Calcium-dependent protease of the cyanobacterium Anabaena: molecular cloning and expression of the gene in Escherichia coli, sequencing and site-directed mutagenesis

Summary It has been suggested that a calcium-dependent intracellular protease of the cyanobacterium, Anabaena sp., participates in the differentiation of heterocysts, cells that are specialized for fixation of N₂. Clones of the structural gene (designated prcA) for this protease from Anabaena variabilis strain ATCC 29413 and Anabaena sp. strain PCC 7120 were identified via their expression in Escherichia coli. The prcA gene from A. variabilis was sequenced. The genes of both strains, mutated by insertion of a drug resistance cassette, were returned to these same strains of Anabaena on suicide plasmids. The method of sacB-mediated positive selection for double recombinants was used to achieve replacement of the wild-type prcA genes by the mutated forms. The resulting mutants, which lacked Ca²⁺-dependent protease activity, were not impaired in heterocyst formation and grew on N₂ as sole nitrogen source.     

Simultaneous phosphate solubilization potential and antifungal activity of new fluorescent pseudomonad strains, Pseudomonasaeruginosa, P. plecoglossicida and P. mosselii

Of 80 fluorescent pseudomonad strains screened for phosphate solubilization, three strains (BFPB9, FP12 and FP13) showed the ability to solubilize tri-calcium phosphate (Ca₃(PO₄)₂). During mineral phosphate solubilization, decrease of pH in the culture medium due to the production of organic acids by the strains was observed. These phosphate solubilizing strains produced indole-3-acetic acid (IAA) and protease as well as exhibited a broad-spectrum antifungal activity against phytopathogenic fungi. When tested in PCR using the gene-specific primers, strain BFPB9 showed the presence of hcnBC genes that encode hydrogen cyanide. On the basis of phenotypic traits, 16S rRNA sequence homology and subsequent phylogenetic analysis, strains BFPB9, FP12 and FP13 were designated as Pseudomonas aeruginosa, P. plecoglossicida and P. mosselii, respectively. Present investigation reports the phosphate solubilization potential and biocontrol ability of new strains that belong to P. plecoglossicida and P. mosselii. Because of the innate potential of phosphate solubilization, production of siderophore, IAA, protease, cellulase and HCN strains reported in this study can be used as biofertilizers as well as biocontrol agents.     

Cleavage of di- and tripeptides by Prevotella ruminicola

The final step in the conversion of protein to amino acids by the common Gram-negative rumen bacterium, Prevotella (formerly Bacteroides) ruminicola , is the cleavage of di- and tripeptides. Dipeptidase and tripeptidase activities were predominantly cytoplasmic, and toluene treatment increased the rate of Ala₂ and Ala₃ hydrolysis by whole cells, suggesting that transport limited the rate of hydrolysis of extracellular di- and tripeptides. The hydrolysis of Ala₂ and Ala₃ by whole cells was not affected by protonophores, ionophores or dicyclohexylcarbodiimide, but Ala₂ hydrolysis by EDTA-treated cells was inhibited by the Ca²⁺/H⁺ ionophore, tetronasin. Ala₃ hydrolysis was not affected by protonophores or ionophores in EDTA-treated cells. The dipeptidase of strain M384 was inhibited > 99% by 1,10-phenanthroline and 39% by EDTA but not other protease inhibitors, consistent with the enzyme being a metalloprotease. Tripeptidase was insensitive to protease inhibitors, except for a 33% inhibition by EDTA. Cleavage of tripeptides occurred at the bond adjacent to the N-terminal amino acid. Distinct di-, tri- and oligopeptidase peaks were obtained by anion-exchange liquid chromatography of disrupted cells. Banding patterns on native PAGE using activity staining also indicated that P. ruminicola M384 had separate single dipeptidase and tripeptidase enzymes which hydrolysed a range of peptides. The dipeptidase of strain M384 was different from other strains of P. ruminicola: strains GA33 and B₁4 had activities which ran at the same R_f; strain GA33 had another band of lower activity; strain 23 had two bands different from those of the other strains. The tripeptidases ran at the same R_f for the different strains. Dipeptidase activity of all strains was inhibited by 1,10-phenanthroline on gels. Gel permeation chromatography indicated that the M_r of the dipeptidases from strains M384 and B₁4 were 115 000 and 114 500 respectively, and 112 500 and 121 500 for the corresponding tripeptidases. Thus the metabolism of small peptides by P. ruminicola involves separate permeases and intracellular peptidases for di- and tripeptides.     

