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PubMed1976

Snake venom proteinase inhibitors. III. Isolation of five polypeptide inhibitors from the venoms of Hemachatus haemachatus (Ringhal's corbra) and Naja nivea (Cape cobra) and the complete amino acid sequences of two of them.

Five proteinase inhibitors which all inhibit the activity of bovine trypsin [EC 3.4.21.4] were isolated from African Elapid venoms of Hemachatus haemachatus (HHV, Ringhal's cobra) and Naja nivea (NNV, Cape cobra). All the inhibitors were essentially homogeneous by polyacrylamide gel electrophoresis in the presence or absence of sodium dodecylsulfate. Amino acid analysis and terminal analysis also supported their chemical homogeneities, except for one of the two inhibitors from Hemachatus haemachatus venom. The isolated inhibitors had a molecular weight of about 6,500, consisting of 52 to 57 amino acid residues, and they were all devoid of tryptophan. However, their amino acid compositions differed from each other. One of the three inhibitors isolated from Naja nivea venom, designated NNV inhibitor Ia, was unique, in that 4 half-cystinyl residues per mole fof the polypeptide were present, whereas all the others contained six residues. Of the isolated proteinase inhibitors, the complete amino acid sequences of two major inhibitors were established by manual and automatic Edman degradations and standard enzymatic techniques. Each of the inhibitors, designated HHV inhibitor II and NNV inhibitor II, consisted of 57 amino acid with arginine and glycine at the NH2- and COOH-termini, respectively. Both contained six half-cystines in disulfide linkages, and their overall amino acid sequences were similar, showing 91% homology. The two inhibitors differed in sequence by only five amino acid replacements, Asp-3 to Arg; Tyr-17 to Arg; Leu-25 to Arg; Gln-32 to Glu; and Arg-52 to His, in the 57 residue peptide chain. Comparing the amino acid sequences of these two cobra venom inhibitors with those of Russell's viper venom inhibitor II and bovine pancreatic trypsin inhibitor (BPTI), about 50% homology was found in their sequences. The 6 half-cystinyl residues of these inhibitors were in the same linear positions. Moreover, the regions which are structurally and functionally important in the well-known BPTI molecule were found with extremely high sequence homology in the cobra venom inhibitors. These findings strongly suggest that the cobra venom inhibitors as well as Russell's viper inhibitor II have very similar conformations to that established for BPTI.

Amino Acid Sequence↗
PubMed2003

Development of the Bowman-Birk inhibitor for oral cancer chemoprevention and analysis of Neu immunohistochemical staining intensity with Bowman-Birk inhibitor concentrate treatment.

