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PubMed2001

The affinity of chick cofilin for actin increases when actin is complexed with DNase I: formation of a cofilin-actin-DNase I ternary complex.

Cofilin, an actin-binding protein, regulates the rate, nature and extent of assembly of the actin cytoskeleton. Native Phast gels show that the addition of cofilin to an actin-DNase I complex (74 kDa) results in the formation of a ternary complex of 94 kDa indicating an equimolar stoichiometry in the ternary complex. Furthermore, native gels show that the addition of cofilin to a solution containing free actin and actin-DNase I and run at pH 8.3 results in cofilin complexing preferentially to the actin-DNase I complex. Conversely, the addition of DNase I to a solution containing an actin-cofilin complex and free actin results in the preferential binding of DNase I to the actin-cofilin complex. These results show that the affinity of cofilin for actin can be increased when actin forms binary complexes. When native gels were run at pH 6.8 the affinity of cofilin for monomeric actin was greater than for the actin-DNase I complex indicating that the cofilin-actin interaction can be regulated by changes in pH. The addition of cofilin to actin resulted in the polymerisation of actin at pH 6.8 whereas at alkaline pH a stable cofilin-actin binary complex could be formed. The biological implications are discussed.

Actin Depolymerizing Factors↗
PubMedDate not supplied

Chlorophyll-protein complexes of brown algae: P700 reaction centre and light-harvesting complexes.

Thylakoid membranes from several brown algae have been fragmented with the non-ionic detergent, Triton X-100. Three intrinsic chlorophyll-protein complexes with different pigment compositions have been isolated by sucrose density gradient centrifugation. Brown algae contain the photosystem 1 reaction-centre complex, a P700-chlorophyll a-protein which has similar spectroscopic and chemical properties to those of higher plants. This complex represents about 10--20% of the total chlorophyll in all species; the Acrocarpia paniculata complex has a chlorophyll/P700 ratio of 38. Two main light-harvesting complexes have also been isolated, which have properties unique to brown algae. The heavier of these, an orange fraction, is a fucoxanthin-chlorophyll a/c-protein; this complex contains most of the fucoxanthin and has only chlorophyll c2. The other, a green fraction, is a chlorophyll a/c-protein enriched in violaxanthin. Neither of these complexes possesses detectable photosystem 1 or photosystem 2 activities. Both of these complexes efficiently transfer light energy to chlorophyll a, indicating that the molecular arrangement of their pigments is similar to that in vivo. Differential extraction of thylakoid membranes indicates that the P700-chlorophyll a-protein is the complex most firmly embedded in the membrane, but the fucoxanthin-chlorophyll a/c-protein is the least firmly bound. We suggest that the fucoxanthin complex is the most variable component of the photosynthetic unit of brown algal chloroplasts.

Cell Membrane↗
PubMed2002

Evaluating hydrogen bond interactions in enzymes containing Mn(III)-histidine complexation using manganese-imidazole complexes.

