Purification and some physico-chemical and enzymic properties of a calcium ion-activated neutral proteinase from rabbit skeletal muscle

Ca²⁺-activated neutral proteinase was purified from rabbit skeletal muscle by a method involving DEAE-Sephacel chromatography, affinity chromatography on organomercurial–Sepharose and gel filtration on Sephacryl S-200 and Sephadex G-150. The SDS (sodium dodecyl sulphate)/polyacrylamide-gel-electrophoresis data show that the purified enzyme contains only one polypeptide chain of mol.wt. 73000. The purification procedure used allowed us to eliminate a contaminant containing two components of mol.wt. about 30000 each. Whole casein or α₁-casein were hydrolysed with a maximum rate at 30°C, pH7.5, and with 5mm-CaCl₂, but myofibrils were found to be a very susceptible substrate for this proteinase. This activity is associated with the destruction of the Z-discs, which is caused by the solubilization of the Z-line proteins. The activity of the proteinase in vitro is not limited to the removal of Z-line. SDS/polyacrylamide-gel electrophoresis on larger plates showed the ability of the proteinase to degrade myofibrils more extensively than previously supposed. This proteolysis resulted in the production of a 30000-dalton component as well as in various other higher- and lower-molecular-weight peptide fragments. Troponin T, troponin I, α-tropomyosin, some high-molecular-weight proteins (M protein, heavy chain of myosin) and three unidentified proteins are degraded. Thus the number of proteinase-sensitive regions in the myofibrils is greater than as previously reported by Dayton, Goll, Zeece, Robson & Reville [(1976) Biochemistry 15, 2150–2158]. The Ca²⁺-activated neutral proteinase is not a chymotrypsin- or trypsin-like enzyme, but it reacted with all the classic thiol-proteinase inhibitors for cathepsin B, papain, bromelain and ficin. Thus the proteinase was proved to have an essential thiol group. Antipain and leupeptin are also inhibitors of the Ca²⁺-activated neutral proteinase.     

Calcium-binding protein regucalcin increases calcium-independent proteolytic activity in rat liver cytosol

The effect of regucalcin, isolated from rat liver cytosol, on neutral proteolytic activity in the hepatic cytosol was investigated. The Ca²⁺-requiring proteinase required 5–10 µM Ca²⁺ for maximal activity in the presence of a protein substrate (globin). The proteinase activity was markedly elevated by the addition of regucalcin (0.25–2.0 µM) in the absence or presence of Ca²⁺ (5.0 µM) added. The effect of regucalcin, however, was the greater in the absence of Ca²⁺ than that in the presence. The pronounced effect of regucalcin on the proteinase activity was also seen in the presence of 1.0 mM EGTA with or without Ca²⁺ (5.0 µM). In the absence of Ca²⁺, the regucalcin-increased proteinase activity was clearly inhibited by the presence of anti-regucalcin antiserum (diluted to 240-fold), leupeptin (20 and 200 µg/ml), and heavy metals (25 µM cadmium or 25 µM zinc), although the inhibition was not complete at the concentration used. The present findings suggest that regucalcin increases proteolytic activity in rat liver cytosol, and that regucalcin may activate Ca²⁺-independent neutral cysteinyl-proteinase.     