OBJECTIVES/HYPOTHESIS: Cancer chemoprevention is a rapidly evolving approach to reverse or inhibit carcinogenesis, and there is active interest in development of effective chemopreventive agents against head and neck cancers. The retinoids are archetypal chemopreventive agents for oral premalignant lesions. They have significant clinical effect, but widespread use is limited by significant clinical toxicity. The Bowman-Birk Inhibitor is one of several nontoxic compounds exhibiting both potent anticarcinogenic activity and minimal toxicity. The purposes of the study were to summarize the preclinical and clinical development of Bowman-Birk Inhibitor and a Bowman-Birk Inhibitor concentrate against oral premalignant lesions and to evaluate Neu immunohistochemical staining intensity for lesions and simultaneously obtained biopsy specimens of normal-appearing mucosa from the Phase IIa Bowman-Birk Inhibitor concentrate oral leukoplakia chemoprevention trial. STUDY DESIGN: Part I is a selected literature review. Part II is a retrospective analysis of pathological specimens prospectively obtained from the Phase IIa clinical trial of Bowman-Birk Inhibitor concentrate. METHODS: Thirty-two sets of biopsy specimens from lesions and uninvolved oral mucosa before and after treatment with Bowman-Birk Inhibitor concentrate in doses ranging from 200 to 1066 chymotrypsin inhibitory units were examined in blinded fashion for Neu immunohistochemical staining intensity using the 3B-5 monoclonal antibody. Staining intensity scores among the lesion and control biopsy specimens before and after Bowman-Birk Inhibitor concentrate treatment were analyzed and compared with previously obtained values for serum Neu, oral mucosal cell Neu, protease activity, and clinical response to treatment. RESULTS: Mean Neu staining score was significantly higher in lesions compared with uninvolved mucosa (P <.001). Pretreatment staining scores for biopsy specimens of lesions and control biopsy specimens of normal-appearing tissues were correlated (Spearman correlation coefficient [r] = 0.375, P =.045), but no correlation between lesion and control biopsy specimen scores was evident after treatment. The change in Neu staining score with Bowman-Birk Inhibitor concentrate treatment in control site biopsy specimens demonstrated an inverse relationship of change in lesion area with Bowman-Birk Inhibitor concentrate treatment (Spearman r = -0.493, P <.007). CONCLUSION: Bowman-Birk Inhibitor concentrate shows promise to become an effective nontoxic chemopreventive agent based on results of extensive preclinical studies, and Phase I and Phase IIa clinical trials. Bowman-Birk Inhibitor concentrate has dose-related clinical activity against oral leukoplakia and modulates levels of Neu and protease activity. The current investigation identified increased Neu staining intensity in hyperplastic lesions compared with simultaneously obtained biopsy specimens of normal-appearing mucosa both before and after Bowman-Birk Inhibitor concentrate treatment. This finding supports prior observations that increased Neu expression is present in a subset of oral premalignant lesions and head and neck cancers. The trend of increased Neu staining score in control biopsy tissues of subjects exhibiting decreased lesion area following Bowman-Birk Inhibitor concentrate treatment raises questions about the mechanisms of Bowman-Birk Inhibitor concentrate action. One possible explanation is that Bowman-Birk Inhibitor stabilizes the extracellular domain of Neu, thereby preventing receptor truncation and internalization. Further study of modulation of Neu and protease activity by Bowman-Birk Inhibitor concentrate treatment may provide insights into the role of proteases and protease inhibitors in oral premalignant lesions and the mechanisms underlying Bowman-Birk Inhibitor concentrate effects. A Phase IIb randomized, placebo-controlled clinical trial to determine the clinical effectiveness of Bowman-Birk Inhibitor concentrate and further evaluate these candidate biomarkers is under way.

Animals↗
PubMedDate not supplied

Inhibition of elastase by a synthetic cotton-bound serine protease inhibitor: in vitro kinetics and inhibitor release.

A cotton-bound serine protease inhibitor of elastase (fiber-inhibitor) has been formulated for in vitro evaluation in chronic wound fluid. As a model to understand the properties of the inhibitor in wound dressings, the kinetic profile and in vitro release of the fiber-inhibitor formulation have been examined. The elastase inhibitor N-Methoxysuccinyl-Ala-Ala-Pro-Val-chloromethylketone was modified onto cotton cellulose fibers and assayed as a colloidal system. Amino acid analysis and reversed phase high performance liquid chromatography were compared as semiquantitative methods to assess elastase inhibitor release from the cotton fibers. The kinetics of inhibition was assessed on treated fibers of synthetic dressings such that a colloidal suspension of the fiber-inhibitor and elastase was employed as an assay. A dose-response relationship was observed in the kinetics of substrate hydrolysis catalyzed by three elastases: porcine pancreatic elastase, which was employed to model this approach; human leukocyte elastase; and elastase in human chronic wound fluid. Both freely dissolved and fiber-bound inhibitors were studied. The initial rates of substrate hydrolysis were inversely linear with freely dissolved inhibitor dose. The apparent first order rate constants, kobs, for the elastase-inhibitor complex were calculated from the kinetic profiles. The kobs for inhibitor bound enzyme varied as a function of inhibitor vs. enzyme concentration and based on the order of mixing of substrate, inhibitor and enzyme in the assay. Enzyme inhibition by the fiber-inhibitor was measured as inhibitor concentration at 50% inhibition (I50). I50 values measured from the colloidal assay with fiber-released inhibitor were within the same range to those for freely dissolved inhibitor. Inhibition of elastase activity in chronic wound fluid was observed with 1-5 mg of fiber-inhibitor formulation. This approach constitutes an in vitro assessment of synthetic serine protease inhibitors on fibers and may be employed to evaluate structure vs. function of elastase inhibition in the modified fibers of wound dressing composites.