It is often difficult to control hydrogen bond interactions in small molecule compounds that model metalloenzyme active sites. The imidazole-containing ligands 4,5-dicarboxyimidazole (H(3)DCBI) and 4,5-dicarboxy- N-methylimidazole (H(2)MeDCBI) allow examination of the effects of internal hydrogen bonding between carboxylate and imidazole nitrogen atoms. A new series of mononuclear manganese imidazole complexes have been prepared using these ligands: Mn(III)(salpn)(H(2)DCBI)(DMF) (1), Mn(III)(salpn)(HMeDCBI) (2), Mn(III)(dtsalpn)(HMeDCBI) (3), [Mn(IV)(dtsalpn)(HMeDCBI)]PF(6) (4), Mn(III)(salpn)(H(2)DCBI) (5), Mn(III)(dtsalpn)(H(2)DCBI) (6), and Mn(IV)(dtsalpn)(H(2)DCBI)PF(6) (8). Complexes 1, 2, 3, 5, and 6 have been prepared by direct reaction of salpn [salpn=(salicylideneaminato)-1,3-diaminopropane)] or dtsalpn [dtsalpn=(3,5-di- t-butylsalicylideneaminato)-1,3-diaminopropane)] and H(3)DCBI and H(2)MeDCBI with Mn(III) acetate, while complexes 4 and 8 were made by bulk electrolysis of complex 3 or 6 in dichloromethane. Complexes 1, 2, and 6 were characterized by X-ray diffraction. The impact of hydrogen bonding interactions of the complexes has been demonstrated by X-ray diffraction, cyclic voltammetry, and EPR spectroscopy. In all complexes the central metal ion is present in a six-coordinate geometry. Magnetic susceptibility measurements confirm the spin and oxidation states of the complexes. The cyclic voltammograms of 3 and 6 in dichloromethane reveal single, reversible redox waves with E(1/2)=600 mV and 690 mV, respectively. The X-band EPR spectrum of 4 shows a broad signal around g=4.4, and the corresponding complex 8 possesses a broad signal at slightly lower field ( g=5.5) than 4. These studies demonstrate that even small changes in the effective charge of the imidazole ligand can have a profound impact on the structure, spectroscopy, and magnetism of manganese(IV) complexes. We use these observations to present a model that may explain the origin of the g=4.1 signal in the S(2) state of photosystem II.

Electrochemistry↗
PubMed1989

Crystal structure of the p-hydroxybenzoate hydroxylase-substrate complex refined at 1.9 A resolution. Analysis of the enzyme-substrate and enzyme-product complexes.

Using synchrotron radiation, the X-ray diffraction intensities of crystals of p-hydroxy-benzoate hydroxylase, complexed with the substrate p-hydroxybenzoate, were measured to a resolution of 1.9 A. Restrained least-squares refinement alternated with rebuilding in electron density maps yielded an atom model of the enzyme-substrate complex with a crystallographic R-factor of 15.6% for 31,148 reflections between 6.0 and 1.9 A. A total of 330 solvent molecules was located. In the final model, only three residues have deviating phi-psi angle combinations. One of them, the active site residue Arg44, has a well-defined electron density and may be strained to adopt this conformation for efficient catalysis. The mode of binding of FAD is distinctly different for the different components of the coenzyme. The adenine ring is engaged in three water-mediated hydrogen bonds with the protein, while making only one direct hydrogen bond with the enzyme. The pyrophosphate moiety makes five water-mediated versus three direct hydrogen bonds. The ribityl and ribose moieties make only direct hydrogen bonds, in all cases, except one, with side-chain atoms. The isoalloxazine ring also makes only direct hydrogen bonds, but virtually only with main-chain atoms. The conformation of FAD in p-hydroxybenzoate hydroxylase is strikingly similar to that in glutathione reductase, while the riboflavin-binding parts of these two enzymes have no structural similarity at all. The refined 1.9 A structure of the p-hydroxybenzoate hydroxylase-substrate complex was the basis of further refinement of the 2.3 A structure of the enzyme-product complex. The result was a final R-factor of 16.7% for 14,339 reflections between 6.0 and 2.3 A and an improved geometry. Comparison between the complexes indicated only small differences in the active site region, where the product molecule is rotated by 14 degrees compared with the substrate in the enzyme-substrate complex. During the refinements of the enzyme-substrate and enzyme-product complexes, the flavin ring was allowed to bend or twist by imposing planarity restraints on the benzene and pyrimidine ring, but not on the flavin ring as a whole. The observed angle between the benzene ring and the pyrimidine ring was 10 degrees for the enzyme-substrate complex and 19 degrees for the enzyme-product complex. Because of the high temperature factors of the flavin ring in the enzyme-product complex, the latter value should be treated with caution. Six out of eight peptide residues near the flavin ring are oriented with their nitrogen atom pointing towards the ring.(ABSTRACT TRUNCATED AT 400 WORDS)

4-Hydroxybenzoate-3-Monooxygenase↗
PubMed1990

31P NMR studies of enzyme-bound substrate complexes of yeast 3-phosphoglycerate kinase: III. Two ADP binding sites and their Mg(II) affinity; effects of vanadate and arsenate on enzymic complexes with ADP and 3-P-glycerate.

