Inhibition of human pancreatic proteinases by human plasma alpha2-antiplasmin and antithrombin

Human plasma alpha1-antitrypsin inhibits human pancreatic trypsin, chymotrypsin and elastase, which are massively released into the blood stream during acute pancreatitis. To examine whether the plasma proteins of individuals with genetic deficiency of alpha1-antitrypsin are protected against the deleterious action of these enzymes by other inhibitors, we have tested their inhibition by alpha2-antiplasmin and antithrombin. We have determined the inhibition rate constants kass and calculated d(t), the in vivo inhibition time. Surprisingly, trypsin is inhibited faster by alpha2-antiplasmin [kass=2.5 x 10(6) M(-1)S(-1), d(t)=2.3 s] and antithrombin [kass=1.7 x 10(5) M(-1)s(-1), d(t)=5.8 s] than by alpha1-antitrypsin [d(t)=17 s or 116 s in alpha1-antitrypsin-sufficient or alpha1-antitrypsin-deficient individuals, respectively]. Low molecular weight heparin accelerates the inhibition of trypsin by antithrombin by a factor of 16 [d(t)=0.36 s]. Antithrombin and alpha2-antiplasmin are not physiological inhibitors of chymotrypsin and elastase. These enzymes are, however, physiologically inhibited by alpha1-antitrypsin and alpha1-antichymotrypsin even in alpha1-antitrypsin-deficient individuals. We conclude that (i) low molecular weight heparin may be helpful in the management of acute pancreatitis, and (ii) genetically determined alpha1-antitrypsin deficiency probably does not lead to a significantly increased risk of plasma protein degradation during this disease.     

Interactions of alpha1-antitrypsin with trypsin and chymotrypsin.

The interaction of alpha1-antitrypsin with trypsin and chymotrypsin has been investigated by protease activity assays, by electrophoretic analysis, by CD and absorption difference spectra, and by gel filtration of reaction mixtures containing excess inhibitor or excess protease. When alpha1-antitrypsin is present in excess, only one stable inhibitor - protease complex is formed. In the presence of excess protease, however, this primary complex is degraded relatively rapidly to one or more secondary complexes. These latter conversions are more pronounced in the case of the antititrypsin-chymotrypsin system. The greater lability of the antitrypsin-chymotrypsin system is evidenced by the relatively rapid release of inactive chymotrypsin from the secondary antitrypsin - chymotrypsin complex. Only minimal amounts of active protease were released from the complexes on the addition of excess protease and one protease could not displace the other from the complex, although competition experiments showed that chymotrypsin reacted more rapidly with the inhibitor than trypsin.     

In vitro interaction of porcine serum and colostrum protease inhibitors with pancreatic trypsin, chymotrypsin and elastase

The partition of 125I-labelled pancreatic trypsin, chymotrypsin and elastase between the inhibitors, alpha 2-macroglobulin f and s, alpha 1-protease inhibitor, alpha 2-antitrypsin, inter-alpha-trypsin inhibitor and the specific sow colostrum protease inhibitor, was studied in vitro by gradually increasing the concentration of these proteases in blood serum from adult and newborn pigs. As revealed by immunoelectrophoresis in combination with autoradiography, differences were noted in the abilities of the various protease inhibitors to interact with and to form complexes with the three proteases, resulting in changes in location, height and numbers of precipitates. Among the serum inhibitors, alpha 2-macroglobulins showed the highest relative affinity to all three proteases, while alpha 1-protease inhibitor showed a high relative affinity only for chymotrypsin. Serum alpha 2-antitrypsin complexed only with trypsin, with a low relative affinity. alpha 2-Antitrypsin also interacted with chymotrypsin and elastase, but without forming complexes. When complexes of sow colostrum protease inhibitor and trypsin were added to the serum from neonatal pigs, these complexes remained stable. The results obtained from these in vitro studies, indicating differences in the relative affinities of the inhibitors to the various proteases, give some information about the role of the inhibitors in vivo, both in adult and in neonatal pigs.     

