Enzymatic characterization of hepatitis C virus NS3/4A complexes expressed in mammalian cells by using the herpes simplex virus amplicon system.

The hepatitis C virus (HCV) NS3 protein possesses three enzymatic activities: an N-terminal serine protease activity, a C-terminal RNA-stimulated NTPase activity, and an RNA helicase activity. To characterize them, the full-length NS3(631)/4A and three C-terminal truncated proteases (NS3(201)/4A, NS3(181)/4A, and NS3(155)/4A were expressed in mammalian cells with HSV amplicon-defective viruses. Our results revealed that all of the NS3/4A proteins produced in mammalian cells (except NS3(155)/4A) are active in processing both cis and trans cleavage sites. Temperature optimization studies revealed that the protease is more active at temperatures ranging from 4 to 25 degrees C and is completely inactive at 42 degrees C. The RNA-stimulated ATPase activity was characterized with a partially purified NS3(631)/4A fraction and has a higher optimal temperature at 37 to 42 degrees C. The effects of detergents on both NS3 protease and RNA-stimulated ATPase were similar. Nonionic detergents such as Triton X-100, Nonidet P-40 and Tween 20 did not affect the activities, while anionic detergents such as sodium dodecyl sulfate and deoxycholic acid were inhibitory. Zwitterionic detergent such as 3-[(3-cholamidopropyl)- dimethyl-ammoniol-1-propanesulfonate (CHAPS) inhibited protease activity at a concentration of 0.5% (8 mM), which had no effect on ATPase activity. Finally, RNA-unwinding activity was demonstrated in the NS3(631)/4A fraction but not in the similarly purified NS3(181)/4A and NS3(201)/4A fractions. NS(363)/4A unwinds RNA duplexes with 3' but not 5' single-stranded overhangs, suggesting that the NS3 RNA helicase functions in a 3'-to-5' direction.

Development of a receptor peptide antagonist to human gamma-interferon and characterization of its ligand-bound conformation using transferred nuclear Overhauser effect spectroscopy.

Polyclonal anti-idiotypic antibody raised to a synthetic discontinuous peptide derived from the human gamma-interferon (huIFN-gamma) sequence recognizes soluble human gamma-interferon receptor (Seelig, G. F., Prosise, W. W., and Taremi, S. S. (1994) J. Biol. Chem. 269, 358-363). We sought to use this reagent to identify a ligand-binding domain within IFN-gamma-receptor. To do this, the neutralizing anti-idiotypic antibody was used to probe overlapping linear peptide octamers of the extracellular domain of the huIFN-gamma receptor. A 22-amino-acid residue receptor segment 120-141 identified by the antibody was synthesized. CD and NMR analysis indicates that peptide 120-141 has no apparent secondary structure in water or in water containing 50% trifluoroethanol. The synthetic receptor peptide inhibited huIFN-gamma induced expression of HLA/DR antigen on Colo 205 cells with an approximate IC50 of 35 microM. Immobilized peptide specifically bound recombinant huIFN-gamma but did not bind human granulocyte-macrophage colony-stimulating factor on a microtiter plate in a direct binding enzyme-linked immunosorbent assay. The binding results are supported by two-dimensional transferred nuclear Overhauser effect (TRNOE) NMR data obtained on the peptide in the presence of recombinant huIFN-gamma. Characterization of the conformation of the bound peptide by TRNOE suggests that this peptide assumes a distinct conformation. Intramolecular interactions within the bound peptide were detected at two non-contiguous regions and at a third region comprising a beta-turn formed by the sequence DIRK. We believe that this represents the structure of the receptor within the ligand-binding domain.

Characterization of recombinant human interleukin 4 receptor from CHO cells: role of N-linked oligosaccharides.