Substance P degrading systems of rat parotid and hypothalamus

Inactivation of substance P and its C-terminal hexapeptide analog [p-Glu⁶]substance P6–11 was studied in rat parotid and hypothalamic slices. It was found that in the parotid slice system the decay of substance P induced K⁺ release occurs concurrently with a decrease in the biologically active concentration of the peptide in the medium. The inactivation was further studied using [p-Glu⁶]substance P6–11 as substrate in the parotid and in the hypothalamic slice systems. In both tissue preparations the hexapeptide is degraded to small peptide fragments by metalloendopeptidase. Separation of the peptide fragments by high performance liquid chromatography and determination of their amino acid composition showed that in the hypothalamic slice system the major cleavage of the hexapeptide analog occurs between Phe⁸-Gly⁹ with minor cleavage sites between Phe⁷-Phe⁸ and Gly⁹-Leu¹⁰. In the rat parotid slice system the major cleavage occurs between Gly⁹-Leu¹⁰ with a minor cleavage site between Phe⁷-Phe⁸. The degradation of the hexapeptide analog in the hypothalamic system was inhibited 77% and 67% by treatment with 1 mM p-chloromercuriphenylsulfonate and p-chloromercuribenzoate, respectively, whereas in the parotid system these reagents inhibited the degradation of the hexapeptide only by 15% and 8%. These results may indicate that different proteases in the parotid and hypothalamus are involved in degradation of substance P. Kinetic studies, including the use of various inhibitors as well as competition by the peptide hormones somatostatin, LHRH, TRH and Leu-enkephalin-NH₂, revealed that in both tissues the hexapeptide analog is a preferred substrate for degradation by protease of considerable specificity towards the C-terminal sequence of substance P. It is suggested that this metalloendopeptidase may be important in the termination of the substance P response.     

The purification and properties of a fibrinolytic neutral metalloendopeptidase from Streptococcus faecalis

A protease has been isolated by affinity chromatography from culture filtrates of a strain of Streptococcus faecalis previously shown to produce a flbrinolytic enzyme. The pH optimum, molecular weight, metal ion chelator sensitivity, and peptidase specificity place this enzyme in the class of bacterial neutral metalloendopeptidase typified by thermolysin and the Bacillus subtilis neutral proteases. Differences with respect to chemical modification and thermal stability exist between the S. faecalis enzyme and the latter proteases. The S. faecalis enzyme (designated EM 19000) renders fibrinogen incoagulable by degradation of the B (β) chains.     

The new fluorogenic substrates of neutrophil proteinase 3 optimized in prime site region

Previously selected by the combinatorial chemistry approach, potent fluorogenic substrate of proteinase 3 was used as the starting structure to design new substrates. The general formula of the synthesized peptides is as follows: ABZ-Tyr-Tyr-Abu-ANB-X-NH₂, where ANB (5-amino-2-nitrobenzoic acid) served as a chromophore and an acceptor of fluorescence, ABZ (aminobenzoic acid) is a donor of fluorescence in these fluorescence resonance energy transfer (FRET) peptides, and X is a proteinogenic amino acid (except Cys). The introduced modifications influenced substrate activity of the synthesized peptides. The highest value of specificity constant for proteinase 3 was obtained for the single peptide with Gln in the discussed position (k_cat/K_M = 275,000 M⁻¹ s⁻¹), which was nearly twice as active as the reference compound (lacking a substituent in the X position). In addition, more efficient energy transfer was observed, due mainly to the bathochromic effect for the introduced modification. This approach opens a new possibility to design potent and highly specific substrates of proteinase 3 and other proteinases optimized in the prime site region.     

Activation of human plasma prekallikrein by Pseudomonas aeruginosa elastase. II. Kinetic analysis and identification of scissile bondof prekallikrein in the activation