New human colorectal carcinoma cell lines that secrete proteinase inhibitors in vitro

Two new human cell lines, RCM-1 and CoCM-1, have been established from primary colorectal adenocarcinomas. Both cell lines were unique in that the cultures secreted trypsin inhibitors in vitro. The activities of these inhibitors were accumulated in serum-free media of both cell lines over a period of several days. Two inhibitors (PI-1 and PI-2) were isolated from serum-free conditioned medium in which RCM-1 was grown by anion-exchange and gel filtration high-performance liquid chromatography. PI-1 inhibited trypsin and chymotrypsin strongly, and pancreatic elastase weakly. Its molecular weight was about 57 kilodaltons (Kd) as determined by gel filtration chromatography. It cross-reacted with the antiserum elicited against human alpha 1-antitrypsin in double immunodiffusion. PI-1 corresponding to alpha 1-antitrypsin was also demonstrated immunohistochemically in both cell lines. PI-2 inhibited trypsin strongly, and chymotrypsin, kallikrein and plasmin weakly. It had higher molecular weight (200-300 Kd) than that of PI-1, and did not cross-react with antisera against human alpha 1-antitrypsin, alpha 2-macroglobulin, alpha 1-antichymotrypsin, alpha 2-plasmin inhibitor, inter-alpha-trypsin inhibitor and urinary trypsin inhibitor. RCM-1 and CoCM-1 are the first colorectal adenocarcinoma cell lines that secrete functionally active trypsin inhibitors, including alpha 1-antitrypsin in vitro, and are useful for the study of tumor-cell derived proteinase inhibitors.     

Inhibition of human factor Xa by various plasma protease inhibitors

The inhibitory effects of the plasma protease inhibitors antithrombin III, alpha 2-macroglobulin and alpha 1-antitrypsin on the activity of human factor Xa have been studied using purified proteins. The rate of inhibition was determined by measuring the residual factor Xa activity at timed intervals utilizing the synthetic peptide susbtrate Bz-Ile-Glu(piperidyl)-Gly-Arg-pNA. Kinetic analysis with varying molar concentrations of inhibitors demonstrated that the inhibition of factor Xa by antithromin III, alpha 2-macroglobulin and alpha 1-antitrypsin followed second-order kinetics. Calculated values of the rate constants for the inhibition of factor Xa by antithrombin III, alpha 2-macroglobulin and alpha 1-antitrypsin were 5.8 . 10(4), 4.00 . 10(4) and 1.36 . 10(4) M -1 . min -1, respectively. The plasma concentrations of the inhibitors can be used to assess their potential relative effectiveness against factor Xa. In plasma this was found as alpha 1-antitrypsin greater than antithrombin III greater than alpha 2-macroglobulin in the ratio 4.64: 2.08: 1.0. Cephalin was shown to inhibit the rate of reaction between factor Xa and antithrombin III.     

Secretory expression of human alpha1 -antitrypsin in Saccharomyces cerevisiae using galactose as a gratuitous inducer

Human alpha1 -antitrypsin (alpha1-AT) was expressed and secreted in Saccharomyces cerevisiae carrying the gal1 and reg1-501 mutations under the control of GAL10 promoter. Increasing galactose from 0 to 0.5 g/l increased the extracellular concentration of alpha1-AT, though the secretion efficiency was decreased. The final titre of alpha1-AT secreted and the secretion efficiency were 17.9 mu g/ml and 24.8% respectively, with 0.5 g galactose/l. © Rapid Science Ltd. 1998     

Xenopus oocytes can synthesise but do not secrete the Z variant of human alpha 1-antitrypsin

Human liver mRNA was prepared from a patient homozygous for alpha 1-antitrypsin deficiency (PiZZ) and from a normal subject (PiMM). Both liver RNAs were microinjected into Xenopus oocytes and alpha 1-antitrypsin identified by immunoprecipitation. The normal M variant of alpha 1-antitrypsin is synthesised and secreted by Xenopus oocytes, the abnormal Z protein is not secreted and an intracellular form accumulates in the oocytes. In the presence of tunicamycin an unglycosylated form of M alpha 1-antitrypsin appears in the incubation medium but no corresponding unglycosylated version of the Z protein is secreted.     