Aged↗
PubMed1976

[The inter-alpha-trypsin inhibitor as precursor of the acid-stable proteinase inhibitors in human serum and urine].

A small amount of antitryptic activity is detectable in the supernatant of deproteinized human serum. Preincubation of serum with trypsin causes an increase in acid-stable antitryptic activity. This rise in activity depends on the inter alpha-trypsin inhibitor concentration. The native inhibitor present in normal sera, and in higher concentrations in sera of patients with nephropathies, and the trypsin-liberated inhibitor show immunological cross reaction with antibodies to the serum inter-alpha-trypsin inhibitor. The two inhibitors differ in molecular weight and electrophoretic mobility. The physiological inhibitor (I-34), with a molecular weight of 34 000 and a high carbohydrate content, can be transformed by trypsin into an inhibitor (I-17) with a molecular weight of 17 000. This inhibitor is identical with the inhibitors liberated by trypsin from serum or from purified inter-alpha-trypsin inhibitor. The acid-stable inhibitor from urine is identical with the physiological serum inhibitor. Analogously, this inhibitor is transformed by trypsin into the inhibitor with a molecular weight of 17 000. We conclude that the inter-alpha-trypsin inhibitor is the precursor of both the physiological and the trypsin-liberated inhibitor. By a mechanism as yet unknown, but most likely a limited proteolysis, the secreted inhibitor is liberated from the high molecular weight precursor. In contrast to the monospecific trypsin-inhibiting precursor, the physiological and artificially liberated inhibitors are trypsin/chymotrypsin/plasmin inhibitors.

Humans↗
PubMed1983

Interaction of F1-ATPase, from ox heart mitochondria with its naturally occurring inhibitor protein. Studies using radio-iodinated inhibitor protein.

The ox heart mitochondrial inhibitor protein may be iodinated with up to 0.8 mol 125I per mol inhibitor with no loss of inhibitory activity, with no change in binding affinity to submitochondrial particles, and without alteration in the response of membrane-bound inhibitor to energisation. Tryptic peptide maps reveal a single labelled peptide, consistent with modification of the single tyrosine residue of the protein. A single type of high-affinity binding site (Kd=96 . 10 (-9)M) for the inhibitor protein has been measured in submitochondrial particles. The concentration of this site is proportional to the amount of membrane-bound F1, and there appears to be one such site per F1 molecule. The ATp hydrolytic activity of submitochondrial particles is inversely proportional to the occupancy of the high-affinity binding site for the inhibitor protein. No evidence is found for a non-inhibitory binding site on the membrane or on other mitochondrial proteins. In intact mitochondria from bovine heart, the inhibitor protein is present in an approx. 1:1 ratio with F1. Submitochondrial particles prepared by sonication of these mitochondria with MgATP contain about 0.75 mol inhibitor protein per mol F1, and show about 25% of the ATPase activity of inhibitor-free submitochondrial particles. Additional inhibitor protein can be bound to these particles to a level of 0.2 mol/mol F1, with consequent loss of ATPase activity. If MgATP is omitted from the medium, or inhibitors of ATP hydrolysis are present, the rate of combination between F1 and its inhibitor protein is very much reduced. The equilibrium level of binding is, however, unaltered. These results suggest the presence of a single, high-affinity, inhibitory binding site for inhibitor protein on membrane-bound F1. The energisation of coupled submitochondrial particles by succinate oxidation or by ATP hydrolysis results in both the dissociation of inhibitor protein into solution, and the activation of ATP hydrolysis. At least 80% of the membrane-bound F1-inhibitor complex responds to this energisation by participating in a new equilibrium between bound and free inhibitor protein. This finding suggests that a delocalised energy pool is important in promoting inhibitor protein release from F1. Dissipation of the electrochemical gradient by uncouplers, or the binding of oligomycin or efrapetin effectively blocks energised release of the inhibitor protein. Conversely, the addition of aurovertin or adenosine 5'--[beta, lambda--imido]triphosphate enhances energy-driven release. The mode of action of various inhibitors on binding and energised release of the protein inhibitor is discussed.

