Protein-protein interactions in blood clotting. The use of polarization of fluorescence to measure the dissociation of plasma factor XIIIa.

1. Human plasma Factor XIII (the precursor of fibrin-glutamine-fibrin-lysine endo-gamma-glutamyltransferase) was randomly labelled by incubation with fluorescein isothiocyanate. The biochemical properties of the system were unaltered by the label. The polarization of the fluorescein fluorescence attached to the plasma protein was measured and the following conclusions were reached. 2. Factor XIII (a'2b2) does not dissociate in the protein-concentration range 10-500 microgram/ml either with or without added Ca2+. 3. Factor XIIIa (a'2b2) does not dissociate in the absence of Ca2+ in the protein-concentration range 10-500 microgram/ml. 4. Additions of Ca2+ to Factor XIIIa result in a decreased polarization of fluorescence as the tetramer dissociates. The decrease in polarization was the same amplitude at protein concentrations 10-500 microgram/ml and Ca2+ concentrations 2-66 mM and indicates that the overall process is essentially irreversible. The decrease in polarization consisted of fast and slow exponential phases. Both the rate of the fast phase and the proportion of the reaction it represented increased with Ca2+ concentration. 5. A comparison of the rate of dissociation measured by fluorescence polarization and the rate of appearance of enzyme activity in the presence of a protein substrate suggests that the Factor XIII is autoactivated by a soluble a-subunit-containing molecular forming a tight complex with the substrate.     

The mononuclear cell in human blood which mediates antibody-dependent cellular cytotoxicity to virus-infected target cells. II. Identification as a K cell.

Studies were carried out to determine whether the mononuclear cell in human blood which mediates antibody-dependent cellular cytotoxicity (ADCC) to herpes simplex virus (HSV)-infected target cells has surface Fc receptors which participate in the reaction. The F (ab')2 fragment of human IgG antibody was inactive both in ADCC and in complement-mediated cytolysis, but retained the capacity to neutralize infectious virus, to agglutinate erythrocytes coated with viral antigens, and to bind to the surface of virus-infected cells. Treatment of sensitized virus-infected target cells with staphylococcus protein A, which has affinity for the Fc epitope of IgG, strongly reduced their susceptibility to lysis by ADCC in a dose-dependent relationship. These findings indicate that the Fc portion of IgG antibody to the virus is necessary for cytotoxicity. Treatment of blood mononuclear cells with either heat-aggregated gamma-globulin or HSV immune complexes inhibited effector cell activity. The presence of "third party" cellular immune complexes also strongly inhibited ADCC. Adsorption of mononuclear cells to plastic surfaces coated with soluble third party immune complexes resulted in a significant reduction in effector cell activity. These findings demonstrate that the ADCC effector cell possesses surface Fc receptors which are utilized in the ADCC reaction. The presence of Fc receptors on the surface of the effector cell indicates that it is a K cell rather than a null cell.     

The electronic structure of Fe2+ in reaction centers from Rhodopseudomonas sphaeroides. I. Static magnetization measurements.

We have measured the static magnetization of unreduced and reduced reaction centers that vary in their quinone content. Measurements were performed in the temperature range 0.7 degrees K less than T less than 200 degrees K and magnetic fields of up to 10 kG. The electronic g-value, crystal field parameters D, E, and the exchange interaction, J, between the quinone spin and Fe2+ were determined using the spin Hamiltonian formalism. The effective moment mu eff/Fe2+ of both reduced and unreduced samples were determined to be 5.35 +/- 0.15 Bohr magnetons. This shows, in agreement with previous findings, that Fe2+ does not change its valence state when the reaction centers are reduced. Typical values of D congruent to +5 cm-1 and E/D congruent to 0.27 are consistent with Fe being in an octahedral environment with rhombic distortion. The values of D and E were approximately the same for reaction centers having one and two quinones. These findings imply that quinone is most likely not a ligand of Fe. The Fe2+ and the spin on the quinone in reduced reaction centers were found to be coupled with an exchange interaction 0 less than /J/ less than 1 cm-1. The validity of the spin Hamiltonian was checked by using an orbital Hamiltonian to calculate energy levels of the 25 states of the S = 2, L = 2 manifold and comparing the magnetization of the lowest five states with those obtained from the spin Hamiltonian. Using the orbital Hamiltonian, we calculated the position of the first excited quintet state to be 340 cm-1 above the ground state quintet. This is in good agreement with the temperature dependence of the quadrupole splitting as determined by Mossbauer spectroscopy.     

NADH binding to porcine mitochondrial malate dehydrogenase.

