Search

BACKGROUND The passive transfer of antibodies specific to blood groups A and B (also called isoagglutinins) contained in immunoglobulin (Ig)G products for intravenous administration (IVIG) is believed to be largely responsible for rare but sometimes serious IVIG-related hemolytic events. We present in this work a modification of the manufacturing process of Privigen—a 10% l-proline–stabilized IVIG product—that allows extensive reduction of isoagglutinin concentrations in the final product. STUDY DESIGN AND METHODS An additional immunoaffinity chromatography (IAC) step was introduced toward the end of the manufacturing process of Privigen. Isoagglutinin titers were measured using the indirect agglutination method and a published flow cytometry–based binding assay. Quality attributes, such as microorganism counts and concentration of endotoxins, IgG, IgA, IgM, aggregates, and so forth were measured using standardized procedures. RESULTS The introduction of an IAC step in the manufacturing process of Privigen resulted in an 88% to 90% reduction in isoagglutinins between the feed of the chromatography column and the flow-through fraction. All other product quality attributes measured were nearly identical before and after IAC. This process modification resulted in a three-titer-step reduction in isoagglutinin levels in the final IgG product compared to Privigen lots produced by the unmodified process. CONCLUSION Introducing an isoagglutinin-specific IAC step in the manufacturing process of Privigen is an efficient strategy for reduction of anti-A and anti-B titers. Such reductions might help minimize the risk of hemolytic events in patients receiving IVIG therapy.

In relation to the recent trials of Intravenous Immunoglobulin (IVIG) in Alzheimer's Disease (AD) it was demonstrated that different IgG preparations contain varying amounts of natural anti-amyloid β (Aβ) antibodies as measured by ELISA. We therefore investigated the relevance of ELISA data for measuring low-affinity antibodies, such as anti-Aβ. We analysed the binding of different commercial Immunoglobulin G (IgG) preparations to Aβ, actin and tetanus toxoid in different binding assays to further investigate the possible cause for observed differences in binding to Aβ and actin between different IgG preparations. We show that the differences of commercial IgG preparations in binding to Aβ and actin in ELISA assays are artefactual and only evident in in vitro binding assays. In functional assays and in vivo animal studies the different IVIG preparations exhibited very similar potency. ELISA data alone are not appropriate to analyse and rank the binding capacity of low-affinity antibodies to Aβ or other endogenous self-antigens contained in IgG preparations. Additional analytical methods should be adopted to complement ELISA data.

Evidence has accumulated that changes in intracellular signaling downstream of desmoglein 3 (Dsg3) may have a significant role in epithelial blistering in the autoimmune disease pemphigus vulgaris (PV). Currently, most studies on PV involve passive transfer of pathogenic antibodies into neonatal mice that have not finalized epidermal morphogenesis, and do not permit analysis of mature hair follicles (HFs) and stem cell niches. To investigate Dsg3 antibody-induced signaling in the adult epidermis at defined stages of the HF cycle, we developed a model with passive transfer of AK23 (a mouse monoclonal pathogenic anti-Dsg3 antibody) into adult 8-week-old C57Bl/6J mice. Validated using histopathological and molecular methods, we found that this model faithfully recapitulates major features described in PV patients and PV models. Two hours after AK23 transfer, we observed widening of intercellular spaces between desmosomes and EGFR activation, followed by increased Myc expression and epidermal hyperproliferation, desmosomal Dsg3 depletion, and predominant blistering in HFs and oral mucosa. These data confirm that the adult passive transfer mouse model is ideally suited for detailed studies of Dsg3 antibody-mediated signaling in adult skin, providing the basis for investigations on novel keratinocyte-specific therapeutic strategies.

Polyvalent Ig preparations, derived from the pooled plasma of thousands of healthy donors, contain a complex mix of both 'acquired' and natural antibodies directed against pathogens as well as foreign and self/auto antigens (Ag). Depending on their formulation, donor pool size, etc., liquid Ig preparations contain monomeric and dimeric IgG. The dimeric IgG fraction is thought to represent mainly idiotype-antiidiotype Ab pairs. Treatment of all IgG fractions at pH 4 effectively monomerizes the IgG dimers resulting in separated idiotype-antiidiotype Ab pairs and thus in a comparable F(ab')(2) binding site availability of the different IgG fractions. Previously, we identified an increased anti-self-reactivity within the monomerized dimer fraction. This study addressed if, among the different IgG fractions, an analogous preferential reactivity was evident in the response against different pathogen-derived protein and carbohydrate antigens. Therefore, we assessed the activity of total unseparated IgG, the monomeric and dimeric IgG fractions against antigenic structures of bacterial and viral antigens/virulence factors. All fractions showed similar reactivity to protein antigens except for exotoxin A of Pseudomonas aeruginosa, where the dimeric fraction, especially when monomerized, showed a marked increase in reactivity. This suggests that the production of antiidiotypic IgG antibodies contributes to controlling the immune response to certain categories of pathogens. In contrast, the monomeric IgG fractions showed increased reactivity towards pathogen-associated polysaccharides, classically regarded as T-independent antigens. Taken together, the differential reactivity of the IgG fractions seems to indicate a preferential segregation of antibody reactivities according to the nature of the antigen.