Adenosine Triphosphate↗
PubMed1989

Inhibition of radiation-induced transformation of C3H/10T1/2 cells by carboxypeptidase inhibitor 1 and inhibitor II from potatoes.

In the current study, the ability of four protease inhibitors to suppress radiation-induced transformation of C3H/10T1/2 cells was investigated. The inhibitors tested included: (i) aprotinin (a serine protease inhibitor), (ii) N-acetyl-L-tyrosine ethyl ester (a chymotrypsin substrate and competitive inhibitor of protein degradation), (iii) carboxypeptidase inhibitor (a metallo-exopeptidase inhibitor) and (iv) Inhibitor II (a chymotrypsin/trypsin inhibitor). While none of the inhibitors were toxic to the cells at the concentrations employed, only carboxypeptidase inhibitor and inhibitor II are internalized radiation-induced transformation in a statistically significant manner. Utilizing fluorescent labeled inhibitors, we found that carboxypeptidase inhibitors and Inhibitor II are internalized by the cells. Fluorescent-labeled inhibitor could be observed in the cells within 15 min of incubation and is present in distinct intercellular vacuoles within 1 h. These results indicate that carboxypeptidase inhibitor and Inhibitor II are internalized by C3H/10T1/2 cells and thus would be able to inhibit intracellular proteases (or other enzymes) involved in the conversion of a cell to the malignant state.

Animals↗
PubMed1986

Wheat germ trypsin inhibitors. Isolation and structural characterization of single-headed and double-headed inhibitors of the Bowman-Birk type.

A number of trypsin inhibitors were isolated from wheat germs by affinity chromatography on immobilized trypsin, gel-filtration, and ion-exchange and reverse-phase chromatography. These inhibitors were classified into two groups, inhibitors I (Mr = 14,500) and II (Mr = 7,000), based on their molecular sizes. Inhibitors I and II inhibited bovine trypsin stoichiometorically at an enzyme to inhibitor ratio of 2 and 1, respectively. Sequence analysis of these inhibitors indicated a high degree of homology and that inhibitors I had a duplicated structure of inhibitors II. They are highly homologous to double-headed proteinase inhibitors (Bowman-Birk inhibitors) of Leguminosae plants. Inhibitors II are the first example of single-headed inhibitor corresponding to one inhibitory domain of the Bowman-Birk type double-headed inhibitors, which suggests that inhibitors II are relic of an ancestral single-headed inhibitor before the gene-duplication that led to the formation of present-day Bowman-Birk type inhibitors.

Amino Acid Sequence↗
PubMed1983

A kinetic analysis of the effects of inhibitor-1 and inhibitor-2 on the activity of protein phosphatase-1.

The steady-state interaction between protein phosphatase-1 and its two inhibitor proteins was studied in vitro at low enzyme concentrations where the assumptions of the Michaelis-Menten equation appeared to be valid. Under these conditions, and in the absence of divalent cations, inhibitor-1 behaved as a mixed inhibitor using phosphorylase alpha as a substrate, whereas inhibitor-2 was a competitive inhibitor. The results demonstrate that inhibitor-1 and inhibitor-2 do not interact with protein phosphatase-1 in an identical manner. Inhibitor-1 was only a substrate for protein phosphatase-1 in the presence of Mn2+, and its dephosphorylation was inhibited competitively by inhibitor-2 (Kis = 8 nM). Inhibitor-1 did not inhibit its own dephosphorylation in the presence of Mn2+. Its Km as a substrate (190 nM) was very much higher than its Ki as an inhibitor (1.5-7.5 nM). The results are consistent with a model in which a single binding site for inhibitor-1 is present on protein phosphatase-1, distinct from the binding site for phosphorylase alpha. It is envisaged that the binding of inhibitor-1 to this site not only inhibits the dephosphorylation of other substrates but permits access of its phosphothreonine to the same catalytic group(s) responsible for the dephosphorylation of other substrates. G-substrate, a protein phosphorylated exclusively on threonine residues, did not inhibit the dephosphorylation of phosphorylase alpha and its dephosphorylation was potently inhibited by inhibitor-1 or inhibitor-2. The role of the phosphothreonine residue in inhibitor-1 is discussed in the light of these results.