Interleukin 4 (IL-4) mediates its biological activities through interaction with its receptor on the cell surface. A recombinant extracellular domain of the alpha subunit of human interleukin 4 receptor was expressed in CHO cells and purified to homogeneity by a combination of ion exchange and immunoaffinity chromatography. Analysis of the purified protein by MALDI MS provided an average mass of 38,241 Da while microsequencing identified the site of the signal sequence processing to be Ser23-Gly24. The receptor was highly glycosylated, containing N-linked complex oligosaccharides with bi-, tri-, and tetraantennary structures. Five of the six potential glycosylation sites could be assigned to Asn residues 53, 98, 128, 134 and 176. N-deglycosylation increased aggregation and reduced solubility of the receptor but did not affect its IL-4 binding activity. These observations provide preliminary insights into the role of N-linked oligosaccharides in IL-4 receptor biosynthesis and function at the cell surface.

A role for the carboxyl terminus of human granulocyte-macrophage colony-stimulating factor in the binding of ligand to the alpha-subunit of the high affinity receptor.

A synthetic segment (110-127) of the carboxyl terminus of recombinant human granulocyte-macrophage colony-stimulating factor (rh-GM-CSF) was used to generate a rabbit polyclonal antibody (345-6), which recognized both peptide and full-length Escherichia coli-derived rh-GM-CSF in a direct enzyme-linked immunosorbent assay. Antibody 345-6 was shown to antagonize the binding of 125I-labeled rh-GM-CSF to its receptor on the KG-1 cell line and to inhibit human GM-CSF-dependent proliferation of the AML-193 cell line. The purified IgG fraction of neutralizing antibody 345-6 was used as immunogen to obtain sheep anti-serum 1418. Antibody 1418 recognized antibody 345-6 on direct enzyme-linked immunosorbent assay but did not recognize rh-GM-CSF or the peptide 110-127 to which antibody 345-6 was raised. Antiserum 1418, as well as a purified IgG fraction of this serum, inhibited both rh-GM-CSF-stimulated cell proliferation and 125I-labeled rh-GM-CSF receptor binding but not 125I-labeled recombinant human interleukin-4 receptor binding. The anti-idiotypic antibody response derived from the anti-(110-127) antibody strongly suggests that the carboxyl-terminal region of rh-GM-CSF may be directly involved in the receptor-ligand interaction of this protein. The high affinity receptor consists of two different components (GM-R alpha beta) a cytokine-specific alpha-subunit and a beta-subunit that is shared by human GM-CSF, interleukin-3, and interleukin-5. In an effort to localize the epitope of antibody 1418 to either GMR alpha or GMR beta, several cell lines containing high, low, or both high and low affinity receptors were examined. Each was specifically and completely inhibited by antibody 1418. Interleukin-3-dependent cell proliferation of the AML-193 cell line was found to be unaffected by the antibody 1418. Thus, the carboxyl-terminal region of rh-GM-CSF is likely to be involved in the interaction of the ligand with the alpha-subunit of the high affinity receptor.

Synthetic mimics of juxtaposed amino- and carboxyl-terminal peptide domains of human gamma interferon block ligand binding to human gamma interferon receptor.

The epitopes of two neutralizing antibodies (47N3-6 and 47N30A35) raised against rhuIFN-gamma each mapped both to amino-terminal regions (22-29 and 12-19, respectively) and to a carboxyl-terminal region 131-139, suggesting the juxtaposition of these two domains in the native protein. Three novel peptides were designed to mimic a conformation of rhuIFN-gamma that places the two regions in close proximity (discontinuous peptides 1 (15-21-GGG-132-138), 2 (15-29...111-118...130-138), and 3 (15-21-CGPGC-130-138)), by bridging the amino- and carboxyl-terminal regions of gamma interferon. Each discontinuous peptide inhibits biological or receptor binding activities with an IC50 of 15-50 microM and produces a neutralizing antibody when used as an immunogen. Neutralizing rabbit polyclonal antibody (P616) raised against discontinuous peptide 1 was used as immunogen to generate an anti-idiotypic response. This anti-idiotypic antibody inhibits receptor binding and recognizes soluble gamma interferon receptor on direct enzyme-linked immunosorbent assay. The anti-idiotypic response suggests that juxtaposed regions at the amino and carboxyl termini serve as the receptor-ligand binding site of human gamma interferon.