Activation of human plasma prekallikrein by a bacterial metalloendopeptidase, Pseudomonas aeruginosa elastase, was reported (Shibuya et al. (1991) Biochim. Biophys. Acta 1097, 23–27). Details of the activation process were presently studied. The activation accompanied limited proteolysis of a peptide bond inside of a disulfide bridge of prekallikrein molecule. Amino acid sequencing analysis of the newly generated amino-terminal revealed that the cleavage site was Arg₃₇₁-Ile₃₇₂ bond which is the scissile bond in the activation of prekallikrein with trypsin-type proteinases. A pentapeptide substrate, 2-aminobenzoyl-Ser-Thr-Ile-Val-4-nitrobenzylamide, which contained the amino acid sequence identical to that around the scissile bond of prekallikrein was synthesized. Pseudomonal elastase, indeed, hydrolyzed the substrate at Arg-Ile bond with the kinetic parameters of K_m = 118 μM, k_cat = 1.56/s and k_cat/K_m = 1.33 · 10⁴/s M. These results indicated that the Arg₃₇₁-Ile₃₇₂ bond was sensitive not only to trypsin-type serine proteinases, but also a bacterial metalloproteinase. Kinetic analysis of the prekallikrein activation by psuedomonal elastase, however, revealed that the activation rate was show, though the K_m values was good enough to expect an occurence of this activation in vivo (K_m = 248 nM, k = 6.8 · 10^?4/s, and k_cat/K_m = 2.7 · 10³/s M. The activation rate of prekallikrein by pseudomonal elastase in Hageman factor deficient plasma was remarkably improved when the plasma was reconstituted with purified Hageman factor molecule. From the results, a biologuical significance of the proteinase cascade in the plasma kinin generation was also indicated. The present in vitro study might support the hypothesis that the Hageman factor/kallikrein-kinin system plays an important role in bacterial infection including the pseudomonal one.     

Partial Isolation and Degradation of Caseins by Cell Wall Proteinase(s) of Streptococcus cremoris HP

The cell wall proteinase fraction of Streptococcus cremoris HP has been isolated. This preparation did not exhibit any activity due to either specific peptidases known to be located near the outside surface of and in the membrane or intracellular proteolytic enzymes. By using thin-layer chromatography for the detection of relatively small hydrolysis products which remain soluble at pH 4.6, it was shown that β-casein is preferentially attacked by the cell wall proteinase. This was also the case when whole casein or micelles were used as the substrate. κ-casein hydrolysis is a relatively slow process, and α_s-casein degradation appeared to proceed at an extremely low rate. These results could be confirmed by using ¹⁴CH₃-labeled caseins. A relatively fast and linear initial progress of ¹⁴CH₃-labeled β-casein degradation is not inhibited by α_s-casein and only slightly by κ-casein at concentrations of these components which reflect their stoichiometry in the micelles. Possible implications of β-casein degradation for growth of the organism in milk are discussed.     

Characterization of the Cell Wall-Bound Proteinase of Lactobacillus casei HN14

Lactobacillus casei HN14, which was isolated from homemade cheese, produces an extracellular, cell wall-bound proteinase. The HN14 proteinase can be removed from the cell envelope by washing the cells in a Ca²⁺-free buffer. The activity of the crude proteinase extract is inhibited by phenylmethylsulfonyl fluoride, showing that the enzyme is a serine-type proteinase. Considering the substrate specificity, the HN14 proteinase is similar to the lactococcal PI-type enzyme, since it hydrolyzes β-casein only. Lactobacillus casei HN14 appeared to be plasmid free, which suggests that the proteinase gene is chromosomally located. Chromosomal DNA of this strain hybridizes with DNA probes Q1 (which contains a fragment of the prtM gene) and Q6 and Q92 (which contain fragments of the prtP gene); all three probes originated from the proteinase gene region of Lactococcus lactis subsp. cremoris Wg2. A restriction enzyme map of the proteinase region of Lactobacillus casei HN14 was constructed on the basis of hybridization experiments. Comparison of the restriction enzyme maps of the Lactobacillus casei HN14 proteinase gene region and those of lactococcal proteinase gene regions studied so far indicates that they are highly similar.     

The irreversible binding of amyloid peptide substrates to insulin-degrading enzyme: A biological perspective

Insulin-degrading enzyme (IDE) is a conserved Zn²⁺metalloendopeptidase involved in insulin degradation and in the maintenance of brain steady-state levels of amyloid β peptide (Aβ) of Alzheimer''s disease (AD). Our recent demonstration that IDE and Aβ are capable of forming a stoichiometric and extremely stable complex raises several intriguing possibilities regarding the role of this unique protein-peptide interaction in physiological and pathological conditions. These include a protective cellular function of IDE as a “dead-end chaperone” alternative to its proteolytic activity and the potential impact of the irreversible binding of Aβ to IDE upon its role as a varicella zoster virus receptor. In a pathological context, the implications for insulin signaling and its relationship to AD pathogenesis are discussed. Moreover, our findings warrant further research regarding a possible general and novel interaction between amyloidogenic peptides and other Zn²⁺metallopeptidases with an IDE-like fold and a substrate conformation-dependent recognition mechanism.Key words: amyloid, insulin-degrading enzyme, peptides, alzheimer''s disease, irreversible binding, metalloproteases     