Antiproteinase activity of alpha 2-macroglobulin

Blood serum separation by the method of gel filtration on Sephadex G-200 with the subsequent immunochemical determination of the quantitative content of basic proteolysis inhibitors permitted isolating the alpha 2-macroglobulin fraction while alpha 1-antitrypsin and alpha 1-antichymotrypsin separation was a failure. The immunochemical analysis of the antienzymic activity of the isolated inhibitors showed that 32.3 +/- 3.5% of the introduced kallikrein, 18.7 +/- 0.6% of trypsin and 14.4 +/- 4.1% of chymotrypsin were bound in the zone of alpha 2-macroglobulin. The rest of antienzymic activity was localized in the zone of alpha 1-antitrypsin and alpha 1-antichymotrypsin. After a preliminary saturation of blood serum with trypsin in the amount equivalent to its antitryptic capacity (200 micrograms/ml) the ability of alpha 2-macroglobulin to bind kallikrein and chymotrypsin lowers considerably (by 69 and 72%, respectively). In the zone of alpha 1-antitrypsin and alpha 1-antichymotrypsin a decrease in the ability to bind kallikrein and chymotrypsin amounted to 44 and 12% respectively. Thus, alpha 2-macroglobulin being bound with trypsin looses considerably its ability to bind other enzymes.     

Phenotype-dependent inhibition of human alpha 1-antitrypsin by 1,6-hexane diamine in vitro

Blood donors and patients from pulmonary wards gave serum specimens for the assay of their alpha 1-antitrypsin activity and phenotype. The same specimens were then incubated at the room temperature overnight with increasing concentrations of 1,6-hexane diamine. The 5 mM amine concentration caused a significant decrease in heterozygous antitrypsins (M1M2, M1M3, M2M3 and M1S) activity while it was less in the homozygous (M1M1 and M2M2) antitrypsin phenotypes. The SS and ZZ phenotypes showed a very low initial activity which was, however, further reduced. Analysis for the antitrypsin protein showed a simultaneous loss of its activity. The data suggest that heterozygous antitrypsin carriers may be more prone to the effects of inhaled amines despite their 'normal' phenotypic constitution.     

Covalent and non-covalent interaction of chymotrypsin with alpha 2-macroglobulin

The pattern of covalent crosslinking between human alpha 2-macroglobulin (alpha 2M) and chymotrypsin has been investigated by chromatography and polyacrylamide gel electrophoresis in denaturing medium. Reaction with a single mol of chymotrypsin per mol alpha 2M results in the formation of a 95% covalent 1:1 chymotrypsin-alpha 2M complex and in the proteolytic cleavage of both 180 kDa monomers in one alpha 2M subunit. Proteolytic cleavage in the other alpha 2M subunit requires the presence of a second mol of chymotrypsin; part (20%) of the protease in the 2:1 chymotrypsin-alpha 2M complex thus formed appears to be non-covalently bound to the alpha 2M chains. Covalent binding is abolished when the reaction of alpha 2M with the protease is carried out in the presence of hydroxylamine. A single mol of the protease is then able to cleave all four 180 kDa monomers in alpha 2M.     

Effects of copper on the growth,photosynthesis and nutrient concentrations of Phaseolus plants

Bean plants (Phaseolus vulgaris L. var. Zargana Kavala) were grown under conditions of increasing Cu concentrations in the growth medium (0.5-160.5 µM). Generally, the Cu concentrations between 0.5-1.5 µM were deficient, 1.5-10.5 µM were optimal, and 10.5-160.5 µM were toxic to plant growth. The Cu toxicity was associated with marked increases in plant tissue Cu concentrations. Under the Cu-deficient and optimal growth conditions, Cu was located primarily in the leaves. Under Cu toxicity, it was primarily sequestered in the roots. With increasing Cu in the growth medium, there was a positive correlation between Cu concentrations in the roots, stems and leaves, Ca in the roots, and K and Mg in the leaves. In contrast, Ca concentrations in the leaves and stems showed a negative correlation. The chlorophyll (Chl) concentration increased with increasing leaf Cu concentration, however, the Chl a/b ratio decreased. Since with an increasing leaf Cu concentration the leaf area decreased more markedly than the leaf dry mass, the net photosynthetic rate (PN) per leaf area increased and per dry mass decreased. The increase in PN per leaf area was almost entirely accounted for by the increase in Chl concentration. The initial Chl fluorescence (F0) increased with increasing leaf Cu concentration. The ratio of variable to maximum fluorescence (Fv/Fm) under Cu toxicity decreased. The half-time for the rise from F0 to Fm (t1/2) remained relatively unchanged with increasing leaf Cu concentration. Therefore the Cu-stress caused a small decrease in the efficiency of photosystem 2 photochemistry, but its primary effect was on growth.     