Carrier Proteins↗
PubMedDate not supplied

Protein tyrosine kinase inhibitors block the stimulatory actions of phosphotyrosine phosphatase inhibitors to increase cell proliferation, alkaline phosphatase activity, and collagen synthesis in normal human bone cells.

The present study sought to test whether inhibition of phosphotyrosine phosphatases (PTPs) would stimulate proliferation and differentiation of normal bone cells, and whether the PTP inhibitor-mediated effects would be blocked by protein tyrosine kinase (PTK) inhibitors. Three inhibitors [phenylarsine oxide (PAO), orthovanadate (VO(4)), and molybdate (MoO(4))] and two normal human bone cells with different basal differentiation status (i.e., mandible- and vertebra-derived bone cells) were used. Cell proliferation was determined with [(3)H]thymidine incorporation, and confirmed by cell counting. Bone cell differentiation was assessed by increases in alkaline phosphatase (ALP) specific activity and collagen synthesis. The three test PTP inhibitors each stimulated [(3)H]thymidine incorporation in both human bone cell types in a biphasic, dose-dependent manner with optimal doses of 20 nM PAO, 1 microM VO(4) and 2 microM MoO(4), respectively. These PTP inhibitors at mitogenic doses each significantly and reproducibly increased ALP specific activity and collagen synthesis. To determine whether the stimulatory effects of PTP inhibitors could be blocked by PTK inhibitors, the effects of tyrphostin A51 and erbstatin, two potent PTK inhibitors, on the actions of PTK inhibitors on [(3)H]thymidine incorporation and ALP specific activity were evaluated. Both tyrphostin A51 and erbstatin, which by themselves alone significantly inhibited human bone cell proliferation and increased ALP specific activity, completely abolished the stimulatory effects of each of the three test PTP inhibitors on bone cell proliferation and ALP specific activity. In conclusion, these findings confirm the premise that inhibition of PTP activities in normal human bone cells could lead to increases in cell proliferation and differentiation, effects that are independent of basal differentiation status of the cells. More importantly, this study demonstrates for the first time that the stimulatory actions of the PTP inhibitor on bone cell proliferation and ALP could be blocked by a PTK inhibitor, suggesting that the osteogenic effects of PTP inhibitors may depend on PTK activities, presumably to increase basal tyrosyl phosphorylation level. Accordingly, one should interpret results of studies using PTK inhibitors with caution in that an inhibition by a PTK inhibitor does not necessarily indicate the requirement of PTK activities, as it could also suggest involvement of an inhibition of PTPs.

Alkaline Phosphatase↗
PubMed1982

Inactivation of factor XIa by plasma protease inhibitors: predominant role of alpha 1-protease inhibitor and protective effect of high molecular weight kininogen.

Factor XIa is a plasma protease that, by activating Factor IX, plays an important role in the early phase of the intrinsic pathway of blood coagulation. Four plasma protease inhibitors, alpha(1)-protease inhibitor, antithrombin III, C1-inhibitor, and alpha(2)-plasmin inhibitor, have been reported to inactivate human Factor XIa, but their quantitative contribution to the inactivation of Factor XIa in plasma has not been fully assessed. Using purified systems, we observed that the second-order rate constants for the reaction of Factor XIa with alpha(1)-protease inhibitor, antithrombin III, and CI-inhibitor were 4.08, 10, and 14.6 M(-1) min(-1) x 10(3), respectively. The pseudo-first-order rate constants, at plasma concentration of the inhibitors, were 1.86 x 10(-1), 4.68 x 10(-2), and 2.4 x 10(-2) min(-1), respectively. These kinetic data predict that alpha(1)-protease inhibitor should account for 68%, antithrombin III for 16%, and C1-inhibitor and the equipotent alpha(2)-plasmin inhibitor each for 8% of the total inhibitory activity of plasma against Factor XIa. The rate of inactivation of Factor XIa in various plasma samples specifically deficient in inhibitors was consistent with these predictions. Factor XI, the zymogen form of Factor XIa, circulates in plasma associated with the contact system cofactor, high molecular weight kininogen (HMW kininogen). Kinetic analysis indicated the existence of a reversible bimolecular Factor XIa-HMW kininogen complex with a dissociation constant (K(d)) = 0.17 muM. The light chain derived from HMW kininogen decreased the inactivation rate of Factor XIa by C1-inhibitor with a K(d) of 0.08 muM for a complex of Factor XIa and the light chain derived from HMW kininogen. The protective effect of HMW kininogen was confirmed by the finding that the inactivation rate of Factor XIa in kininogen-deficient plasma was increased over normal plasma. The present study confirms that alpha(1)-protease inhibitor is the major inhibitor of Factor XIa in plasma, and that the formation of a reversible complex between Factor XIa and HMW kininogen decreases the rate of inactivation of the enzyme by its inhibitors.