Effect of king cobra venom on α2-macroglobulin and proteases in human blood plasma

Normal human blood plasma showed hydrolytic activities on several synthetic substrates for proteases, the most effective being H-D-Ile-Pro-Arg-p-nitroanilide, H-D-Pro-Phe-Arg-p-nitroanilide and H-D-Val-Leu-Arg-p-nitroanilide. When plasma was preincubated for 12 h at 37°C, there was no significant alteration of the hydrolytic activities. On incubation for 12 h with king cobra venom (2 μg for 0.1 ml plasma), there was considerable decrease in the activities and complete abolition of the protease binding capacity of α₂-macroglobulin. On chromatography on Sephadex G-200, α₂-macroglobulin activity and bulk of the protease activity of normal plasma were eluted in the void volume region. A minor protease peak was eluted with aVe/Vo value of 2.5. With venom treated plasma, there was no decrease with this peak. The major protease peak and α₂-macroglobulin activity were drastically reduced. Chromatography on red Sepharose showed that all the α ₂ ⁻ acroglobulin activity and bulk of the protease activity in normal plasma were bound to the column. In venom treated plasma there was marked reduction in the bound fraction. The data suggest that cobra venom proteases directly or through proteases generated in plasmain situ causes limited cleavage of α₂-macroglobulin as well as α₂-macroglobulin bound proteases, inactivating them.     

Screening of antagonistic bacterial strains against Meloidogyne incognita using protease activity

Twenty-six bacterial strains that had demonstrated antagonism to some fungal and bacterial pathogens were evaluated for their ability to inhibit Meloidogyne incognita Kofoid & White. The inhibition rates of egg-hatching and second-stage juveniles (J2) mortality of M. incognita by these strains ranged from ?16.5 to 87.4% and from 1.3 to 77.8%, respectively. The 12 strains causing J2 mortality over 40% were chosen for greenhouse experiments in which their biocontrol efficacy reached 33.3–65.6%. On the other hand, among the 26 strains, 20 demonstrated in vitro protease activity and 14 revealed chitinase activity. Significantly, strains Bacillus sp. AR156 and GJ24 in greenhouse tests showed the strongest protease activities. The analyses of the relationships of the efficacy of the 12 strains with their protease and chitinase activities, respectively, indicated that biocontrol efficacy was highly correlated with protease activity (r=0.92, P<0.001) but barely correlated with chitinase activity. The strong positive correlation between protease activity and efficacy suggests that in vitro protease activity could be used as a parameter for selecting biological control agents (BCAs) against root-knot nematodes. Consistently, the biocontrol efficacy of AR156, GJ24, abamectin reached 74.3, 73.4, and 40.9% in the field in Huai-an, Jiangsu; and 71, 69.9, and 37% in Zao-zhuang, Shandong, respectively. The fact that the strains with high protease activities also had significantly higher biocontrol efficacy than abamectin in the field implies that in vitro protease activity may be adopted as a reliable new parameter for speeding up the process of screening the biological control agents (BCAs).     

Development of a high yielding E. coli periplasmic expression system for the production of humanized Fab' fragments

Humanized Fab′ fragments may be produced in the periplasm of Escherichia coli but can be subject to degradation by host cell proteases. In order to increase Fab′ yield and reduce proteolysis we developed periplasmic protease deficient strains of E. coli. These strains lacked the protease activity of Tsp, protease III and DegP. High cell density fermentations indicated Tsp deficient strains increased productivity two fold but this increase was accompanied by premature cell lysis soon after the induction of Fab′ expression. To overcome the reduction in cell viability we introduced suppressor mutations into the spr gene. The mutations partially restored the wild type phenotype of the cells. Furthermore, we coexpressed a range of periplasmic chaperone proteins with the Fab′, DsbC had the most significant impact, increasing humanized Fab′ production during high cell density fermentation. When DsbC coexpression was combined with a Tsp deficient spr strain we observed an increase in yield and essentially restored “wild type” cell viability. We achieved a final periplasmic yield of over 2.4g/L (final cell density OD₆₀₀ 105), 40 h post Fab′ induction with minimal cell lysis.The data suggests that proteolysis, periplasm integrity, protein folding and disulphide bond formation are all potential limiting steps in the production of Fab′ fragments in the periplasm of E. coli. In this body of work, we have addressed these limiting steps by utilizing stabilized protease deficient strains and chaperone coexpression. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:212–220, 2017     