Selective proteolytic cleavage of recombinant human interleukin 4. Evidence for a critical role of the C-terminus.

Human interleukin 4 is a 129 amino acid lymphokine secreted by activated T cells that exerts pleiotropic biological effects on B and T lymphocytes and other hematopoietic cells. Structure-function relations were studied by employing selective proteolytic cleavage of purified recombinant human interleukin 4 (rhuIL-4). Limited proteolysis with endoprotease Glu-C from Staphylococcus aureus (V8) produced two digestion products that were observed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with apparent molecular weight values of 19K (I) and 15K (II), respectively. These species were isolated by reversed-phase HPLC. Amino acid sequencing indicated that species II was an 84 amino acid core fragment extending from Gln-20 to Glu-103 and containing a hydrolyzed peptide bond at Glu-26. On the basis of known disulfide bond assignments, it was concluded that species II was stabilized by two disulfide bonds (Cys-24/Cys-65 and Cys-46/Cys-99). Analysis of its secondary structure by circular dichroism revealed a high content of alpha helix. Species I was the full-length rhuIL-4 with selective cleavage at Glu-26 and Glu-103. Both species I and II were inactive in an in vitro assay based on proliferation of peripheral blood lymphocyte blasts and lacked the ability to bind to teh rhuIL-4 receptor on Daudi cells. In order to elucidate further the role of the residues removed by S. aureus V8 protease, rabbit antisera were raised to synthetic peptides corresponding to residues 1-26 at the N-terminus and 104-129 at the C-terminus. Only antisera directed to the C-terminal peptide inhibited binding of 125I-rhuIL-4 to Daudi cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of functionally distinct domains of human granulocyte-macrophage colony-stimulating factor using monoclonal antibodies.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a glycoprotein that is required for the survival, growth, and differentiation of hematopoietic progenitor cells. Although the primary structure of GM-CSF is known from cDNA cloning, the relationship between structure and function of GM-CSF is not fully understood. Fifteen different monoclonal antibodies (MoAbs) to human GM-CSF were generated to map immunologically distinct areas of the molecule. Each of the MoAbs was biotinylated and shown by enzyme-linked immunosorbent assay to bind to recombinant GM-CSF that had been affixed to a solid phase. Each of the 15 unconjugated MoAbs was then used to compete with each biotinylated MoAb for binding to GM-CSF. These cross-blocking studies identified eight distinct epitopes of native GM-CSF. Seven of these epitopes were also present in denatured GM-CSF by Western blotting, and four of the epitopes were at least partially conserved on GM-CSF that was reduced in beta-mercaptoethanol. MoAbs to four of eight epitopes neutralized both recombinant (glycosylated and nonglycosylated) and natural human GM-CSF in a GM colony-forming unit (CFU-GM) assay and blocked GM-CSF-induced activation of neutrophils. For most of the antibodies there was a good correlation between neutralizing activity and the capacity to block binding of 125I-GM-CSF to neutrophils or blasts. Non-neutralizing antibodies to one epitope partially blocked binding of 125I-GM-CSF to neutrophils. None of the MoAbs neutralized interleukin-3, G-CSF, or M-CSF. The locations of seven of the epitopes could be partially mapped with regard to the amino acid structure by determining reactivity to GM-CSF synthetic peptides or to human-mouse chimeric GM-CSFs. The neutralizing antibodies were found to map to amino acids 40-77, 78-94, or 110-127. Thus, these MoAbs are useful to identify functional domains of GM-CSF and in identifying regions that are likely to be involved in receptor interaction.

Evidence for a polypeptide segment at the carboxyl terminus of recombinant human gamma interferon involved in expression of biological activity.