Antithrombin III↗
PubMed1984

Hepatocyte uptake of alpha 1-proteinase inhibitor-trypsin complexes in vitro: evidence for a shared uptake mechanism for proteinase complexes of alpha 1-proteinase inhibitor and antithrombin III.

In vivo clearance studies have indicated that the clearance of proteinase complexes of the homologous serine proteinase inhibitors alpha 1-proteinase inhibitor and antithrombin III occurs via a specific and saturable pathway located on hepatocytes. In vitro hepatocyte-uptake studies with antithrombin III-proteinase complexes confirmed the hepatocyte uptake and degradation of these complexes, and demonstrated the formation of a disulfide interchange product between the ligand and a cellular protein. We now report the results of in vitro hepatocyte uptake studies with alpha 1-proteinase inhibitor-trypsin complexes. Trypsin complexes of alpha 1-proteinase inhibitor were prepared and purified to homogeneity. Uptake of these complexes by hepatocytes was time and concentration-dependent. Competition experiments with alpha 1-proteinase inhibitor, alpha 1-proteinase inhibitor-trypsin, and antithrombin III-thrombin indicated that the proteinase complexes of these two inhibitors are recognized by the same uptake mechanism, whereas the native inhibitor is not. Uptake studies were performed at 37 degrees C with 125I-alpha 1-proteinase inhibitor-trypsin and analyzed by sodium dodecyl sulfate-gel electrophoresis in conjunction with autoradiography. These studies demonstrated time-dependent uptake and degradation of the ligand to low molecular weight peptides. In addition, there was a time-dependent accumulation of a high molecular weight complex of ligand and a cellular protein. This complex disappeared when gels were performed under reducing conditions. The sole cysteine residue in alpha 1-proteinase inhibitor was reduced and alkylated with iodoacetamide. Trypsin complexes of the modified inhibitor were prepared and purified to homogeneity. Uptake and degradation studies demonstrated no differences in the results obtained with this modified complex as compared to unmodified alpha 1-proteinase inhibitor-trypsin complex. In addition, the high molecular weight disulfide interchange product was still present on sodium dodecyl sulfate-polyacrylamide gel electrophoresis of solubilized cells. Clearance and clearance competition studies with alpha 1-proteinase inhibitor-trypsin, alkylated alpha 1-proteinase inhibitor-trypsin, antithrombin III-thrombin, and anti-thrombin III-factor IXa further demonstrated the shared hepatocyte uptake mechanism for all these complexes.

Alkylation↗
PubMed1986

Demonstration of modified inactive first component of complement (C1) inhibitor in the plasmas of C1 inhibitor-deficient patients.

The first component of complement (C1) inhibitor plays a critical role in the regulation of the classical complement pathway and the contact system, and the deficiency of C1 inhibitor protein or function is associated with recurrent angioedema. In this study we evaluated the size of the C1 inhibitor antigens present in the plasmas of C1 inhibitor-deficient patients. We found that the C1 inhibitor in the plasmas existed in three forms: high molecular weight forms in complex with proteases, native 110-kD C1 inhibitor, and a modified inactive 94-kD form. The proportion of the total C1 inhibitor in the 94-kD form was 28% in nine hereditary angioedema patients, 92% in five acquired C1 inhibitor-deficiency patients, and 1.2% in five normal controls. In vitro activation of normal plasma with kaolin, but not heat-aggregated gamma-globulin generated 94-kD C1 inhibitor from 110-kD C1 inhibitor. Neither kaolin activation nor heat-aggregated gamma-globulin activation generated 94-kD C1 inhibitor in Hageman factor-deficient plasma. These results suggest that 94-kD C1 inhibitor is generated in vitro by activation of the contact system. The in vivo mechanism of 94-kD C1 inhibitor generation in C1 inhibitor-deficient patients is not known.