31P nuclear magnetic resonance (NMR) measurements (at 121.5 MHz and 5 degrees C) were made on complexes of 3-phosphoglycerate kinase with ADP and 3-P-glycerate. Addition of Mg(II) to E.ADP shifts the alpha-P signal downfield by 3.8 ppm such that the alpha-P signal superimposes that for beta-P(E.MgADP). Such a shift is atypical among the Mg(II)-nucleotide complexes with other ATP-utilizing enzymes. This shift allowed the determination that enzyme bound ADP is saturated with Mg(II) for [Mg(II)]/[ADP] = 3.0--similar to that reported for ATP complexes with this enzyme (B.D. Ray and B.D. Nageswara Rao, Biochemistry 27, 5574 (1988]. This parallel behavior suggests that ADP binds at two sites on the enzyme as does ATP with disparate Mg(II) affinities. 31P relaxation times in E.MnADP.vanadate.3-P-glycerate and E.CoADP.vanadate.3-P-glycerate complexes indicate that these are long-lived, tightly bound complexes. 31P chemical shift measurements on diamagnetic complexes (with Mg(II] revealed three signals in the 2-5 ppm region (attributable to 3-P-glycerate) only upon addition of all the components necessary to form the E.MgADP.vanadate.3-P-glycerate complex. Subsequent sequestration of Mg(II) from the complex with excess EDTA reversed the Mg(II) induced effects on the ADP signals but did not cause coalescence of the three signals seen in the 2-5 ppm region. Addition of excess sulfate to dissociate these complexes from the enzyme resulted in a single resonance of 3-P-glycerate. The use of arsenate in place of vanadate yielded very similar results. These results suggest that, in the presence of MgADP, vanadate or arsenate, and 3-P-glycerate, the enzyme catalyzed the formation of multiple structurally distinguishable complexes that are stable on the enzyme and labile off the enzyme.

Adenosine Diphosphate↗
PubMed1992

Contribution of a measure of disease complexity (COMPLEX) to prediction of outcome and charges among hospitalized patients.

Attention has been focused on the need to adjust hospital reimbursement and outcomes of hospital care for level of illness. Extant measures of disease severity, however, fail to consider the contribution of disease complexity. We developed an easily retrievable measure of disease complexity (COMPLEX) by modifying an existing severity system, computerized Disease Staging. The contribution of COMPLEX (the number of body systems affected with a Disease Staging score of 2 or more) to the prediction of outcome was assessed in two studies: (1) a population-based analysis of readmission and mortality after hospitalization and (2) an analysis of hospital charges among patients who were in an intensive-care unit. The amount of variation in mortality explained by factors included in the Health Care Financing Administration model was significantly improved when COMPLEX was added to the model (adjusted odds ratio per body system, 1.83; 95% confidence interval, 1.61 to 2.08). A significant association was also observed between COMPLEX score and hospital readmission after adjustment for age, sex, case-mix, and disease severity (adjusted odds ratio, 1.31; 95% confidence interval, 1.20 to 1.44). When COMPLEX was added to case-mix and disease severity in a model for predicting hospital charges, the percentage of variation in hospital charges explained by the model increased from 25% to 38%. These findings demonstrate the important contribution of disease complexity to the analysis of outcome of medical care and utilization of resources. Outcome or reimbursement models that do not incorporate disease complexity may negatively affect institutions with a high proportion of patients who have complex conditions.

Aged↗
PubMed1997

The interaction of plasmid DNA with polyamidoamine dendrimers: mechanism of complex formation and analysis of alterations induced in nuclease sensitivity and transcriptional activity of the complexed DNA.