Effects of Antifungal Agents in Sap Activity of <Emphasis Type="Italic">Candida albicans</Emphasis> Isolates

Some antifungal agents have shown to exert effects on expression of virulent factors of Candida as the production of secretory aspartyl proteinase (Sap). In this study, we sought to determine and to compare the influence of fluconazole and voriconazole in proteinase activity of this microorganism. Thirty-one isolates obtained from oral mucosa of human immunodeficiency virus positive (HIV⁺) patients were used in this study. The minimal inhibitory concentrations (MIC) of fluconazole and voriconazole were determined using the broth microdilution method with RPMI 1640 medium and with yeast carbon base–bovine serum albumin (YCB–BSA) medium. The Sap activity following by digestion of BSA as substrate was determined for four Candida albicans strains arbitrarily chosen according to susceptibility (susceptible or resistant) to fluconazole or voriconazole. Besides, the SAP1 to SAP7 genes were screened by PCR for the same isolates that were determined by the Sap activity. In vitro susceptibility testing using the two media presented similar MIC values. Increased Sap activity was observed in resistant isolates on presence of drugs, but the Sap activity by susceptible isolates to azoles showed different behavior on the presence of drug. We detected the presence of SAP1 to SAP7 genes from all susceptible or resistant C. albicans isolates. The present study provides important data about the proteinase activity and the presence of genes of SAP family in fluconazole and voriconazole susceptible or resistant C. albicans isolates.     

Evidence for conversion of N-Tyr-MIF-1 into MIF-1 by a specific brain aminopeptidase

N-Tyr-MIF-1 (Tyr-Pro-Leu-Gly·NH₂), an immunoreactive neuropeptide exhibiting saturable high affinity binding in rat brain was found to be converted into MIF-1 (Pro-Leu-Gly·NH₂) by a specific brain aminopeptidase present in rat brain homogenates or cytosol, but with low activity associated with synaptosomal plasma membranes and microsomes. Conversion occurred at a rate of 16 μmol per g w/wt per h and was unaffected by puromycin but inhibited by bestatin (I₅₀, 5 × 10^?5 M). Aminopeptidases purified from cytosolic fractions of rat brain (arylamidase), mouse brain (Mn²⁺-activated aminopeptidase) or porcine kidney (leucine aminopeptidase) were inactive towards N-Tyr-MIF-1 but degraded MIF-1 with release of Leu-Gly·NH₂ as detected by RP-HPLC procedures. Morphiceptin (Tyr-Pro-Phe-Pro·NH₂), a μ opioid agonist, also acted as a substrate for the N-Tyr-MIF-1 converting enzyme with cleavage of the Tyr-Pro bond. These tetrapeptides, but not MIF-1 or its N-blocked analogs, were degraded in vitro by a metalloendopeptidase purified from kidney membranes. Since dipeptide products were not detected for crude extracts, a significant role for brain metalloendopeptidase on turnover can be excluded. Thus the results point to the presence of a specific (X-Pro-degrading) aminopeptidase in brain cytosol as an enzyme responsible for converting N-Tyr-MIF-1 and inactivating morphiceptin.     

Some Properties of Neutral Proteinases I and II of Aspergillus sojae as Zinc-containing Metalloenzyme

Some experiments were carried out with purified neutral proteinases I and II of Aspergillus sojae in relation to their characteristics as metalloenzyme.