Angioedema↗
PubMed1984

Amidolytic and immuno-nephelometric determination of alpha 1-proteinase inhibitor and alpha 2-macroglobulin in serum with calculation of specific inhibitor activities in health and disease.

In sera of healthy persons (n = 50) and patients with a variety of diseases (n = 197) the two major proteinase inhibitors, alpha 1-proteinase inhibitor (alpha 1-antitrypsin) and alpha 2-macroglobulin, were measured by two methods: a chromogenic (amidolytic) substrate assay to assess the functional activities, and a laser nephelometric method to determine the immunoreactive concentrations of the respective proteins. The specific proteinase inhibitor activities defined as the number of inhibitor units per g inhibitor protein were calculated. The precision and accuracy of both assays were found to be similar, showing a satisfactory correlation of results for the sera of healthy persons (r = 0.916 for alpha 2-macroglobulin and 0.988 for alpha 1-proteinase inhibitor). In diseased individuals the correlation was lower than in normal persons (0.862 for alpha 2-macroglobulin and 0.907 for alpha 1-proteinase inhibitor). A poor correlation was obtained in patients with liver diseases (r = 0.586 for alpha 1-proteinase inhibitor and 0.852 for alpha 2-macroglobulin). Reference ranges were established for functional and immunological concentrations and for specific inhibitor activities, respectively. Normal values followed a Gaussian distribution. In patients with various diseases including those with acute phase response, the specific inhibitor activities of alpha 1-proteinase inhibitor are reduced significantly; this is because inhibitor activity shows a smaller relative increase than immunoreactivity. Among the various diseases, no significant differences were noted. The specific inhibitor activity of alpha 2-macroglobulin changed significantly only in patients with carcinoma, liver diseases and trauma. Follow up of some patients shows also intraindividual variation of specific proteinase inhibitor activities.

Blood Proteins↗
PubMed1993

C1-inhibitor-serine proteinase complexes and the biosynthesis of C1-inhibitor by Hep G2 and U 937 cells.

The biosynthesis of the serpin alpha 1-proteinase inhibitor is regulated by a feedback mechanism whereby complexes between alpha 1-proteinase inhibitor and serine proteinases bind to liver cells and monocytes, a reaction that activates alpha 1-proteinase-inhibitor gene transcription. Such a mechanism may form the basis for the development of new therapeutic strategies for serpin deficiency states with reduced levels of otherwise normally functioning serpins. This issue was addressed for C1-inhibitor, the missing serpin in hereditary angioedema. C1-inhibitor biosynthesis by Hep G2 hepatoma cells was assessed by enzyme-linked immunosorbant assay, by metabolic labeling followed by immunoprecipitation, and by Northern blotting. C1-inhibitor biosynthesis was stimulated by gamma-interferon (100 U/mL) but not by cell exposure to C1-inhibitor-kallikrein (1 mumol/L), C1-inhibitor-C1s (1 mumol/L), and C1-inhibitor-plasmin complexes (1 mumol/L) or to reactive site-cleaved C1-inhibitor (1 mumol/L). Moreover, radioiodinated C1s-C1-inhibitor complex did not bind to Hep G2 cells. C1-inhibitor-kallikrein complex was also without effect on C1-inhibitor mRNA in U 937 cells. Therefore, the proposed mechanism, by which serpin-enzyme complex or reactive site-cleaved serpin binding to a specific receptor provides a signal for the stimulation of the biosynthesis of that serpin, is not operative for the biosynthesis of C1-inhibitor by Hep G2 or U 937 cells.

Amino Acid Sequence↗
PubMed2003

Overexpression, purification, and characterization of human and bovine mitochondrial ATPase inhibitors: comparison of the properties of mammalian and yeast ATPase inhibitors.