A panel of 18 murine monoclonal antibodies was raised in BALB/c mice to the full-length, 146 amino acid residue recombinant human gamma interferon (rHuIFN gamma-A). Two monoclonal antibodies, designated 47N3-6 and 30N47-1, were purified from ascites tumors and further characterized. Antibody 47N3-6 neutralized both the antiviral and antiproliferative activities of rHuIFN gamma-A. Both Western blotting and enzyme-linked immunosorbent assays indicated that antibody 47N3-6 could bind to rHuIFN gamma-A as well as to a genetically engineered truncated form lacking the first three amino-terminal residues (rHuIFN gamma-D) but did not recognize a genetically engineered variant terminating at residue 131 (rHuIFN gamma-B). This antibody also demonstrated binding to a 15 amino acid residue oligopeptide, designated F-1, corresponding to residues 132-146 at the carboxyl terminus of rHuIFN gamma-A. Chemical cleavage of peptide F-1 with cyanogen bromide produced two fragments that were separated by reversed-phase high-pressure liquid chromatography. Dot-blot analysis indicated that antibody 47N3-6 could bind to a fragment, KRKRSQHse, derived from residues 132-137 of rHuIFN gamma-A, but could bind only weakly to the cyanogen bromide fragment corresponding to residues 138-146. It was consistent with these results that antibody 47N3-6 demonstrated binding to a form lacking the five carboxyl-terminal amino acids (rHuIFN gamma-D') but did not bind to a synthetic polypeptide corresponding to residues 138-146. Peptide F-1 exhibited neither antiviral nor antiproliferative activity, and it did not antagonize the antiviral activity of rHuIFN gamma-A.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunochemical mapping of alpha-2 interferon.

A panel of five monoclonal antibodies, designated U1-U5, produced by murine hybridoma clones has been raised to recombinant interferon (IFN) alpha-2, and one monoclonal antibody, designated U6, has been raised to a mixture of cyanogen bromide fragments of IFN alpha-2. These antibodies have been characterized with respect to (1) neutralization of IFN antiviral and antiproliferative activities, (2) binding to four cloned IFN alpha subtypes (alpha-1, alpha-2, alpha-4, and alpha-7) that are naturally occurring and to two novel products of recombinant DNA technology (delta-4 alpha-1 and delta-4 alpha-2/alpha-1 hybrid), and (3) binding to three cyanogen bromide fragments of IFN alpha-2. Four of the six monoclonal antibodies inhibited IFN antiviral activity. In conjunction with the previously reported monoclonal antibodies III/21 [Arnheiter, H., Thomas, R. M., Leist, T., Fountoulakis, M., & Gutte, B. (1981) Nature (London) 294, 278-280] and NK-2 [Secher, D. S., & Burke, D. C. (1980) Nature (London) 285, 446-450], eight unique epitopes have been described. Analysis of cross-reactivity patterns with IFN alpha fragments and subtypes indicated that monoclonal antibodies U1 and NK-2, which neutralized both antiviral and antiproliferative activities, and U2, which was nonneutralizing in these assays, were directed to distinct epitopes located in a polypeptide consisting of the amino-terminal 15 amino acid residues linked to residues 60-110 by a disulfide bond. The epitope recognized by U1 was determined to reside, at least in part, between residues 5 and 15. Competitive binding studies indicated that neutralizing monoclonal antibody U3, which did not bind to any of the cyanogen bromide fragments, was directed to an epitope partially overlapping that of NK-2. Epitopes to which neutralizing monoclonal antibodies U3, U4, and U5 and nonneutralizing antibody U6 were directed were readily distinguished by cross-reactivity with IFN alpha subtypes. The nonneutralizing monoclonal antibody U6 was determined to be directed to an epitope between residues 22 and 58. The fact that delta-4 alpha-1 and the delta-4 alpha-2/alpha-1 hybrid were active in an antiviral assay indicated a lack of direct functional significance for the first four amino-terminal amino acid residues and the Cys1-Cys98 disulfide bond. However, reduction with 2-mercaptoethanol of IFN alpha-2 altered the integrity of four of the eight epitopes. These data support a critical role for disulfide linkages in maintaining the native conformation of IFN alpha-2 and provide a potential basis for predicting the location of functionally important domains.

[Intrapericardial administration of drugs in the animal experiment]. / Intraperikardiale Zuleitung von Wirkstoffen im Tierexperiment.