Kosmotropic salt activation and substrate specificity of poliovirus protease 3C

Picornaviruses produce a large polyprotein, which is cleaved by virally encoded cysteine peptidases, picornain-2A and -3C. Picornain-3C has characteristics of both the serine peptidase chymotrypsin and the cysteine peptidase papain in that the 3D structure resembles chymotrypsin, but its nucleophile is a cysteine SH rather than a serine OH group. We investigated the specificity of poliovirus picornain-3C (PV3C) protease and the influence of kosmotropic salts on catalytic activity, using FRET peptides related to a cleavable segment of the virus polyprotein. The peptidase activity of PV3C was found to be 100-fold higher in the presence of 1.5 M sodium citrate. This activation was anion-dependent, following the Hofmeister series citrate(3-) > SO4(2-) > HPO4(2-) > acetate- > HCO3(-) > Cl-. The activation appeared to be independent of substrate sequence and arose primarily from an increase in kcat. A shift to higher pH was also observed for the pK1 of the enzyme pH-activity profile. Experiments with the fluorescent probe ANS (1-anilino-8-naphthalene sulfonate) showed that the protease bound the dye in the presence of 1 M sodium citrate but not in its absence or in the presence of 1 M NaCl. Structural changes in PV3C protease were detected using circular dichroism and the thermodynamic data indicated a more organized active site in the presence of sodium citrate. PV3C protease was also activated in D2O, which was added to the activation by citrate. These effects seem to be related to nonspecific interactions between the solvent and the protein. Our data show that the catalytic efficiency of PV3C protease is modulated by the composition of the environment and that this modulation may play a role in the optimal processing of polyprotein for the virus assembly that occurs inside specific vesicles formed in poliovirus-infected cells.     

Inhibition of prothrombinase complex by plasma proteinase inhibitors

The rate of inactivation of human coagulation factor Xa by the plasma proteinase inhibitors antithrombin III and alpha 1-antitrypsin has been studied in the presence of the accessory components which constitute the prothrombinase complex. The rate of inactivation of factor Xa by antithrombin III was found to be decreased in the presence of phospholipid vesicles with high affinity for factor Xa. The second-order rate constant for the reaction fell from 6.21 X 10(4) to 3.40 X 10(4) M-1 min-1 in the presence of 20 microM phospholipid. Purified factor Va had no effect on the rate of inactivation of factor Xa in the absence of phospholipid. In the presence of phospholipid, factor Va increased the protective effect displayed by phospholipid, further reducing the rate constant to 2.20 X 10(4) M-1 min-1. The rate of inactivation of factor Xa by alpha 1-antitrypsin was unaffected under these conditions. Platelet-bound prothrombinase complex was formed by incubation of factor Xa with washed human platelets activated by a mixture of collagen and thrombin. The prothrombinase activity was inhibited by antithrombin III was a second-order rate constant of 0.85 X 10(4) M-1 min-1. This rate was obtained in both the presence and absence of exogenous factor Va. Platelet factor 3 vesicles, isolated from platelet aggregation supernatants, also formed prothrombinase complex in the presence of factor Va, and this was inhibited by antithrombin III at the same rate as the platelet-bound complex. There was no protection of the platelet-bound prothrombinase complex from inhibition by alpha 1-antitrypsin.     