The application of synthetic vectors for gene transfer has potential advantages over virus-based systems. However, little is known about the mechanisms involved in binding of synthetic materials to DNA and the nature of the DNA complexes that result from this interaction. Polyamidoamine (PAMAM) dendrimers are unique polymers with defined spherical structure. Dendrimers bind DNA to form complexes that efficiently transfect cells in vitro. We examined the formation of DNA/dendrimer complexes and found it based entirely on charge interaction. Electronmicroscopic examination of the complexes indicated that the majority of the plasmid DNA is contracted into isolated toroids, but also revealed larger, irregular aggregates of polymer and DNA. The binding of plasmid DNA to dendrimer appears to alter the secondary and tertiary structure, but does not fragment the DNA or alter its primary structure. Complexed DNA is protected against degradation by either specific nucleases or cellular extracts containing nuclease activity. While the initiation of transcription in vitro from promoters (for either T7 polymerase or eukaryotic RNA polymerase II) in dendrimer-complexed DNA is inhibited, elongation of the RNA transcript and translation do not appear to be affected. These resemble alterations of the DNA function when complexed with naturally-occurring polycations like non-acetylated histones. However, DNA complexed to dendrimer appears to maintain transcriptional activity while histone complexes at similar charge ratios do not. These results elucidate some aspects of the interaction between PAMAM dendritic polymers and DNA, and could lead to improvements in the design of polymers or formation of DNA complexes that will increase the efficiency of non-viral gene transfer.

DNA↗
PubMed1986

Metal anthracycline complexes as a new class of anthracycline derivatives. Pd(II)-adriamycin and Pd(II)-daunorubicin complexes: physicochemical characteristics and antitumor activity.

Pd(II) complexes of two anthracyclines, adriamycin and daunorubicin, have been studied. Using potentiometric absorption, fluorescence, and circular dichroism measurements, we have shown that adriamycin can form two complexes with Pd(II). The first complex (I) involves two molecules of drug per Pd(II) ion; one of the molecules is chelated to Pd(II) through the carbonyl oxygen on C12 and the phenolate oxygen on C11, and the other one is bound to Pd(II) through the nitrogen of the amino sugar. This complexation induces a stacking of the two molecules of drug. In the second complex (II), two Pd(II) ions are bound to two molecules of drug (A1 and A2). One Pd(II) is bound to the oxygen on the carbons C11 and C12 of molecule A1 and the amino sugar of molecule A2 whereas the second Pd(II) ion is bound to the oxygen on C11 and C12 of molecule A2 and the amino sugar of molecule A1. The same complexes are formed between Pd(II) and daunorubicin. The stability constant for complex II is beta = (1.3 +/- 0.5) X 10(22). Interaction with DNA has been studied, showing that almost no modification of the complex occurred. This complex displays antitumor activity against P-388 leukemia that compares with that of the free drug. Complex II, unlike adriamycin, does not catalyze the flow of electrons from NADH to molecular oxygen through NADH dehydrogenase.

Animals↗
PubMed1996

Infrared spectroscopy of the cyanide complex of iron (II) myoglobin and comparison with complexes of microperoxidase and hemoglobin.

The cyanide complex of FeIIMb prepared and maintained at temperatures below 0 degrees C is sufficiently stable to permit spectroscopic characterization and allow comparison with free HCN and other ferric and ferrous CN complexes. The visible absorption spectrum of FeIIMb-CN has a split alpha band maxima at 571 and 563 nm, suggesting distortion in the x-y plane of the porphyrin. FeIIMb-CN, like the CO complex, was found to be optically active by circular dichroism. The C-N stretching frequencies for the CN-ferrous complexes are very sensitive to parameters within the heme pocket. The values are as follows: FEIIMb at pH 8, 2057 cm-1 with a shoulder appearing at 2078 cm-1 at pH 5.6; FeIIMp, 2034 cm-1. In contrast, the frequencies for C-N stretch differ little among ferric heme complexes, ranging from 2123 to 2125 cm-1 for myoglobin, hemoglobin, and microperoxidase. These values compare with free HCN (2094 cm-1) or CN- (2080 cm-1). Quantum chemical modeling of the neutral iron-porphyrin complex with imidazole and cyanide and of its anion was used to explain the effects of the cyanide coordination and of iron reduction on the C-N stretching frequencies. The lower nu C-N for FeIIMb-CN relative to the ferric complex is attributed to the appearance of additional electron density on all the anti-bonding CN orbitals. The extra electron density was also used to explain that the band width of C-N stretching mode was greater in the ferrous complexes than in the ferric complex. Finally, the calculation shows that sigma donation weakens the Fe-C bond, in qualitative agreement with the spontaneous dissociation of CN- from FeIIMb at -5 degrees C. The sensitivity of CN complexes of ferrous heme proteins to the heme pocket environment and the ability to correlate spectroscopic parameters with calculated electron density suggest that infrared spectroscopy of the CN ligand is an appropriate tool to study ferrous heme proteins.