Lack of virulence of bovine type IIIStreptococcus agalactiae strains for mice correlates with reduced in vitro production of extracellular type-specific antigen

Six strains of type IIIStreptococcus agalactiae isolated from milk samples from cases of bovine mastitis were examined for in vitro production of three potential extracellular virulence factors: neuraminidase, protease, and extracellular type-specific antigen. Virulence in mice, expressed as LD₅₀ values, was examined for these six strains to determine if a relationship existed between the in vitro production of any of the three extracellular products and mouse lethality. Only in vitro production of extracellular type-specific antigen showed a correlation with virulence of these organisms in the mouse model. All six bovine strains were relatively avirulent in the mouse while producing reduced levels in vitro of extracellular typespecific antigen as compared to nine human isolates. The bovineS. agalactiae strains were an average of 538-fold less virulent for the mouse than were the high type-specific antigen producers isolated from human sources.     

Biocontrol and PGPR Features in Native Strains Isolated from Saline Soils of Argentina

A bacterial collection of approximately one thousand native strains, isolated from saline soils of Cordoba province (Argentina), was established. From this collection, a screening to identify those strains showing plant growth promotion and biocontrol activities, as well as salt tolerance, was performed. Eight native strains tolerant to 1 M NaCl and displaying plant growth promotion and/or biocontrol features were selected for further characterization. Strains MEP₂ 18, MRP₂ 26, MEP₂ 11a, MEP₃ 1, and MEP₃ 3b significantly increased the growth of maize seedlings under normal and saline conditions, whereas isolates ARP₂ 3, AEP₁ 5, and ARP₂ 6 were able to increase the root dry weight of agropyre under saline conditions. On the other hand, strains MEP₂ 18 and ARP₂ 3 showed antagonistic activity against phytopathogenic fungi belonging to Sclerotinia and Fusarium genus. Antifungal activity was found in cell-free supernatants, and it was heat and protease resistant. Strains MEP₂18 and ARP₂3 were identified as Bacillus sp. and strains MEP₂11a and MEP₃3b as Ochrobactrum sp. according to the sequence analysis of 16S rRNA gene.     

北黄海沉积物可培养产蛋白酶细菌分离鉴定

【目的】揭示北黄海沉积物中可培养产胞外蛋白酶细菌及蛋白酶多样性,增加人们对北黄海生态系统中产蛋白酶菌多样性的认识,为海洋产蛋白酶微生物的挖掘提供菌群资源。【方法】分别将5个北黄海沉积物样品梯度稀释涂布至酪蛋白明胶筛选平板,选择性分离产蛋白酶细菌;并通过分析基于16S rRNA基因序列的系统发育关系,揭示这些细菌的分类地位和遗传多样性;分别测定胞外蛋白酶活性并对酶活较高的39株菌进行基于苯甲基磺酰氟(PMSF,丝氨酸蛋白酶抑制剂)、邻菲罗啉(o-phenanthroline,O-P,金属蛋白酶抑制剂)、E-64(半胱氨酸蛋白酶抑制剂)和pepstatin A(天冬氨酸蛋白酶抑制剂)4种抑制剂的酶活抑制实验以及所有菌株对3种底物(酪蛋白、明胶、弹性蛋白)的水解能力;分析这些细菌所产胞外蛋白酶的特性及多样性。【结果】从5个北黄海沉积物样品中分离获得66株产蛋白酶细菌,这些菌株隶属于Bacteroidetes、Proteobacteria、Actinobacteria和Firmicutes 4个门的7个属,其中Pseudoalteromonas(69.9%)、Sulfitobacter(12.1%)和Salegentibacter(10.6%)是优势菌群;沉积物中可培养的产蛋白酶细菌的丰度为104 CFU/g;蛋白酶酶活抑制实验表明所有测定菌株产生的胞外蛋白酶属于丝氨酸蛋白酶和/或金属蛋白酶,仅有少数菌株所产蛋白酶具有半胱氨酸蛋白酶或天冬氨酸蛋白酶活性。【结论】北黄海沉积物中可培养产蛋白酶细菌类群较为丰富,Pseudoalteromonas、Sulfitobacter和Salegentibacter菌株是优势菌群,测定菌株所产胞外蛋白酶主要是丝氨酸蛋白酶和/或金属蛋白酶。     