Sequestration of mutated alpha1-antitrypsin into inclusion bodies is a cell-protective mechanism to maintain endoplasmic reticulum function

A variant alpha1-antitrypsin with E342K mutation has a high tendency to form intracellular polymers, and it is associated with liver disease. In the hepatocytes of individuals carrying the mutation, alpha1-antitrypsin localizes both to the endoplasmic reticulum (ER) and to membrane-surrounded inclusion bodies (IBs). It is unclear whether the IBs contribute to cell toxicity or whether they are protective to the cell. We found that in hepatoma cells, mutated alpha1-antitrypsin exited the ER and accumulated in IBs that were negative for autophagosomal and lysosomal markers, and contained several ER components, but not calnexin. Mutated alpha1-antitrypsin induced IBs also in neuroendocrine cells, showing that formation of these organelles is not cell type specific. In the presence of IBs, ER function was largely maintained. Increased levels of calnexin, but not of protein disulfide isomerase, inhibited formation of IBs and lead to retention of mutated alpha1-antitrypsin in the ER. In hepatoma cells, shift of mutated alpha1-antitrypsin localization to the ER by calnexin overexpression lead to cell shrinkage, ER stress, and impairment of the secretory pathway at the ER level. We conclude that segregation of mutated alpha1-antitrypsin from the ER to the IBs is a protective cell response to maintain a functional secretory pathway.     

Effect of varying citrate levels on C4 compound formation and on enzyme levels in Leuconostoc mesenteroides subsp. cremoris grown in continuous culture

Summary The effects of citrate on diacetyl, acetoin and 2,3-butylene glycol (2,3-BG) production by Leuconostoc mesenteroides subsp. cremoris grown in continuous culture at pH 5.2 were studied. In glucose alone end-product production agreed with the theoretical stoichiometry. In the presence of citrate, lactate and acetate production was higher than the theoretical stoichiometry from glucose. Lactate production was constant when the initial citrate concentration was increased whereas ethanol production strongly decreased. In the absence of citrate, citrate lyase (CL) exhibited weak activity. Diacetyl reductase (DR) and acetoin reductase (AR) exhibited basal activity. When varying citrate concentrations ranging from 10 to 75 mm were added to glucose broth, DR, AR, lactate dehydrogenase, NADH oxidase and alcohol dehydrogenase decreased as the initial citrate concentration increased suggesting that they were partly repressed by citrate. In contrast, CL increased and the specific citrate utilization rate also increased in the same way, indicating no saturation of the first step of citrate metabolism. Acetate kinase (AK) was slightly higher in the presence of citrate and increased when the initial citrate concentration increased. This result was correlated with an increase of acetate from the acetyl phosphate pathway. More ATP was produced in the presence of citrate, which could explain the increase in biomass formation. Citrate bioconversion into diacetyl, acetoin and 2,3-BG increased as the initial citrate increased. Correspondence to: C. Diviès     

Disassembly kinetics of thick filaments in rabbit skeletal muscle fibers. Effects of ionic strength, Ca2+ concentration, pH, temperature, and cross-bridges on the stability of thick filament structure.

The kinetics of dissociation from both ends of thick filaments in a muscle fiber was investigated by an optical diffraction method. The dissociation velocity of thick filaments at a sarcomere length of 2.75 microns increased with increasing the KCl concentration (from 60 mM to 0.5 M), increasing the pH value (from 6.2 to 8.0) or decreasing the temperature (from 25 to 5 degrees C) in the presence of 10 mM pyrophosphate and 5 mM MgCl2. Micromolar concentrations of Ca2+ suppressed the dissociation velocity markedly at shorter sarcomere lengths. The dissociation velocity, v, decreased as thick filaments became shorter, and v = -db/dt = vo exp (alpha b), where b is the length of the thick filament at time t and vo and alpha are constants. The vo value was largely dependent on the KCl concentration but the alpha value was not. The stiffness of a muscle fiber decreased nearly in proportion to the decrease of overlap between thick and thin filaments induced by the dissociation of thick filaments. This indicates that cross-bridges are uniformly distributed and contribute independently to the stiffness of a muscle fiber during the dissociation of thick filaments.     

Identification of divalent metal ion-dependent inhibition of activated protein C by alpha 2-macroglobulin and alpha 2-antiplasmin in blood and comparisons to inhibition of factor Xa, thrombin, and plasmin.