Mitochondrial ATP synthase (F1F0-ATPase) is regulated by an intrinsic ATPase inhibitor protein. In this study, we overexpressed and purified human and bovine ATPase inhibitors and their properties were compared with those of a yeast inhibitor. The human and bovine inhibitors inhibited bovine ATPase in a similar way. The yeast inhibitor also inhibited bovine F1F0-ATPase, although the activity was about three times lower than the mammalian inhibitors. All three inhibitors inhibited yeast F1F0-ATPase in a similar way. The activities of all inhibitors decreased at higher pH, but the magnitude of the decrease was different for each combination of inhibitor and ATPase. The results obtained in this study show that the inhibitory mechanism of the inhibitors was basically shared in yeast and mammals, but that mammalian inhibitors require unique residues, which are lacking in the yeast inhibitor, for their maximum inhibitory activity. Common inhibitory sites of mammalian and yeast inhibitors are suggested.

Amino Acid Sequence↗
PubMed1990

Bovine plasma protein C inhibitor with structural and functional homologous properties to human plasma protein C inhibitor.

Bovine plasma protein C inhibitor was purified; it was then characterized in comparison with human protein C inhibitor. The specific inhibitory activity of the purified inhibitor for bovine activated protein C was 8,500 times that of the inhibitor in plasma. The purified inhibitor showed a single band with Mr 56,000 by SDS-PAGE at pH 7.0, and two bands at pH 8.8, a major one with Mr 56,000 and a minor one with Mr 105,000, under both unreduced and reduced conditions. The pI range of the inhibitor was between 4.4 and 6.1. The Mr of the inhibitor was reduced by treatment with neuraminidase, O-glycanase, and also with glycopeptidase-A, suggesting that the inhibitor has both Asn-linked and Ser/Thr-linked carbohydrate chains. Twenty-seven of the NH2-terminal 49 amino acid residues of the bovine inhibitor, which lacks the first 4 residues from the NH2-terminal amino acid sequence of human inhibitor, were identical to those of the human inhibitor. The bovine inhibitor inhibited bovine and human activated protein C, human thrombin, Factor Xa, Factor XIa, and plasma kallikrein with Ki = 1.0, 5.2, 2.6, 3.0, 1.3 X 10(-8) M, and 4.5 X 10(-9) M, respectively. The inhibitory rates for activated protein C and thrombin were accelerated significantly in the presence of heparin or negatively charged dextran sulfate. However, the acceleration by heparin or dextran sulfate for the inhibition of Factor Xa, Factor XIa, and plasma kallikrein was not significant. The bovine inhibitor did not inhibit human Factor XIIa or plasmin.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed2001

Sequencing of protease inhibitor therapy: insights from an analysis of HIV phenotypic resistance in patients failing protease inhibitors.

OBJECTIVE: To characterize the pattern of HIV-1 susceptibility to protease inhibitors in patients failing an initial protease inhibitor-containing regimen. DESIGN: A cross-sectional analysis of antiretroviral susceptibility. SETTING: HIV clinics in six metropolitan areas. PATIENTS: Eighty-eight HIV-infected adults with HIV RNA > 400 copies/ml after > or = 6 months of antiretroviral therapy, including the use of one protease inhibitor for > or = 3 months. MEASUREMENTS: The frequency and magnitude of decreased susceptibility, measured with a phenotypic assay using recombinant constructs, to five protease inhibitors. Decreased susceptibility was defined as > 2.5-fold increase in the 50% inhibitory concentration (IC50) compared with drug sensitive control virus. RESULTS: At study entry, patients were being treated with nelfinavir (63%), indinavir (25%), or another protease inhibitor (11%). HIV isolates from these patients were susceptible (fold change < 2.5) to all five protease inhibitors in 18% of patients and to none in 8%. Isolates from patients receiving nelfinavir were less likely to have reduced susceptibility to other protease inhibitors than isolates from patients treated with indinavir (P < 0.001) or one of the other three agents (P < 0.001), even after adjustment for the duration of prior protease inhibitor use. Reduced susceptibility to saquinavir and amprenavir was observed significantly less frequently than for the other protease inhibitors. CONCLUSION: The frequency of protease inhibitor cross-resistance and the magnitude of changes in susceptibility varied according to the initial protease inhibitor used in the failing treatment regimen. Significantly less protease inhibitor cross-resistance was demonstrated for isolates from patients failing a nelfinavir-containing regimen compared with those from patients receiving other protease inhibitors.

Adult↗