Liposome Aggregation: Promoted by Trypsin and Papain in the Absence of Cations

Abstract

Phospholipid vesicle aggregation is usually mediated by phospholipid-binding proteins such as the annexins in a Ca²⁺-dependent manner. Here, we describe aggregation of unilamellar liposomes by trypsin and papain in the absence of cations. Cations including Ca²⁺ inhibited the aggregation. While both trypsin and papain promoted aggregation of liposomes made of phosphatidylcholine and phosphatidylglycerol, only papain elicited aggregation of liposomes made of exclusively phosphatidylcholine. Incubation of trypsin for 30 min at 37°C destroyed its liposome aggregating activity, similar treatment had no effect on papain's. Chymotrypsin and pepsin had no liposome aggregating activity.     

Membrane coarctation by calcium as a regulator for bound enzymes

The enzyme activity of spherical membranes formed by conjugates of trypsin and chymotrypsin with a polycarboxylic polymer decreases with increasing Ca²⁺ concentration in the surrounding solution. This phenomenon is reversible and attributed to the coarctation of the membrane structure rather than to changes in the intrinsic behavior of the bound enzymes. Coarctation decreases the swelling and increases the virtual cross-linking of the membrane so that the diffusion rate of the substrate to the catalytic sites is reduced. As a result the overal enzymic activity decreases and the observed reaction departs from the Michaelis-Menten kinetics. The activity of the trypsin conjugate decreases with increasing Ca²⁺ concentration unlike that of trypsin in free solution, because the effect of membrane coarctation masks the enhancement of tryptic activity by Ca²⁺. The physical and chemical properties of these polycarboxylic membranes, which contain about 40% enzyme protein, resemble those of some cell membranes such as erythrocyte ghosts. The results suggest that a similar indirect regulation of the activity of bound enzymes via membrane coarctation by Ca²⁺ or other multivalent metal ions may occur in living systems also.     

Comparison of phospho- and dephospho-ATP citrate lyase

The dephospho- form of rat liver citrate lyase has been prepared by treating purified [³²P]-ATP citrate lyase with a partially purified phosphatase. A comparison of the properties of the phospho- and dephosphoenzyme has been performed. The pH optima were the same for both forms of the enzyme in four different buffer systems although the optimum values varied identically for both enzyme forms with the buffer. Both the phospho- and dephosphoenzymes show the same kinetic properties except for the K_m observed for ATP in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer system where it was 54 μm for the phosphoenzyme and 292 μm for the dephosphoenzyme. The present study also indicates that both enzymes are cleaved by trypsin and lysosomal proteases in a similar manner. Both forms of the enzyme tend to associate with mitochondria to the same extent and both enzymes have identical temperature stability curves.     

红螯螯虾胚胎发育期主要消化酶和同工酶的活性变化

采用生物化学方法测定了红螯螯虾(Cherax quadricarinatus)胚胎发育各期主要消化酶(胃蛋白酶、胰蛋白酶、淀粉酶、纤维素酶和脂肪酶)的比活力及主要同工酶(乳酸脱氢酶、苹果酸脱氢酶、葡萄糖-6-磷酸脱氢酶和酯酶)的活力。结果显示,5种消化酶各自表现出不同的变化模式,胃蛋白酶和胰蛋白酶的比活力早期均逐渐上升,到发育后期胃蛋白酶出现快速下降,而胰蛋白酶却仍保持较高水平;淀粉酶比活力呈“V”字型变化趋势,晚期活性较高;纤维素酶和脂肪酶的比活力则均较低。4种同工酶酶谱随胚胎的发育渐趋复杂,酶活性也随之增强。结果表明,消化酶和同工酶活力的高低均受其基因的调控,并随胚胎发育适时表达,为胚胎组织、器官和系统的形成以及未来仔虾的开口摄食提供物质保证。     

猪血药用物质基础研究(Ⅱ)- 酶种类的选择

通过体外模拟体内条件,选取主要消化酶胃蛋白酶、胰蛋白酶对猪血脱色液进行酶解,以高效酶木瓜蛋白酶和1394酶作为对照,结合酶解后多肽总含量以及<3 kDa的多肽得率进行酶解效果的综合评价,得到胃蛋白酶为最佳酶解酶。     

New Anthracycline Antibiotic CG12 Obtained by Microbial Glycosidation of α-Citromycinone