The both enzymes contained one gram atom of zinc and about two gram atoms of calcium per mole (molecular weights of 41,700 for I and 19,800 for II were estimated by gel filtration) of enzyme protein, and the zinc was essential for the activity. Some metal-chelating agents, such as ethylenediaminetetraacetic acid (EDTA), o-phenanthroline, 8-hydroxyquinoline and α,α′-dipyridyl, inhibited the activity of the both enzymes. In the inactivation of neutral proteinase II by EDTA a distinct pH-dependency was observed. The EDTA-inactivated enzymes were reactivated fully or partially by the addition of some metal ions such as Zn²⁺, Co²⁺, Mn²⁺, Cu²⁺ (only neutral proteinase II) and Ni²⁺. Zinc-free apo-enzymes were prepared from the native enzymes by the dialysis against EDTA solution. The apo-enzyme of neutral proteinase I still contained calcium, while that of neutral proteinase II did not. The apo-enzymes restored their activity for the most part either by the addition of excess amount of zinc or by mixing with a stoichiometric amount of zinc in the presence of calcium at an alkaline condition.     

Degradations of human immunoglobulins and hemoglobin by a 60 kDa cysteine proteinase of Trichomonas vaginalis

The present study was undertaken to investigate the role of cysteine proteinase of Trichomonas vaginalis in escaping from host defense mechanism. A cysteine proteinase of T. vaginalis was purified by affinity chromatography and gel filtration. Optimum pH for the purified proteinase activity was 6.0. The proteinase was inhibited by cysteine and serine proteinase inhibitors such as E-64, NEM, IAA, leupeptin, TPCK and TLCK, and also by Hg²⁺, but not affected by serine-, metallo-, and aspartic proteinase inhibitors such as PMSF, EDTA and pepstatin A. However, it was activated by the cysteine proteinase activator, DTT. The molecular weight of a purified proteinase was 62 kDa on gel filtration and 60 kDa on SDS-PAGE. Interestingly, the purified proteinase was able to degrade serum IgA, secretory IgA, and serum IgG in time- and dose-dependent manners. In addition, the enzyme also degraded hemoglobin in a dose-dependent manner. These results suggest that the acidic cysteine proteinase of T. vaginalis may play a dual role for parasite survival in conferring escape from host humoral defense by degradation of immunoglobulins, and in supplying nutrients to parasites by degradation of hemoglobin.     

Two cytosolic,Ca2+-dependent,neutral proteinases from rabbit liver: Purification and properties of the proenzymes

Two Ca²⁺-requiring proteinases have been purified from rabbit liver cytosol and shown to be present in isolated hepatocytes. They differ in relative molecular mass, with the major and minor forms, M_r = 150,000 and M_r = 200, 000, accounting for 75 and 18% of the total cytosolic neutral proteinase activity, respectively. Both are recovered as inactive proenzymes that can be converted to the active, low-Ca²⁺-requiring proteinases by incubation with Ca²⁺ and substrate [S. Pontremoli, E. Melloni, F. Salamino, B. Sparatore, M. Michetti, and B. L. Horecker (1984) Proc. Natl. Acad. Sci. USA81, 53–56. Each proenzyme is composed of two subunits, with molecular masses of 80 and 100 kDa, respectively. Activation of the proenzymes was found to correlate with their dissociation into subunits. The optimum pH for conversion of the proenzymes to the active proteinases in the presence of 5 mm Ca²⁺ and 2 mg/ml of denatured globin was approximately 7.5, and the same pH optimum was observed for the digestion of denatured globin by the activated proteinases. Following activation, each proteinase was observed to undergo autolytic inactivation at rates that were dependent on the concentration of both Ca²⁺ and the digestible substrate. A model is proposed for the activation of the proenzymes and the subsequent inactivation of the active proteinases.     