马蜂肠道菌的分离鉴定及产消化酶功能菌株的筛选

【背景】马蜂(Vespa mandarinia Smith)可以防治多种田间害虫,还具有药用价值,其肠道菌群结构和功能还有待研究。【目的】获得马蜂肠道可培养细菌并筛选出具有产消化酶功能的菌株,为理解肠道菌对宿主的影响机理及功能菌株的利用提供科学依据和研究材料。【方法】采用传统细菌分离培养法获得马蜂肠道菌,结合形态学以及16S rRNA基因序列分析进行鉴定;利用水解圈法分别筛选产蛋白酶、脂肪酶、淀粉酶和纤维素酶菌株;通过测量水解圈D与菌落直径d的比值,比较不同细菌的产酶能力。【结果】在马蜂肠道中共分离出6属10种细菌,其中芽孢杆菌属5种,肠球菌属、葡萄球菌属、明串珠菌属、乳球菌属和不动杆菌属各1种。从获得的61个菌株中筛选出6个具有产消化酶功能的菌株。其中,苏云金芽孢杆菌V44具有产蛋白酶、淀粉酶、脂肪酶和纤维素酶4种消化酶的能力;粪肠球菌V6具有产淀粉酶、蛋白酶和脂肪酶3种消化酶的能力;蜡样芽孢杆菌V43具有产蛋白酶、淀粉酶和纤维素酶3种消化酶的能力;粪肠球菌V20、蜡样芽孢杆菌V19和维德曼氏芽孢杆菌V22均具有产蛋白酶的能力。【结论】马蜂肠道细菌资源较丰富,部分有产消化酶的功能,可帮助马蜂消化食物,对宿主健康有一定影响。本研究筛选的6个菌株都能产蛋白酶,其中菌株V43和V44分别具有最强产淀粉酶和脂肪酶的能力,是可进一步开发利用的肠道功能菌株资源。     

Studies on Bacterial Protease

Some physicochemical properties and amino acid composition of the alkaline protease of B. amylosacchariticus were determined. The molecular weight and sedimentation coefficient were estimated to be 22,700 and 2.89 s, respectively, and the amino terminal amino acid was identified to be alanine. The enzyme contained 15.9% of nitrogen and was composed of 220 residues of amino acid: lys₆, his₅, arg₃, asp₂₀, thr₁₄, ser₃₇, glu₁₂, pro₁₀, gly₂₅ ala₂₇ val₂₀, met₃, isoleu₁₂, leu₁₂, tyr₉, phe₂, try₃ and amide ammonia₁₆ The results indicate that protein nature and chemical properties of the alkaline protease presented here are distinct from those of alkaline proteases obtained from the other strains of B. subtilis, such as subtilopeptidase A, B and BPN’     

碱性蛋白酶SubC在枯草芽孢杆菌中的高效异源表达

【背景】碱性蛋白酶是工业用酶中占比最大的酶类,广泛应用于清洁、食品、医疗等行业。近期研究发现碱性蛋白酶在生产生物活性肽方面有巨大潜力,这将进一步拓宽其在保健食品领域中的应用。【目的】利用枯草芽孢杆菌异源表达地衣芽孢杆菌来源的碱性蛋白酶SubC。【方法】通过筛选3种枯草芽孢杆菌宿主菌株(Bacillus subtilis 1A751、MA07、MA08)和6种信号肽(AmyE、AprE、NprE、Pel、YddT、YoqM),同时优化诱导剂浓度、发酵培养基和发酵时长,最终得到最优重组菌株MA08-AmyE-subCopt。【结果】重组菌株MA08-AmyE-subCopt的胞外酶活力为3.33×103 AU/mL,胞外蛋白分泌量为胞内可溶蛋白表达量的4倍,与携带野生型信号肽的对照组菌株WT相比,酶活提高了73.4%。【结论】异源碱性蛋白酶SubC在枯草芽孢杆菌中成功表达,为碱性蛋白酶SubC的表达和在保健食品领域的工业化应用提供了理论基础。     