We studied the effects of peptidylarginine deiminase, a Ca²⁺-dependent protein-modulating enzyme that catalyzes the deimination of arginyl residues in protein on two major trypsin inhibitors in mouse plasma. One of these inhibitors was α-1-antitrypsin and the other was a recently characterized trypsin inhibitor termed contrapsin (H. Takahara and H. Sinohara, J. Biol. Chem., 257, 2438 (1982)). The enzyme abolished the trypsin-inhibiting activity of contrapsin in a process that was pseudo-first order with the rate dependent on enzyme concentration (second order rate constant = 3.4 × 10³ m^-1 · s^-1), but no detectable changes in the activity was noted for α-1-antitrypsin. Millimolar Ca²⁺ and dithiothreitol were absolutely required for the inactivation of contrapsin by peptidylarginine deiminase. Although no significant alterations of the charge and the size in modified contrapsin were observed, the modified inhibitor indicated that 1 arginyl residue was converted to a citrullyl residue. Modified contrapsin whose anti-tryptic activity was lost inhibited chymotrypsin much more strongly than the native inhibitor. These data suggest that a vital amino acid residue for the anti-tryptic activity of mouse contrapsin is an arginyl residue and the conversion of this arginyl residue to a citrullyl residue causes the functional changes of the inhibitor.     

Spatial changes in the concentrations and activities of amylase and trypsin in Euphausia superba. A comparison between activity measurement and immunoquantitation

Summary Two digestive enzymes: alpha amylase and trypsin were purified by affinity chromatography from the antarctic euphausiid Euphausia superba and the corresponding antibodies developed in male New-Zealand rabbits. Electrophoresis of purified enzymes yielded 4 isozymes for amylases and 6 for trypsin. The purification level was relatively low for amylase because of a high molecular weight contaminant with a low level of activity. Rocket immunoelectrophoresis of amylase showed three precipitin lines; a single line was obtained for trypsin. The antiserum obtained at a dilution of 1% presented a good linearity of the response between 60 to 1000 ng of pure trypsin. Both enzyme concentrations were measured by the quantitative method of rocket immunoelectrophoresis for different stations of the FIBEX cruise. Enzyme activities and concentration displayed significant spatial changes and a certain degree of association with the developmental stages sampled. Several inconsistencies in the relationships between growth stages and enzyme rates or quantities suggested that the spatial variations were not solely related to specific growth requirements but to combine effects of both physiological and environmental factors. Comparison of the spatial changes in concentration and activity showed that the changes in enzyme concentration were in a large part (60% of the total variance) responsible for the activity changes. The results strongly suggest that enzyme synthesis and degradation ensured the gross regulation of activity while modifications of the kinetic properties could be implicated in the fine tuning.     

The effects of biogenic phosphates on proteinase-induced protein cleavage and functioning of plasminogen activator

We showed, using the method of lysis of fibrin plates and five substrate proteins in a thin layer of agar gel, that inorganic orthophosphate (0.001–0.06 M) enhances by 50–250% the activatory functions of streptokinase, urokinase, and tissue plasminogen activator and, in general, by 1.2–12.0 times enhances protein lysis by trypsin, α-chymotrypsin, subtilisin, papain, bacterial metalloprotease, and even pepsin at a concentration < 4 mM. At higher concentrations, phosphate sharply inhibited pepsin activity and inhibited by 40–50% gelatin lysis by papain and gelatin (at a peak concentration) and casein lysis by metalloprotease. Inorganic pyrophosphate ions at concentrations of 10^?8–10^?1 M enhanced the cleavage of a number of proteins by serine proteinases and, at concentrations of 10^?5–10^?3 M, the activities of pepsin, plasminogen tissue activator, and streptokinase by 100 and 40%, respectively. The pyrophosphate concentrations of >10^?3 and >10^?4 M inhibited pepsinand metalloproteinase-catalyzed lysis of vritually all proteins. ATP increased casein lysis by serine proteinases, metalloproteinase, and pepsin by 20–60% at concentration of >10^?3 M and by 30–260% at 10^?2 M concentration. At concentrations of 10^?2 M, it inhibited the cleavage of some proteins by trypsin, chymotrypsin, papain, and metalloproteinase by 20–100%, and, at concentrations of 10^?3 M, lysis of albumin by pepsin and other proteins (except for fibrinogen) by metalloproteinase. A GTP concentration of 10^?7–10^?2 M increased protein degradation by serine proteinases, papain, and gelatin lysis by pepsin by 20–90%, whereas albumin lysis was inhibited by 40–70%. The presence of 10^?6–10^?5 M GTP led to a slightly increased degradation of hemoglobin and casein by bacterial metalloproteinase, while ≥10^?3 M GTP induced a drop in the activity of the metalloproteinase by 20–50%. ADP enhanced gelatin lysis by trypsin, casein lysis by pepsin and papain, and inhibited metalloproteinase activity by 20–100% (at ≥10^?3 M). Peculiarities of the effects of AMP and GD(M)P on gelatin lysis were found.     