The binding of NADH to porcine mitochondrial malate dehydrogenase in phosphate buffer at pH 7.5 has been studied by equilibrium and kinetic methods. Hyperbolic binding was obtained by fluorimetric titration of enzyme with NADH, in the presence or absence of hydroxymalonate. Identical results were obtained for titrations of NADH with enzyme in the presence or absence of hydroxymalonate, measured either by fluorescence emission intensity or by the product of intensity and anisotropy. The equilibrium constant for NADH dissociation was 3.8 +/- 0.2 micrometers, over a 23-fold range of enzyme concentration, and the value in the presence of saturating hydroxymalonate was 0.33 +/- 0.02 micrometer over a 10-fold range of enzyme concentration. The rate constant for NADH binding to the enzyme in the presence of hydroxymalonate was 3.6 X 10(7) M-1 s-1, while the value for dissociation from the ternary complex was 30 +/- 1 s-1. No limiting binding rate was obtained at pseudo-first order rate constants as high as 200 s-1, and the rate curve for dissociation was a single exponential for at least 98% of the amplitude. In addition to demonstrating that the binding sites are independent and indistinguishable, the absence of effects of enzyme concentration on the KD value indicates that NADH binds with equal affinity to monomeric and dimeric enzyme forms.     

In vitro synthesis of a putative precursor to the mitochondrial enzyme, carbamyl phosphate synthetase.

A simple and rapid procedure is described for purification of carbamyl phosphate synthetase from the matrix fraction of rat liver mitochondria. Antibodies to the enzyme were raised in sheep and purified from antiserum by affinity chromatography on enzyme-bound Sepharose columns. When membrane-free polyribosomes, isolated from a cytosolic fraction of rat liver, were incubated in a messenger-dependent rabbit reticulocyte protein-synthesizing system in the presence of [35S]methionine, the purified antibody precipitated a product of translation representing 0.2% of total trichloroacetic acid-insoluble radioactivity. It demonstrated mobility characteristics in sodium dodecyl sulfate-polyacrylamide gels expected for a polypeptide of molecular mass approximately 5500 daltons larger than the mature mitochondrial form of the enzyme (160,000 daltons). Proteolysis of both the mature and presumptive in vitro precursor forms of the enzyme yielded respective sets of peptide fragments which gave similar patterns upon gel electrophoresis. Excess mitochondrial enzyme effectively competed with the in vitro product for interaction with anti-carbamyl phosphate synthetase antibody.     

Spectral evidence for tyrosine ionization linked to a conformational change in liver alcohol dehydrogenase ternary complexes.

Resonance energy transfer from Trp-314 to ionized Tyr-286 was proposed (Laws, W. R., and Shore, J. D. (1978) J. Biol. Chem. 253, 8593-8597) as the mechanism for the observed decrease in protein fluorescence of liver alcohol dehydrogenase seen with alkaline pH, or with the formation of a ternary complex with NAD+ and trifluoroethanol. In the present study, ultraviolet difference spectra confirm the presence of ionized tyrosine not only in these two cases but also in the ternary complex with NADH and isobutyramide. Our results indicate that ternary complex formation, with either oxidized or reduced coenzyme, causes a conformational change leading to partial ionization of tyrosine residues in regions of the enzyme far from the active site.     

Preparative size-exclusion chromatography of human serum apolipoproteins using an analytical liquid chromatograph

Apolipoproteins, extracted from human serum high-density lipoproteins, can be resolved and recovered with high yield from a preparative MicroPak TSK Type 3000SW size-exclusion column using Tris-buffered 6 m urea or 6 m guanidinium chloride mobile phases. Adequate resolution of some apolipoprotein pairs is only achieved at low flow velocities and low sample loads, necessitating repetitive injections of small amounts of material for preparative isolation. An analytical high-performance liquid chromatograph equipped with a simplified sample introduction scheme and low-pressure switching valves for fraction collection was used to isolate milligram quantities of HDL apolipoproteins.     

Granulosa cells as a source and target organ for tumor necrosis factor-alpha

Tumor necrosis factor (TNF-alpha), a 17 kDa cytokine, is a product of activated macrophages which was recently shown to be produced by rat and bovine granulosa cells. In the present work, human granulosa cells derived from preovulatory follicles were used. It was demonstrated that human granulosa cells produce TNF-alpha (5-10 units/300,000 cells per 15 h). This production was increased by addition of follicle-stimulating hormone or by a combination of human chorionic gonadotrophin and CSF to the culture media. TNF was also found in bovine follicular fluid and the concentration was higher in the periovulatory than mid-cycle follicles. TNF-alpha was found to increase prostaglandin F-2 alpha production by human granulosa cells (P less than 0.001). We conclude that granulosa cells are both a source and target organ for TNF-alpha.     

Immunocytochemistry of cytoskeletal proteins in adult Schistosoma mansoni

Antisera to vertebrate actin and actin-binding proteins were used to characterize the cytoskeleton of adult Schistosoma mansoni. Actin, alpha-actinin and tropomyosin immunoreactivities were detected in the cytoplasm of the apical tegument. Antiserum to alpha-actinin bound to the tegumental spines and this protein may be involved in cross-linking of spine actin filaments. Actin, alpha-actinin and tropomyosin antisera bound to the musculature. Strongest immunoreactivity was seen in the parenchyma. Antisera to actin, alpha-actinin, tropomyosin and spectrin bound to parenchyma cells including those of the tubercles, suggesting that these proteins are located in muscle cell bodies. The distribution of cytoskeletal proteins is discussed in relation to tegumental repair processes.