Quaternary structure of a SPATE autotransporter protein

The temperature-sensitive hemagglutinin (Tsh) is a representative of the growing subfamily of secreted bacterial virulence factors, known as serine protease autotransporters of the Enterobacteriaceae (SPATEs). Expressed by avian and human pathogenic strains of Escherichia coli Tsh acts as a serine protease and an adhesin to erythrocytes, hemoglobin, and extracellular matrix proteins. Mature Tsh is comprised of a 106-kDa secreted domain (Tsh_s) and a 33-kDa outer membrane β-domain (Tsh_β). Based on the size of β-domains and functional properties of their passenger domains, all SPATEs are considered to be conventional autotransporters. However, it is unsettled if the conventional autotransporters exist as monomers, oligomers, or multimers (e.g., hexamers). To determine the quaternary structure of Tsh in vitro, we purified Tsh_β from the outer membranes and showed that it is natively folded because it is heat modifiable and resistant to protease digestion. Blue-native polyacrylamide gel electrophoresis of Tsh_β indicated that Tsh_β exists as a monomer or a dimer. The cross-linking analysis demonstrated that purified Tsh_β exists as a monomer. The size-exclusion chromatography and cross-linking analyses of purified Tsh_s also showed that the passenger domain of Tsh is a monomer. Overall, our data indicated that Tsh is a monomeric protein in vitro and support the concept that the SPATE autotransporters exist as monomers rather than as multimers. Implications of our findings on the mechanism of autotransporter secretion across the outer membrane are discussed.     

IgA Protease Produced by Streptococcus sanguis and Antibody Production against IgA Protease in Patients with Behçet's Disease

The production of IgA protease in twelve strains of Streptococcus sanguis isolated from patients with Behçet's disease (BD) was examined. Protease activity was detected in 10 out of 12 strains. The protease was purified from one representative strain, S. sanguis 113–20, by employing Rotofor and DEAE-Sephacel chromatography. The molecular mass of the purified protease was approximately 100 kDa, and it cleaved the proline-threonine site of the IgA. Both IgG and IgA titers against the cells (113–20) and the purified IgA protease in the sera of BD patients and healthy controls, 36 each, were assayed. The IgG titers against the cells and protease were not significant in the BD patients or controls, but the IgA titers against the cells and protease in the BD patients were significantly higher than those of the controls. These data indicate that the BD patients are infected with IgA protease-producing S. sanguis strains, which cause an increase of IgA titer against these organisms and IgA protease antigen. Since the organisms can proliferate in BD patients for a long period of time (years), it seems that IgA antibodies cannot effectively eliminate the organisms.     

Purification and characterization of a propanol-tolerant neutral protease from Bacillus sp. ZG20

Abstract

A propanol-tolerant neutral protease was purified and characterized from Bacillus sp. ZG20 in this study. This protease was purified to homogeneity with a specific activity of 26,655?U/mg. The recovery rate and purification fold of the protease were 13.7% and 31.5, respectively. The SDS-PAGE results showed that the molecular weight of the protease was about 29?kDa. The optimal temperature and pH of the protease were 45?°C and 7.0, respectively. The protease exhibited a good thermal- and pH stability, and was tolerant to 50% propanol. Mg²⁺, Zn²⁺, K⁺, Na⁺ and Tween-80 could improve its activity. The calculated K_m and V_max values of the protease towards α-casein were 12.74?mg/mL and 28.57?µg/(min mL), respectively. This study lays a good foundation for the future use of the neutral protease from Bacillus sp. ZG20.     

A novel screening strategy to identify biocontrol fungi using protease production or chitinase activity against Meloidogyne root-knot nematodes