Studies on some enzymes relevant to citric acid accumulation by Aspergillus niger

Enzymatic studies have been performed on a local strain of Aspergillus niger to find a correlation with citric acid accumulation. The activity of aconitase [aconitate hydratase, citrate(isocitrate) hydrolyase, EC 4.2.1.3] and isocitrate dehydrogenase (NADP⁺) [threo-d_s-isocitrate:NADP⁺ oxidoreductase (decarboxylating) EC 1.1.1.42] decreased after 4 days whereas that of citrate synthase [citrate oxaloacetate-lyase (pro-3S-CH₂COO^?→acetylCoA), EC 4.1.3.7] did so after 8 days, when citric acid accumulation in the medium reached a maximum (45.9 mg ml^?1). In vitro studies with mycelial cell-free extracts demonstrated inhibition of citrate synthase activity by sodium azide and potassium ferricyanide on both the 4th and 8th days. Aconitase was inhibited by sodium arsenate, sodium fluoride, iodoacetic acid and potassium ferricyanide only on the 4th day. Isocitrate dehydrogenase (NADP⁺) activity on the 4th and 8th days was inhibited by iodoacetic acid but was stimulated by potassium ferricyanide. The possible existence of isozyme species of these enzymes is discussed.     

The tricarboxylic acid cycle in L₃ Teladorsagia circumcincta: metabolism of acetyl CoA to succinyl CoA

Nematodes, like other species, derive much of the energy for cellular processes from mitochondrial pathways including the TCA cycle. Previously, we have shown L₃Teladorsagia circumcincta consume oxygen and so may utilise a full TCA cycle for aerobic energy metabolism. We have assessed the relative activity levels and substrate affinities of citrate synthase, aconitase, isocitrate dehydrogenase (both NAD⁺ and NADP⁺ specific) and α-ketoglutarate dehydrogenase in homogenates of L₃T. circumcincta. All of these enzymes were present in homogenates. Compared with citrate synthase, low levels of enzyme activity and low catalytic efficiency was observed for NAD⁺ isocitrate dehydrogenase and especially α-ketoglutarate dehydrogenase. Therefore, it is likely that the activity of these to enzymes regulate overall metabolite flow through the TCA cycle, especially when [NAD⁺] limits enzyme activity. Of the enzymes tested, only citrate synthase had substrate affinities which were markedly different from values obtained from mammalian species. Overall, the results are consistent with the suggestion that a full TCA cycle exists within L₃T. circumcincta. While there may subtle variations in enzyme properties, particularly for citrate synthase, the control points for the TCA cycle in L₃T. circumcincta are probably similar to those in the tissues of their host species.     

卤虫投喂下长吻(鱼危)仔稚鱼消化酶发育和口宽变化的研究

采用酶学和形态学测定方法, 研究在投喂卤虫条件下长吻(鱼危)仔鱼4种主要消化酶: 胃蛋白酶、胰蛋白酶、脂肪酶和淀粉酶的活性变化以及长吻(鱼危)仔鱼口宽、全长变化。实验共进行13d, 实验结果表明: (1)长吻(鱼危)仔鱼全长、口宽的发育与其日龄表现出明显的线性正相关(R_TL²=0.974, R_MW²=0.964)。口宽与全长比值(MW/TL)在仔鱼开口后急剧下降, 并自7日龄开始维持在0.07—0.08, 口宽和全长处于同步发育期并表现出明显的相关性(R²=0.948), 说明7日龄(/h, days post hatching)后口宽和全长处于同步发育期, 标志仔鱼转食的开始。(2)长吻(鱼危)仔鱼初次开口时即可检测出四种消化酶的活性。5—7/h时胰蛋白酶显著高于初孵仔鱼, 与此时仔鱼开始开口摄食的行为相一致。胃蛋白酶、脂肪酶活性在仔鱼孵化后第7天即开口的第3天, 淀粉酶活性在孵化后第6天, 显著高于初次孵化出来的仔鱼。8—13/h时, 胃蛋白酶、胰蛋白酶、脂肪酶和淀粉酶活性均在较高水平平稳的波动, 标志着消化道发育逐渐健全。     

A Thermostable phytase from <Emphasis Type="Italic">Neosartorya spinosa</Emphasis> BCC 41923 and its expression in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