Intrapericardially supplied agents for instance cardioactive drugs were transferred via lymph vessels in the myocardium likely. In case of dye application the myocardium and also the cardiac valves were coloured additionally. The effect on the heart should be more intensive and faster than after systemic application.

[Experimental studies on the problem of improving the myocardial blood circulation in an intraoperatively resuscitated heart]. / Experimentelle Untersuchungen zur Frage der Verbesserung der Herzmuskeldurchblutung eines intraoperativ reanimierten Herzens.

A pumping system for supporting an intraoperatively failured heart is presented. This system consists of a bell-shaped receptacle for the heart and a pump with a working volume of 49 ml and a working pressure of 220 Torr. The pumping power is transmitted to the heart by means of an external pneumatic pressure line. The efficacy of the pump in the high pressure system amounted to 20-30 per cent. An effective coronary perfusion in case of a cardiac failure is guaranteed by a venous underpressure pump of this pumping system. A favourable influence was also observed in case of the low pressure system.

Gamma-glutamylcysteine synthetase from erythrocytes.

gamma-Glutamylcysteine synthetase was isolated by means of a three-step method in highly active (specific activity, about 1400 units/mg) and apparently homogeneous form from rat erythrocytes. The enzyme has a molecular weight of about 100,000, and is composed of two subunits (Mr approximately 75,000 and 25,000). The erythrocyte enzyme exhibits physicochemical, catalytic, and immunological properties that closely resemble those displayed by rat kidney gamma-glutamylcysteine synthetase. The isolation procedure described here, which was also successfully applied to isolation of the enzyme from sheep erythrocytes, may be useful in exploring the properties of mutant forms of the enzyme.

Reversible dissociation of gamma-glutamylcysteine synthetase into two subunits.

gamma-Glutamylcysteine synthetase (rat kidney; Mr approximately 104,000) is composed of 2 nonidentical subunits. In the present work, a procedure was developed for the reversible dissociation of the enzyme into its subunits (Mr = 73,000 and 27,700) under nondenaturing conditions. Students in which gel electrophoresis was used, in conjunction with an enzyme activity stain and elution and re-electrophoresis of protein bands, showed that the heavy subunit contains all of the structural requirements for enzymatic activity and also for feedback inhibition of the enzyme activity by glutathione. The light subunit, which may be formed from a precursor protein, has a significantly lower content of Trp, Phe, Tyr, Val, and Ala residues than the heavy subunit, while its content of Lys, His, Met, and Asx residues is higher.

Gamma-glutamylcysteine synthetase. Interactions of an essential sulfhydryl group.

gamma-Glutamylcysteine synthetase (isolated from rat kidney) has one sulfhydryl group that reacts with 5,5'-dithiobis-(2-nitrobenzoate). This single exposed sulfhydryl group is not required for enzyme activity. The enzyme is potently inactivated by cystamine, which apparently interacts with a sulfhydryl group at the active site to form a mixed disulfide. 5,5'-Dithiobis-(2-nitrobenzoate) does not interact with the sulfhydryl group that reacts with cystamine. After the enzyme was 90% inactivated by reaction with cystamine, 3.4 mol of 5,5'-dithiobis-(2-nitrobenzoate) reacted per mol of enzyme, indicating that binding of cystamine exposes sulfhydryl groups which are apparently buried or unreactive in the native enzyme. L-Glutamate (but not D-glutamate or L-alpha-aminobutyrate) protected against inactivation by cystamine. In contrast, ATP enhanced the rate of inactivation by cystamine, and the apparent Km value for this effect is similar to that for ATP in the catalytic reaction. Studies on the structural features of cystamine that facilitate its interaction with the enzyme showed that selenocystamine, monodansylcystamine, and N-[2[2-aminoethyl)-dithio)ethyl]-4-azido-2-nitrobenzeneamine are also good inhibitors. Whereas S-(S-methyl)cysteamine-Sepharose does not interact with the enzyme (Seelig, G. F., and Meister, A. (1982) J. Biol. Chem. 257, 5092-5096), S-(S-methyl)cysteamine is a potent inhibitor; 1 mol of this compound completely inactivated 1 mol of enzyme. In the course of this work, a useful modification of the method for isolating this enzyme from kidney was developed.