Characteristics of the Chromaffin Granule Aspartic Proteinase Involved in Proenkephalin Processing

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

Partial purification and characterization of trypsin-like proteinases from insecticide-resistant and -susceptible strains of the maize weevil,Sitophilus zeamais

Serine proteinases from three strains of Sitophilus zeamais (Coleoptera: Curculionidae), one susceptible and two resistant to insecticides — one exhibiting fitness cost (resistant cost strain) and the other lacking it (resistant no-cost strain), were partially purified using an aprotinin–agarose affinity column providing purification factors ranging from 36.5 to 51.2%, with yields between 10 and 15% and activity between 529 and 875 µM/min/mg protein with the substrate N-α-benzoyl-l-Arg-p-nitroanilide (L-BApNA). SDS-PAGE of the purified fraction revealed a 56,000 Da molecular mass band in all strains and a 70,000 Da band more visible in the resistant no-cost strain. The purified proteinases from all strains were inhibited by phenylmethyl sulphonyl fluoride (PMSF), N-α-tosyl-l-lysine chloromethyl ketone (TLCK), aprotinin, benzamidine and soybean trypsin inhibitor (SBTI) characterizing them as trypsin-like serine proteinases. Trypsin-like proteinases from the resistant strains exhibited higher affinity for L-BApNA. The resistant no-cost strain exhibited V_max-values 1.5- and 1.7-fold higher than the susceptible and resistance cost strains, respectively. A similar trend was also observed when using N-α-p-tosyl-L-Arg methyl ester (L-TAME) as substrate. These results provide support to the hypothesis that the enhanced serine proteinase activity may be playing a role in mitigating physiological costs associated with the maintenance of insecticide resistance mechanisms in some maize weevil strains.     

Partial purification and characterization of endoproteinases from senescing barley leaves

Two major endoproteinases were purified from senescing primary barley leaves. The major enzyme (EP₁) appeared to be a thiol proteinase and accounted for about 85% of the total proteolytic activity measured in vitro. This proteinase was purified 5,800-fold and had a molecular weight of 28,300. It was highly unstable in the absence of dithiothreitol or at a pH greater than 7.5. Leupeptin, at a concentration of 10 micromolar, inhibited this enzyme 100%. A second proteinase (EP₂) was purified approximately 50-fold and had a molecular weight of 67,000. It was inhibited 20% by 1 millimolar dithiothreitol and 50% by 1 millimolar phenylmethyl sulfonylfluoride. EP₂ contributed about 15% of the total proteolytic activity measured in vitro. Both proteinases hydrolyzed a variety of artificial and protein substrates, and both had pH optima of 5.5 to 5.7 when either azocasein or [¹⁴C]ribulose-1,5-bisphosphate carboxylase ([¹⁴C]RuBPCase) was the substrate. The thiol endoproteinase hydrolyzed azocasein linearly but hydrolyzed [¹⁴C]RuBPCase biphasically. A third endoproteinase (EP₃), not detected by standard proteolytic assays, was observed when [¹⁴C]RuBPCase was the substrate.     

Substrate specificity of two cationic lipases with high phospholipase A1 activity purified from guinea pig pancreas I. Studies on neutral glycerides

The substrate specificity of two cationic lipases with high phospholipase A₁ activity purified from guinea pig pancreas has been tested towards various neutral glycerides. Triolein hydrolysis proceeded in the absence of di- and monoolein accumulation. Optimal conditions for di- and monoolein hydrolysis included an alkaline pH (9–10), a substrate concentration of 10 mM, and the presence of sodium deoxycholate (12 and 24 mM, respectively). Pancreatic colipase (bovine) had no effect on the activity of the two lipases. The comparison between the rates of hydrolysis of various substrates revealed the following order of decreasing enzyme activity: diolein > 1(3)-monoolein > tributyrin = triacetin ⩾ triolein = 2-monoolein. No hydrolysis of p-nitrophenylacetate and cholesteryloleate could be detected. Using 1-[³H]palmitoyl-2-[¹⁴C]linoleoyl-sn-glycerol, both enzymes displayed a strong preference for the 1-position, leading to the accumulation of 2-[¹⁴C]linoleoyl-sn-glycerol. Identical activities were found for the two lipases. It is concluded that the two cationic lipases from guinea pig pancreas represent a unique group of lipolytic enzymes different from other previously described enzymes, including classical pancreatic lipase, gastric and lingual enzymes, mold lipases and carboxylesterhydrolase.     