Animals↗
PubMed2003

Investigation of the extraction complexes of light lanthanides(III) with bis(2,4,4-trimethylpentyl)dithiophosphinic acid by EXAFS, IR, and MS in comparison with the americium(III) complex.

The structure of the extraction complexes of light lanthanides (La(III), Nd(III), Eu(III)) with bis(2,4,4-trimethylpentyl)dithiophosphinic acid (HBTMPDTP) have been characterized with extended X-ray absorption fine structure spectroscopy (EXAFS), IR, and MS; the IR spectrum of the extraction complex of (241)Am with HBTMPDTP has been studied too. The molecular formula of the extraction complexes of lanthanides is deduced to be HML(4).H(2)O (M = La, Nd, Eu; L = anion of HBTMPDTP). The coordination number of Ln(III) in the complexes is 8; the coordinated donor atoms are 7 sulfur atoms from 4 HBTMDTP molecules and 1 O atom from a hydrated water molecule. With the increase of the atomic number of Ln, the coordination bond lengths of Ln-O and Ln-S decrease in the complexes. For La(III), Nd(III), and Eu(III), the coordination bond lengths of Ln-O are 2.70, 2.56, and 2.50, respectively, the coordination bond lengths of Ln-S are 3.01, 2.91, and 2.84, respectively, and the average distances between Ln and P atoms are 3.60, 3.53, and 3.46, respectively. The structure of the extraction complexes of Ln(III) with HBTMDTP is different from that of the Am(III) extraction complex. The results of IR show that there is no water coordinated with Am in the extraction complex. The molecular formula of the complex of Am(III) is deduced as being HAmL(4), and there are 8 S atoms from 4 HBTMPDTP molecules coordinated with Am. Composition and structure differences of the extraction complexes may be one of the most most important factors affecting the excellent selectivity of HBTMPDTP for Am(III) over Ln(III).

Journal Article↗
PubMed2001

Investigation of the mechanism of alkane reductive elimination and skeletal isomerization in Tp'Rh(CNneopentyl)(R)H complexes: the role of alkane complexes.

Experiments are described that provide indirect evidence for the involvement of alkane sigma-complexes in oxidative addition/reductive elimination reactions of Tp'Rh(L)(R)H complexes (Tp' = tris-3,5-dimethylpyrazolylborate, L = CNCH(2)CMe(3)). Reductive elimination rates in benzene-d(6) were determined for loss of alkane from Tp'Rh(L)(R)H, where R = methyl, ethyl, propyl, butyl, pentyl, and hexyl, to generate RH and Tp'Rh(L)(C(6)D(5))D. The isopropyl hydride complex Tp'Rh(L)(CHMe(2))H was found to rearrange to the n-propyl hydride complex Tp'Rh(L)(CH(2)CH(2)CH(3))H in an intramolecular reaction. The sec-butyl complex behaves similarly. These same reactions were studied by preparing the corresponding metal deuteride complexes, Tp'Rh(L)(R)D, and the scrambling of the deuterium label into the alpha- and omega-positions of the alkyl group monitored by (2)H NMR spectroscopy. Inverse isotope effects observed in reductive elimination are shown to be the result of an inverse equilibrium isotope effect between the alkyl hydride(deuteride) complex and the sigma-alkane complex. A kinetic model has been proposed using alkane complexes as intermediates and the selectivities available to these alkane complexes have been determined by kinetic modeling of the deuterium scrambling reactions.