A phytase gene was cloned from Neosartorya spinosa BCC 41923. The gene was 1,455 bp in size, and the mature protein contained a polypeptide of 439 amino acids. The deduced amino acid sequence contains the consensus motif (RHGXRXP) which is conserved among phytases and acid phosphatases. Five possible disulfide bonds and seven potential N-glycosylation sites have been predicted. The gene was expressed in Pichia pastoris KM71 as an extracellular enzyme. The purified enzyme had specific activity of 30.95 U/mg at 37°C and 38.62 U/mg at 42°C. Molecular weight of the deglycosylated recombinant phytase, determined by SDS-PAGE, was approximately 52 kDa. The optimum pH and temperature for activity were pH 5.5 and 50°C. The residual phytase activity remained over 80% of initial activity after the enzyme was stored in pH 3.0 to 7.0 for 1 h, and at 60% of initial activity after heating at 90°C for 20 min. The enzyme exhibited broad substrate specificity, with phytic acid as the most preferred substrate. Its K _m and V _max for sodium phytate were 1.39 mM and 434.78 U/mg, respectively. The enzyme was highly resistant to most metal ions tested, including Fe²⁺, Fe³⁺, and Al³⁺. When incubated with pepsin at a pepsin/phytase ratio of 0.02 (U/U) at 37°C for 2 h, 92% of its initial activity was retained. However, the enzyme was very sensitive to trypsin, as 5% of its initial activity was recovered after treating with trypsin at a trypsin/phytase ratio of 0.01 (U/U).     

Influence of lithium on dopamine-stimulated adenylate cyclase activity in rat brain

Dopamine-stimulated adenylate cyclase activity from various rat brain areas was inhibited $\text{in vitro}$ by lithium. The inhibition was dose-dependent and non-competitive. In lithium-treated rats no changes in enzyme activity could be demonstrated.     

The effect of alpha-lactalbumin and beta-lactoglobulin hydrolysates on the metabolic activity of Escherichia coli JM103

Bovine milk proteins alpha-lactalbumin (alpha-la) and beta-lactoglobulin (beta-lg) were hydrolysed with seven different proteolytic enzymes, and the effect of various hydrolysates on a genetically modified luminous Escherichia coli JM103 was tested in vitro with a bioluminescence assay for bacterial growth and metabolism. Undigested proteins did not inhibit the activity of tested E. coli JM103 at a concentration as high as 0.1 g ml-1. At the same concentrations, alpha-la hydrolysed with pepsin or trypsin and beta-lg hydrolysed with alcalase, pepsin or trypsin, showed a lower metabolic activity during the first 8 h of growth. The activity of E. coli JM103 in the presence of 25 mg ml-1 alpha-la or beta-lg hydrolysed with pepsin and trypsin was only 21% of the control after incubation for 6 h. The preliminary results indicated that ultrafiltration through 10 kDa and 1 kDa molecular mass cut-off membranes may be used to enrich bacteriostatic properties.     

Investigation and comparison of the binding between tolvaptan and pepsin and trypsin: Multi‐spectroscopic approaches and molecular docking

Tolvaptan (TF), a selective arginine vasopressin V2 receptor antagonist, was approved by the Food and Drug Administration in 2009. This study mainly investigated the differences between the binding of TF with pepsin and trypsin by using a series of spectroscopy and molecular modeling methods. Thermodynamic parameters and molecular docking results suggested that the binding of TF to pepsin and trypsin were both spontaneous but driven by different forces. For pepsin, the binding was driven by hydrogen bonds and van der Waals forces; but for trypsin, it was driven by electrostatic forces and hydrophobic forces. The quenching mechanism between TF and pepsin and trypsin was investigated by fluorescence experiments and time‐resolved fluorescence spectroscopy. Synchronous fluorescence and 3‐dimensional fluorescence were used to investigate the micro‐environmental and conformational changes of pepsin and trypsin after the insertion of TF. In addition, activity‐measurement results showed that both the pepsin and trypsin activities increased with increasing TF concentration, which may help to understand the possible effect of TF on the digestion and absorption of nutrients in vivo.     