News & Notes: Specificity of a Neutral Zn-Dependent Proteinase from Thermoactinomyces sacchari Toward the Oxidized Insulin B Chain

The proteolytic specificity of the neutral Zn-dependent proteinase from Thermoactinomyces sacchari was determined by analysis of the peptides obtained after incubation with the oxidized insulin B chain as a substrate. The enzyme is an endopeptidase with broad specificity. In total, 12 peptide bonds in the B chain of insulin were hydrolyzed. The major requirement is that a hydrophobic residue such as Leu, Val, or Phe should participate with the α-amino group in the bond to be cleaved. However, hydrolysis of bonds at the N-terminal side of His, Thr, and Gly was also observed. The peptide bond Leu 15–Tyr 16 in the oxidized insulin B chain, which is the major cleavage site for the alkaline microbial proteinases, is resistant to the attacks of the enzyme from Thermoactinomyces sacchari and other neutral proteinases. The proteolytic activity of the Zn-dependent proteinase from T. sacchari is different from those of other metalloendopeptidases from microorganisms. Received: 10 November 1999 / Accepted: 15 December 1999     

Degradation of ribulose-1,5-bisphosphate carboxylase by proteolytic enzymes from crude extracts of wheat leaves

In crude extracts from the primary leaf of wheat seedlings, Triticum aestivum L., cv. Olympic, maximum proteinase activity, as determined by measuring the rate of release of amino nitrogen from ribulose-bisphosphate carboxylase (RuBPCase), was found to be obtained only when EDTA and L-cysteine were included in the extraction buffer. Highest proteinase activity was obtained by grinding at pH 6.8, although the level of activity was similar in the pH range 5.6 to 8.0; this range also coincided with maximum extractability of protein. The lower amount of RuBPCase degrading proteinase extracted at low pH was not due to an effect of pH on enzyme stability. The optimum temperature of reaction was 50° C and reaction rates were linear for at least 120 min at this temperature. In the absence of substrate the proteinase was found to be very sensitive to temperatures above 30° C, with even short exposures causing rapid loss of activity. The relation between assay pH and RuBPCase degradation indicated that degradation was restricted to the acid proteinase group of enzymes, with a pH optimum of 4.8, and no detectable activity at a pH greater than 6.4. The levels of extractable RuBPCase proteinase exhibited a distinct diurnal variation, with activity increasing during the latter part of the light period and then declining once the lights were turned off. The effect of leaf age on the level of RuBPCase, RuBPCase proteinase and total soluble protein was investigated. Maximum RuBPCase activity occurred 9 days after sowing as did soluble protein. After the maximum level was obtained, the pattern of total soluble protein was shown to be characterised by three distinct periods of protein loss: I (day 9–13) 125 ng leaf^-1 day^-1; II (day 15–27) 11 ng leaf^-1 day^-1; III (day 29–49) 22 ng leaf^-1 day^-1. Comparison of the pattern of RuBPCase activity and total protein suggest that the loss of RuBPCase may be largely responsible for the high rate of protein loss during period I. Proteinase activity increased sharply during the period of most rapid loss of RuBPCase activity, and because the specific activity of RuBPCase also declined, we concluded that RuBPCase was being degraded more rapidly than the other proteins. Once the majority of the RuBPCase was lost, there did not appear to be a direct relation between RuBPCase proteinase activity and rate of total soluble protein loss, since the proteinase exhibited maximum activity during the slowest period of protein loss (II), and was declining in activity while the rate of protein loss remained stable during the third and final period of total protein loss.Abbreviations RuBPCase ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39) - TCA trichloroacetic acid Supported by the Wheat Industry Research Council of Australia and the Australian Research Grants Committee D2 74/15052