Journal Article↗
PubMed1972

The transformation of the cytoplasmic oestradiol-receptor complex into the nuclear complex in a uterine cell-free system.

Experiments performed with a cell-free system in tris-EDTA buffer, pH 7.4, indicate that the high-speed supernatant fraction of the rat uterus contains all the factors necessary to transform the 8S cytoplasmic oestradiol-receptor complex to the nuclear complex. The transformation is temperature-dependent. This nuclear complex was extracted in the form of a 5S particle with 0.4m-KCl from sediments of either uterine or heart nuclei that had been incubated together with the cytoplasmic soluble fraction of the uterus at 2 degrees C for 30min. This complex can also be obtained similarly from the soluble fraction of the uterus, incubated in the absence of nuclei. Previous warming of the soluble fraction to 37 degrees C for 7min was necessary for the successful extraction of the nuclear particle under these conditions of incubation. After an incubation of the transformed complex with the nuclear sediment at 37 degrees C for 7min, the 5S complex was extractable from the uterine nuclear sediment but not from the heart nuclear sediment, which may indicate the tissue specificity of the nuclear acceptor sites for the transformed complex. The extracted uterine nuclear complex sediments in the 5S region, but whether it is the native complex or a subunit or other part of the native complex resulting from the extraction with salt is unknown.

Animals↗
PubMed1996

Role of the proposed serpin-enzyme complex receptor recognition site in binding and internalization of thrombin-heparin cofactor II complexes by hepatocytes.

Several serpin-enzyme complexes bind to a receptor on hepatocytes that mediates their endocytosis and lysosomal degradation. Joslin et al. (Joslin, G., Fallon, R. J., Bullock, J., Adams, S. P., and Perlmutter, D. H.(1991) J. Biol. Chem. 266, 11282-11288) proposed that a sequence near the C-terminal end of the serpin (e.g. FVFLM in alpha1-antitrypsin) binds to the serpin-enzyme complex receptor (SEC receptor). In experiments with synthetic peptides, they found that substitution of alanine at the fourth or fifth position in this sequence reduced the affinity of peptide binding to Hep G2 cells. To test the hypothesis that the corresponding sequence in heparin cofactor II (HCII), FLFLI (residues 456-460), mediates binding and uptake of the thrombin-HCII complex by Hep G2 cells, we constructed five recombinant HCII variants, F456A, L457A, F458A, L459A, and I460A. At 4 degrees C, the 125I-thrombin-HCII(native) complex bound reversibly to 0.6-2.6 x 10(5) sites per Hep G2 cell with a Kd of 19-32 nM. Binding was inhibited by excess unlabeled thrombin-HCII(native), thrombin-antithrombin, or elastase-alpha1-antitrypsin, but not by free HCII or thrombin, which is consistent with the reported specificity of the SEC receptor. However, complexes of thrombin with each of the HCII variants inhibited binding as effectively as the complex with native HCII. Competitive binding experiments with various concentrations of unlabeled thrombin-HCII(native) or thrombin-HCII(I460A) indicated that these complexes bind to Hep G2 cells with equal affinity. At 37 degrees C, complexes of 125I-thrombin with each of the five HCII variants were internalized and degraded at the same rate as the complex with native HCII. Our data suggest that the pentapeptide FLFLI in HCII is not involved in binding, internalization, and degradation of thrombin-HCII complexes by Hep G2 cells.

Amino Acid Sequence↗
PubMed1991

A complex between prostate-specific antigen and alpha 1-antichymotrypsin is the major form of prostate-specific antigen in serum of patients with prostatic cancer: assay of the complex improves clinical sensitivity for cancer.