We isolated 137 fungal strains from females and eggs of Meloidogyne spp. collected from plant roots and infested soil in Jiangsu, China, and examined their in vitro protease production and chitinase activities. We then selected 20 strains, each with a protease production higher than 2 (protease production was measured as the ratio of diameters of the zone of clearing to that of the colony) and a chitinase activity higher than 0.2 U/min mL. For these strains, we examined their egg parasitic rates, mortality rates of stage 2 juvenile (J2), egg hatching rates and evaluated their biocontrol efficacies under greenhouse conditions. The biocontrol efficacies of these 20 potential biocontrol fungal strains ranged from 29 to 58%. The actual biocontrol efficacy against Meloidogyne root-knot was highly correlated with the in vitro protease production (r=0.82), chitinase activity (r=0.80), and mortality of J2 (r=0.76), but poorly correlated with the egg parasitic rate (r=0.51) and the egg hatch rate (r=0.52). Based on these results, we suggest that in vitro protease production or chitinase activities of the fungal strains could be an applicable indicator for assessing biocontrol efficacy against nematodes. This notion was also supported by our field experiments on tomatoes with Paecilomyces lilacinus strains XT18, XCS46 and Pochonia chlamydosporia strain XT124, which reached the biocontrol efficacies of 60.7, 51.5 and 48.1% in 2007; and 67.8, 57.3 and 53.2% in 2008, respectively.     

Analysis of nucleotide pools during protease production with Bacillus licheniformis

Summary During development of a fed-batch procedure for protease production with Bacillus licheniformis the nucleotide pools of the culture were assayed. Transitions between different growth phases or different nutrient limitations could easily be discerned by alterations in the nucleotide pool. As already described for B. subtilis, induction of sporulation was marked by a drop in the guanosine triphosphate (GTP) pool of the cells, which always preceded protease production. This was also valid for closed-loop controlled fed-batch processes in which the concentration of ammonium, which repressed protease biosynthesis, was kept constant at low levels. A marked decrease in the GTP content of the cells, e.g. after addition of mycophenolic acid during the exponential phase, increased protease formation during the stationary phase. During protease production the energy charge was lower (0.6–0.8) than during the exponential and early stationary phases (0.8–0.95) although very low energy charges (<0.5) did not support protease formation. Offprint requests to: G. Bierbaum     

Studies on the Proteolytic Enzymes of Black Aspergilli

In order to investigate the production of acid protease, the cultivation conditons were studied with black Aspergilli belonging to Kuro-Koji mold group by solid cultivation (Koji-culture).

It was observed that the production of acid-protease by A. saitoi R-3813 mut. UV-13 was markedly increased by adding an adequate amount of an inorganic nitrogen compound to the cultivation medium. Concerning the production of acid protease by adding an inorganic nitrogen compound, one hundred and eighty-four strains of Kuro-Koji mold group and eleven strains of other Aspergilli were tested.     

Restriction site polymorphism in genes encoding type 2 but not type 1 gonococcal IgA1 proteases

Neisseria gonorrhoeae produces two phenotypically distinct types of IgA1 proteases, each of which cleaves a specific peptide bond in the hinge region of the human IgA1 heavy chain. The genes encoding IgA1 protease from twenty-eight different strains of N. gonorrhoeae, including twelve which produce type 1 enzyme, thirteen which produce type 2 enzyme, and three which are protease negative, were analyzed. Nine restriction site patterns were found in the iga genes. All twelve type 1 strains showed identical restriction maps of the iga gene, which differed from all the iga-2 variants. The three protease negative strains each contained DNA homologous to the probe. While strain to strain variation in restriction maps of specific genes is not unique and has been reported in N. gonorrhoeae previously, the existence of such restriction site polymorphism among iga-2 genes contrasts strongly with the lack of such variation among iga-1 genes. The basis for this lack of diversity among the iga-1 genes is under further investigation.     

On the turnover and proteolysis of catalase in tissues of the guinea pig and acatalasemic mice.

Turnover characteristics (half-lives and rate constants for synthesis and degradation) have been determined for the catalases of guinea pig and three different strains of mice by means of the kinetics of return of enzyme activity after inhibition with 3-amino-1,2,4-triazole. The catalase of hypocatalasemic mice (strain C_sD) did not display an appreciably different half-life to that of the wild-type mice, but catalase in the tissues of acatalasemic mice (strain C_sB) exhibited a half-life which was only half that of the wild type, while the half-life of guinea pig catalase was more than twice that of wild-type mice. Significant differences were also noticed in regard to the in vitro susceptibility of the catalases of these animals to protease inactivation. Large-granule (lysosomal, mitochondrial and peroxisomal) extracts proved far more susceptible to protease inactivation than cytosol extracts, and marked changes in the heteromorph pattern of mouse liver cytosol catalase were observed to accompany limited proteolysis. These results support the conclusion that the in vitro susceptibility of proteases may be an important determining factor in the rate of degradation of an enzyme in vivo.