abdominal gland of the male red-bellied newt, Cynops pyrrhogaster. Sequence analysis of a cDNA encoding sodefrin revealed that the peptide is located in the C-terminal region of its precursor protein (residues 177-186 of preprosodefrin) and extended from its C-terminus by the tripeptide sequence Ile(187)-Ser(188)-Ala(189) and flanked at its N-terminus by Leu(174)-Gly(175)-Arg(176). This suggests that sodefrin is generated by enzymatic cleavage at monobasic (Lys and Arg) sites within the precursor molecule. To demonstrate the presence in the abdominal gland of proteolytic enzymes capable of generating sodefrin, an enzymatic assay was developed using t-butoxycarbo-nyl (Boc)-Leu-Gly-Arg-4methylcoumaryl-7-amide (MCA) and Boc-Leu-Leu-Lys-MCA as synthetic substrates. A crude extract of the abdominal gland hydrolyzed both substrates to liberate 7-amino-4- methylcoumarin, suggesting that enzymes that generate sodefrin from its precursor molecule are present in the gland. The activity in the extract for cleaving Boc-Leu-Gly-Arg-MCA was optimal at pH 9.0 and 45 degrees C and for Boc-Leu-Leu-Lys-MCA at pH 9.0 and 40 degrees C. The effects of a range of specific inhibitors on activities in the extract suggest an involvement of enzymes belonging to the serine protease family. It was also demonstrated that enzymatic activity in an extract of the abdominal glands of sexually developed males was significantly (three- to six-fold; p<0.01) higher than that of sexually undeveloped males.     

Purification and some properties of phospholipase C (alpha-toxin) of Clostridium perfringens.

1. Phospholipase C[EC 3.1.4.3] was purified from the culture filtrate of Clostridium perfringens by successive chromatographies on CM-Sephadex, DEAE-Sephadex, and Sephadex G-100. During the purification it was noted that, beside the monomer form of the enzyme which was purified, a part of the enzyme existed in active polymerized forms. 2. The purified preparation gave a single band on polyacrylamide gel electrophoresis and gave a single precipitin line in immunodiffusion with the National Standard gas gangrene (C. perfringens) antitoxin, indicating the homogeneity of the preparation. 3. The specific lecithin-hydrolyzing activity of the purified preparation was comparable to that of a preparation obtained by affinity chromatography, which had the highest specific activity previously reported. 4. The molecular weight of the purified enzyme was estimated to be 43,000 by SDS-polyacryl-amide gel electrophoresis, although the same preparation gave a molecular weight of 31,000 as determined by gel filtration on Sephadex G-150. From this and the above finding that a part of the enzyme exists in active polymerized forms, the discrepancy among reported values for the molecular weight of C. perfringens phospholipase C can be accounted for. 5. For maximum hydrolytic activity toward lecithin, the enzyme required sodium deoxycholate (SDC) and Ca2+ ions. In the presence of 6 mM Ca2+, the optimal molar ratio of SDC to lecithin for maximal hydrolytic activity was about 0.5 for dipalmitoyl lecithin and about 1.0 for egg lecithin. The effects of various divalent cations on the enzymatic hydrolysis were also investigated. 6. The effects of sodium deoxycholate and Ca2+ ions on the enzymatic hydrolysis are discussed, based on their possible roles in mixed micelle formation.     

Kinetic characterization of gyroxin,a serine protease from Crotalus durissus terrificus venom

This work describes for the first time the characterization of the enzymatic features of gyroxin, a serine protease from Crotalus durissus terrificus venom, capable to induce barrel rotation syndrome in rodents. Measuring the hydrolysis of the substrate ZFR-MCA, the optimal pH for proteolytic cleavage of gyroxin was found to be at pH 8.4. Increases in the hydrolytic activity were observed at temperatures from 25 °C to 45 °C, and increases of NaCl concentration up to 1 M led to activity decreases. The preference of gyroxin for Arg residues at the substrate P1 position was also demonstrated. Taken together, this work describes the characterization of substrate specificity of gyroxin, as well as the effects of salt and pH on its enzymatic activity.     

A biologically active thrombin cleavage product of human serum spreading factor

Purified human serum spreading factor preparations consisting of two immunologically-related, biologically-active proteins of molecular weights approximately 65,000 and 75,000 were incubated with purified hydrolytic enzymes: papain, neuraminidase and thrombin. Biologically active products of the enzymatic digestions were obtained in each case. Digestion of serum spreading factor preparations with thrombin produced a single active form of molecular weight approximately 57,000. Generation of a single molecular weight form of serum spreading factor by thrombin cleavage of the two higher molecular weight forms should simplify studies of the biochemistry and biology of this protein, and may represent a reaction of physiological significance.