We have studied the forms of prostate-specific antigen (PSA) in serum of patients with prostatic cancer and benign prostatic hyperplasia. Fractionation of serum by gel filtration and assay of the fractions for PSA showed that a considerable part of the PSA immunoreactivity in serum consisted of complexes that were larger than PSA. The complexes were assayed by time-resolved immunofluorometric assays based on an antibody against PSA on the solid phase and europium-labeled antibodies against various protease inhibitors as indicator antibodies. In addition to its monomeric form, PSA was found to occur in complex with alpha 1-antichymotrypsin. The proportion of the alpha 1-antichymotrypsin complex was a major form of PSA and it increased with increasing PSA concentrations, being over 85% at PSA levels exceeding 1000 micrograms/liter. A complex with alpha 1-protease inhibitor was also observed in serum of patients with prostatic cancer and very high levels of PSA. Complexes with alpha 2-macroglobulin and inter-alpha-trypsin inhibitor were detected, but their concentrations were low and similar in sera of cancer patients, normal men, and normal women, suggesting that they were not prostate derived. Commercial immunoradiometric assays for PSA were found to measure free PSA and its complexes with alpha 1-antichymotrypsin but not the complexes with alpha 2-macroglobulin and inter-alpha-trypsin inhibitor. The proportion of the PSA-alpha 1-antichymotrypsin complex was higher in patients with prostatic cancer than in those with benign hyperplasia. Therefore, assay of the complex had a higher sensitivity for cancer than assay of total PSA immunoreactivity.

Antigens, Neoplasm↗
PubMed1990

Endocytosis and degradation of alpha 1-antitrypsin-protease complexes is mediated by the serpin-enzyme complex (SEC) receptor.

Alpha 1-Antitrypsin (alpha 1-AT) is similar to other members of the serine protease inhibitor (serpin) supergene family in that it undergoes structural rearrangement during the formation of a covalently stabilized inhibitory complex with its cognate enzyme, neutrophil elastase. We have recently demonstrated an abundant, high-affinity cell surface receptor on human hepatoma cells and human mononuclear phagocytes which recognizes a conformation-specific domain of the alpha 1-AT-elastase complex as well as of other serpin-enzyme complexes (Perlmutter, D. H., Glover, G. I., Rivetna, M., Schasteen, C. S., and Fallon, R. J. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 3753-3757). Binding to this serpin-enzyme complex (SEC) receptor activates a signal transduction pathway for increased expression of the alpha 1-AT gene and may be responsible for clearance of serpin-enzyme complexes. In this study, we show that there is time-dependent and saturable internalization of alpha 1-AT-elastase and alpha 1-AT-trypsin complexes in human hepatoma HepG2 cells. Internalization is mediated by the SEC receptor as defined by inhibition by synthetic peptides corresponding to residues 359-374 of alpha 1-AT. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of intracellular radioactivity demonstrated that intact 75- and 66-kDa alpha 1-AT-trypsin complexes were internalized. Kinetic analysis of internalization at 37 degrees C showed that a single cohort of 125I-alpha 1-AT-trypsin complexes, prebound to cells at 4 degrees C, disappeared from the cell surface and accumulated intracellularly within 5-15 min at 37 degrees C. The intracellular concentration of radiolabeled complexes then decreased rapidly coincident with appearance of acid-soluble degradation products in the extracellular culture fluid. Intracellular degradation was inhibited by internalization at 18 degrees C or by internalization at 37 degrees C in the presence of weak bases ammonium chloride, primaquine, and chloroquine, indicating that degradation is lysosomal. These results indicate that in addition to its role in signal transduction the SEC receptor participates in internalization and delivery of alpha 1-AT-protease complexes to lysosome for degradation.

Carcinoma, Squamous Cell↗
PubMed1993

Complex I and complex III of mitochondria have common inhibitors acting as ubiquinone antagonists.