[Experimental studies on the effect of the administration of oxygen into the pericardial cavity in acute myocardial ischemia]. / Experimentelle Untersuchungen zur Frage der Einflussnahme auf ein akut ischämisches Myokard mittels Applikation von Sauerstoff in den perikardialen Raum.

Artificial myocardial ischaemia caused by ligation of the left anterior descending coronary artery was treated in minipigs by the application of oxygen into the pericardial cavity. The results show that in this way acute myocardial ischaemia may be reduced. The application of oxygen into the pericardial cavity brought about, within 15 min, an improvement in the haemodynamic parameters. Several electrolytes, which were estimated from the blood of the sinus venosus, returned to normal.

Cystamine-Sepharose. A probe for the active site of gamma-glutamylcysteine synthetase.

gamma-Glutamylcysteine synthetase, previously known to be potently inhibited by cystamine, has been found to bind covalently to cystamine-Sepharose. ATP facilitates, whereas glutamate plus magnesium ions inhibit, binding of the enzyme to cystamine-Sepharose. A large fraction of the enzyme applied to columns of cystamine-Sepharose binds by forming a disulfide bond between cysteamine-Sepharose and a sulfhydryl group at or near the active site of the enzyme. The enzyme may be released by treatment with dithiothreitol. Some of the enzyme applied to such columns is inactivated and not bound covalently to the column. That the enzyme does not bind to columns of S-(S-methyl)cysteamine-Sepharose, whereas free S-(S-methyl)cysteamine is a potent inhibitor, indicates that a cysteamine-S disulfide moiety derived from the external cysteamine residue of cystamine-Sepharose is the critical group recognized by the enzyme. The observed partitioning of the enzyme on columns of cystamine-Sepharose between covalently column-bound enzyme and nonbound inactivated enzyme suggests that the reactive enzyme sulfhydryl group forms a disulfide linkage with the sulfur atom at the immobilized end of cystamine to link the enzyme to the column and to liberate free cysteamine, and also that the enzyme interacts with the external cysteamine moiety of the bound cystamine. The latter may occur if the free cysteamine released is spontaneously oxidized to free cystamine followed by its inhibition of the enzyme, or if there is a direct reaction between the enzyme-reactive sulfhydryl group and the sulfur atom of the external cysteamine moiety of cystamine-Sepharose.

Half-of-the-sites and all-of-the-sites reactivity in human plasma blood coagulation factor XIIIa.

The reactivities of human plasma factor XIIIa toward iodoacetic acid and toward alpha-bromo-4-hydroxy-3-nitroacetophenone have been studied under conditions where this dimeric enzyme reacts with the reagents in half-of-the-sites fashion and under conditions where it reacts with the reagents in all-of-the-sites fashion. Direct measurements of alkylation of active site -- SH groups in the apparently identical subunits of the enzyme as functions of remaining catalytic activity are in agreement with the observed reactivities. In addition to extending earlier evidence for half-of-the-sites reactions in factor XIIIa (Chung, S. I., Lewis, M. S., and Folk, J. E. (1974) J. Biol. Chem. 249, 940-950), the present findings suggest that the all-of-the-sites reactivity results from a positively cooperative interaction between enzyme subunits.

[The reaction of some enzymatic and coagulative physiologic parameters after electrically induced thrombosis in the arterial vascular system in dog and pygmy pig]. / Das Verhalten einiger enzymatischer und gerinnungsphysiologischer Parameter nach elektrisch induzierter Thrombose in der arteriellen Strombahn beim Hund und Zwergschwein.

Changes of enzyme and coagulation parameters were observed after electrically induced thrombosis in the arterial vascular system. These changes where only significant if the thrombosis was induced in the coronary arteries and a myocardial infarction was following. On the contrary anodic direct current (D. C. 10 mA about 15 minutes) in the femoral artery of dog or pygmy pig did not produce any significant changes of the examined parameters.