Collagenase in the immunohistochemical demonstration of laminin,fibronectin and factor VII/RAg in nervous tissue after fixation

Summary The effects of collagenase on the immunohistochemical demonstrability of laminin, fibronectin and Factor VIII/RAg in human nervous tissue have been studied. The influence of this, and other proteolytic enzymes such as pepsin and trypsin, has been investigated in relation to different fixatives. Collagenase gave better results with Carnoy fixed material than after formalin fixation; unlike trypsin and pepsin, it did not produce tissue digestion.Partially supported by CNR Special Project Control of Neoplastic Growth, Grant N^o 82.00410.96     

Controls of citrate synthase activity

The inhibition of citrate synthase by a variety of nucleotides and polycarboxylate compounds is not unexpected since many of the compounds are substrate analogs of citrate synthase. These effectors are interesting by virtue of the fact that many of them are intermediates and/or end products in the metabolic path of which citrate synthase can be considered the first committed step. As a consequence, it is possible to propose regulation of citrate synthase by ATP (or phosphorylation potential) by acyl CoA (acylation level) and NADH (redox potential). Aside from these putative controls, it is possible that the major control of citrate synthase activity is by changes in the concentration of its substrates acetyl CoA and oxalacetate.I discuss in this review the many factors that must be considered before one can decide whether or not interactions between metabolites and enzymes observed in an $\text{in vitro}$ catalytic situation have metabolic relevance. These factors include 1) the concentrations of substrates at the enzyme site, 2) the concentrations of effectors at the enzyme site, 3) the presence of modifying substances, and 4) the difference in behavior of an enzyme at its concentration $\text{in vivo}$ compared to its concentration $\text{in vitro}$. In the case of citrate synthase as is generally true for other enzymes, no accurate knowledge of these factors are available $\text{in vitro}$ so that little can be said concerning the $\text{in situ}$ control of citrate synthase, which may be the result of all the factors acting in concert. The studies of effectors on enzymes $\text{in vitro}$ can only serve as a guideline for parameters to study when techniques are available to study control of enzymes $\text{in situ}$.     

CONVERSION OF TRYPSIN TO A COPPER ENZYME: TYROSINASE/CATECHOL OXIDASE BY CHEMICAL MODIFICATION

New active sites can be introduced into naturally occurring enzymes by the chemical modification of specific amino acid residues in concert with genetic techniques. Chemical strategies have had a significant impact in the field of enzyme design such as modifying the selectivity and catalytic activity which is very different from those of the corresponding native enzymes. Thus, chemical modification has been exploited for the incorporation of active site binding analogs onto protein templates and for atom replacement in order to generate new functionality such as the conversion of a hydrolase into a peroxidase. The introduction of a coordination complex into a substrate binding pocket of trypsin could probably also be extended to various enzymes of significant therapeutic and biotechnological importance.

The aim of this study is the conversion of trypsin into a copper enzyme: tyrosinase by chemical modification. Tyrosinase is a biocatalyst (EC.1.14.18.1) containing two atoms of copper per active site with monooxygenase activity. The active site of trypsin (EC 3.4.21.4), a serine protease was chemically modified by copper (Cu⁺²) introduced p-aminobenzamidine (pABA- Cu⁺²: guanidine containing schiff base metal chelate) which exhibits affinity for the carboxylate group in the active site as trypsin-like inhibitor. Trypsin and the resultant semisynthetic enzyme preparation was analysed by means of its trypsin and catechol oxidase/tyrosinase activity. After chemical modification, trypsin-pABA-Cu⁺² preparation lost 63% of its trypsin activity and gained tyrosinase/catechol oxidase activity. The kinetic properties (K_cat, K_m, K_cat/K_m), optimum pH and temperature of the trypsin-pABA-Cu⁺² complex was also investigated.     

Purification and kinetics of a protease-resistant,neutral, and thermostable phytase from Bacillus subtilis subsp. subtilis JJBS250 ameliorating food nutrition

Abstract

A novel protease-resistant and thermostable phytase from Bacillus subtilis subsp. subtilis JJBS250 was purified 36-fold to homogeneity with a combination of ammonium sulfate precipitation followed by Q-Sepharose and Sephadex G-50 chromatographic techniques. The estimated molecular mass of the purified phytase was 46?kDa by electrophoresis with optimal activity at pH 7.0 and 70?°C. About 19% of original activity was maintained at 80?°C for 10?min. Phytase activity was stimulated in presence of surfactants like Tween-20, Tween-80, and Triton X-100 and metal ions like Ca⁺², K⁺, and Co⁺² and it was inhibited by SDS and Mg⁺², Al⁺², and Fe⁺². Purified enzyme showed specificity to different salts of phytic acid and values of K_m and V_max were 0.293?mM and 11.49 nmoles s^?1, respectively for sodium phytate. The purified enzyme was resistant to proteases (trypsin and pepsin) that resulted in amelioration of food nutrition with simultaneous release of inorganic phosphate, reducing sugars, and soluble protein.