Mitochondrial complex I and complex III have common inhibitors with ubiquinone-like structure. The tridecyl analog of stigmatellin, which inhibits mitochondrial complex III at nanomolar concentrations, also inhibits the NADH:ubiquinone reductase activity of complex I at micromolar concentrations. The inhibitor titer depends upon the concentration of the mitochondrial particles and extrapolates to 0.2 microM at zero particle concentration. The stigmatellin analog is more powerful than its parent compound and is noncompetitive with exogenous ubiquinones, rotenone and piericidin. Myxothiazol, which is another potent inhibitor of complex III, is also found to inhibit the activity of complex I with a titer comparable to that of the tridecyl analog of stigmatellin. Additionally, piericidin, which is the most powerful inhibitor of complex I, inhibits the ubiquinol:cytochrome c reductase activity of complex III at micromolar concentrations in mitochondrial particles and at submicromolar concentrations in the isolated enzyme complex.

Animals↗
PubMed1988

Electron-transfer complexes of Ascaris suum muscle mitochondria. III. Composition and fumarate reductase activity of complex II.

Complex II of the anaerobic respiratory chain in Ascaris muscle mitochondria showed a high fumarate reductase activity when reduced methyl viologen was used as the electron donor. The maximum activity was 49 mumol/min per mg protein, which is much higher than that of the mammalian counterpart. The mitochondria of Ascaris-fertilized eggs, which require oxygen for its development, also showed fumarate reductase activity with a specific activity intermediate between those of adult Ascaris and mammals. Antibody against the Ascaris flavoprotein subunit reacted with the mammalian counterparts, whereas those against the Ascaris iron-sulfur protein subunit did not crossreact, although the amino acid compositions of the subunits in Ascaris and bovine heart were quite similar. Cytochrome b-558 of Ascaris complex II was separated from flavoprotein and iron-sulphur protein subunits by high performance liquid chromatography with a gel permeation system in the presence of Sarkosyl. Isolated cytochrome b-558 is composed of two hydrophobic polypeptides with molecular masses of 17.2 and 12.5 kDa determined by gradient gel, which correspond to the two small subunits of complex II. Amino acid compositions of these small subunits showed little similarity with those of cytochrome b-560 of bovine heart complex II. NADH-fumarate reductase, which is the final enzyme complex in the anaerobic respiratory chain in Ascaris, was reconstituted with bovine heart complex I, Ascaris complex II and phospholipids. The maximum activity was 430 nmol/min per mg protein of complex II. Rhodoquinone was essential for this reconstitution, whereas ubiquinone showed no effect. The results clearly indicate the unique role of Ascaris complex II as fumarate reductase and the indispensability of rhodoquinone as the low-potential electron carrier in the NADH-fumarate reductase system.

Amino Acid Sequence↗
PubMed1993

Unexpected similarities of the B800-850 light-harvesting complex from Rhodospirillum molischianum to the B870 light-harvesting complexes from other purple photosynthetic bacteria.

The B800-850 light-harvesting complex (also called LH2) was isolated from photosynthetic membranes of Rhodospirillum molischianum DSM 119 using molecular sieve and ion-exchange chromatography. Its two bacteriochlorophyll a-binding polypeptides (alpha-subunit and beta-subunit) were purified with a reverse-phase HPLC system. The complete amino acid sequences of both subunits have been determined. The alpha- and beta-subunits consist of 56 and 45 amino acids, respectively, corresponding to molecular weights of 5939 and 5133. In contrast to the B800-850 complexes from other photosynthetic bacteria, the native B800-850 complex from Rs. molischianum is most likely an octamer of monomers with a stoichiometry of three bacteriochlorophyll a and 1.5 lycopenes per alpha,beta-subunit. Resonance Raman spectra provide evidence for a 5-coordinated Mg2+ in the BChl, and a carotenoid mainly in the all-trans configuration. A comparison between resonance Raman data from different photosynthetic bacteria indicates that the BChl a-binding site of the B800-850 complex from Rs. molischianum is more similar to the B870 complexes (also called LH1) than to the B800-850 complexes of other photosynthetic bacteria. Sequence similarities especially between the beta-subunits of the B800-850 complex of Rs. molischianum and the B870 and B800-850 complexes of other photosynthetic bacteria agree with this result and provide information on the mode of pigment binding in bacterial antenna complexes.

Amino Acid Sequence↗