The half-life of activated protein C (APC) was 31 min in citrated blood and 18 min in whole blood. Immunoblotting analysis of citrated blood identified APC-protein C inhibitor (APC-PCI) and APC-alpha 1-antitrypsin complexes. Whole blood contained two additional APC-inhibitor complexes, one stimulated by Ca2+ and another by Mg2+. The former was identified as APC-alpha 2-macroglobulin (APC-alpha 2M) while the latter was not identified. APC-alpha 2-antiplasmin complexes (APC-alpha 2AP) were identified, comigrating with APC-PCI complexes. Purified alpha 2M and alpha 2AP inhibited APC in the presence of Ca2+ (k2 = 99 and 100 M-1 S-1, respectively. Inhibition of APC and Factor Xa by alpha 2M and inhibition of APC by alpha 2AP was stimulated by Ca2+, Mn2+, and Mg2+. Inhibition of thrombin by alpha 2M and of plasmin by alpha 2AP was not altered by EDTA or Ca2+, suggesting divalent metal ions affect APC and Factor Xa rather than the inhibitors. k2 values for the APC inhibitors and their plasma concentrations suggest that PCI and alpha 1-antitrypsin are the more important APC inhibitors and that alpha 2M and alpha 2AP are metal ion-dependent auxiliary inhibitors. Inhibitors can account for the in vivo half-life of APC.     

Modulation of recombinant human prostate-specific antigen: activation by Hofmeister salts and inhibition by azapeptides. Appendix: thermodynamic interpretation of the activation by concentrated salts

Prostate specific antigen (PSA, also known as human kallikrein 3) is an important diagnostic indicator of prostatic disease. PSA exhibits low protease activity (>10(4)-fold less than chymotrypsin) under the usual in vitro assay conditions. In addition, PSA does not react readily with prototypical serine protease inactivators. We expressed human PSA (rh-PSA) in Escherichia coli and have demonstrated that rh-PSA has properties similar to those of native PSA isolated from human seminal fluid. Both PSA and rh-PSA are >10(3)-fold more active in the presence of 1.3 M Na(2)SO(4). This activation is anion-dependent, following the Hofmeister series when normality is considered: SO(4)(2)(-) approximately citrate > Ac(-) > Cl(-) > Br(-) > I(-). The nature of the cation has little effect on salt activation. The rate of inactivation of rh-PSA by DFP is 30-fold faster in the presence of 0.9 M Na(2)SO(4), and the rate of inactivation by Suc-Ala-Ala-Pro-Phe-CK is >20-fold faster under these conditions. Azapeptides containing Phe or Tyr at position P(1) also inactivate rh-PSA in the presence of high salt concentrations. These compounds represent the first described inhibitors designed to utilize the substrate binding subsites of PSA. CD spectroscopy demonstrates that the conformation of rh-PSA changes in the presence of high salt concentrations. Analytical ultracentifugation and dynamic light scattering indicate that PSA remains monomeric under high-salt conditions. Interestingly, human prostatic fluid contains as much as 150 micro mol citrate/g wet weight, which suggests that salt concentrations may regulate PSA activity in vivo.     

Efficient removal of albumin from human serum by monosize dye-affinity beads

Cibacron Blue F3GA was covalently attached onto monosize poly(glycidyl methacrylate) [poly(GMA)] beads for removal of human serum albumin (HSA) from human serum. Monosize poly(GMA) beads, 1.6 microm in diameter, were produced by dispersion polymerization. Cibacron Blue F3GA loading was 1.73 mol/g. HSA adsorption experiments were performed by stirred-batch adsorption. The non-specific adsorption of HSA was low (0.8 mg/g polymer). Dye attachment onto the monosize beads significantly increased the HSA adsorption (189.8 mg/g). The maximum HSA adsorption was observed at pH 5.0. With an increase of the aqueous phase concentration of sodium chloride, the adsorption capacity decreased drastically. The equilibrium adsorption of HSA significantly decreased with increasing temperature. The elution studies were performed by adding 0.1 M Tris/HCl buffer containing 0.5 M NaSCN to the HSA solutions in which adsorption equilibria had been reached. The elution results demonstrated that the adsorption of HSA to the adsorbent was reversible. The depletion efficiencies for HSA were above 87% for all studied concentrations. To test the efficiency of HSA removal from human serum, proteins in the serum and eluted portion were analyzed by two-dimensional gel electrophoresis. Eluted proteins include mainly albumin, and a small number of nonalbumin proteins such as apo-lipoprotein A1, sero-transferrin, haptoglobulin and alpha1-antitrypsin were bound by the dye-affinity beads. IgA was not identified in eluted fraction.