Subcutaneous administration of human IgG is an alternative to intravenous replacement therapy that is associated with more stable serum IgG levels and fewer systemic adverse events. Highly concentrated IgG solutions are most convenient to minimize infusion volume, but their preparation and stability presents substantial technical difficulties. We report on the stability and local tolerance of IgPro20, an l -proline-stabilized, 20% polyvalent human IgG developed for subcutaneous administration. Stability was tested according to ICH guidelines. Local tolerance and vasoactivity were examined in rabbit and rat models, respectively. The presence of l -proline in IgPro20 reduced viscosity and addition of Polysorbate 80 and inert gassing improved the appearance of the solution. After storage at 25 °C for 24 months, monomer + dimer content, aggregates, and fragments were within specification (≥90.0%, ≤4.0%, and ≤10.0%, respectively), and Fc function and antibody activities were maintained. In rats, intravenous injection of IgPro20 produced mild and transient hypotension comparable to that seen with intravenous IgG products. Local tolerance of IgPro20 in rabbits was comparable to that of a marketed subcutaneous IgG, Beriglobin ® P. Functionality and quality of IgPro20 are maintained during storage at 25 °C for at least 24 months. The product is well tolerated as assessed in animal models.

Intravenous immunoglobulin (IVIg) preparations are derived from pooled plasma from up to 60,000 healthy human donors and reflect the immunologic experience of the donor population. IVIg contains monomeric and dimeric IgG populations which are in a dynamic equilibrium depending on concentration, pH, temperature, donor pool size, time and stabilizers added in order to keep the portion of dimeric IgG below a certain level. In the present study, monomeric and dimeric fractions were isolated by size exclusion chromatography. The dimeric fractions, however, showed a dynamic instability and tended to dissociate. Both dimeric and monomeric IgG fractions were acid treated (pH 4) in order to dissociate the dimeric IgG. Western-blot analysis identified a sub-population of SDS resistant IgG dimers. Furthermore, the reactivities of the fractions were tested against a panel of self- and exo-antigens. There was a marked increase in activity of the dimeric compared to the monomeric IgG fraction against various intracellular self-antigens. Our data indicates that the increased reactivities of pH 4-treated fractions can mainly be attributed to dimer dissociation, as pH 4-treated monomers do not show significantly increased activities against a range of antigens.

Therapeutic intravenous immunoglobulin (IVIg) preparations contain antibodies reflecting the cumulative antigen experience of the donor population. IVIg contains variable amounts of monomeric and dimeric IgG, but there is little information available on their comparative antibody specificities. We have isolated highly purified fractions of monomeric and dimeric IgG by size-exclusion chromatography. Following treatment of all fractions at pH4, analyses by immunodot and immunocytology on human cell lines showed a preferential recognition of autoantigens in the dimeric IgG fraction. Investigation of the HEp-2 cytoplasmic proteome by 2D-PAGE, Western blot, and subsequent identification of IVIg reactive spots by mass spectrometry (LC-MS/MS) showed that IVIg recognized only a restricted set of the total proteins. Similar experiments showed that more antigens were recognized by the dimeric IgG fraction, especially when the dissociated dimer fraction was used, as compared to its monomeric counterpart. These observations are consistent with idiotype-anti-idiotype masking of auto-specific Abs in the dimeric fraction of IVIg.

The passive transfer of antibodies specific to blood groups A and B (also called isoagglutinins) contained in immunoglobulin (Ig)G products for intravenous administration (IVIG) is believed to be largely responsible for rare but sometimes serious IVIG-related hemolytic events. We present in this work a modification of the manufacturing process of Privigen-a 10% l-proline-stabilized IVIG product-that allows extensive reduction of isoagglutinin concentrations in the final product.An additional immunoaffinity chromatography (IAC) step was introduced toward the end of the manufacturing process of Privigen. Isoagglutinin titers were measured using the indirect agglutination method and a published flow cytometry-based binding assay. Quality attributes, such as microorganism counts and concentration of endotoxins, IgG, IgA, IgM, aggregates, and so forth were measured using standardized procedures.The introduction of an IAC step in the manufacturing process of Privigen resulted in an 88% to 90% reduction in isoagglutinins between the feed of the chromatography column and the flow-through fraction. All other product quality attributes measured were nearly identical before and after IAC. This process modification resulted in a three-titer-step reduction in isoagglutinin levels in the final IgG product compared to Privigen lots produced by the unmodified process.Introducing an isoagglutinin-specific IAC step in the manufacturing process of Privigen is an efficient strategy for reduction of anti-A and anti-B titers. Such reductions might help minimize the risk of hemolytic events in patients receiving IVIG therapy.

Background: We have developed a novel liquid intravenous immunoglobulin (IVIG-F10). It consists of a nanofiltered 12% immunoglobulin G (IgG) solution, stabilized with nicotinamide, L-proline and L-isoleucine. The efficacy, tolerability, safety, and pharmacokinetics of this product were assessed in patients with chronic immune thrombocytopenic purpura (ITP) and primary humoral immunodeficiencies (PID). Patients and Methods: 33 chronic ITP patients with platelet counts of 9/l were treated with IVIG-F10 or Sandoglobulin at doses of 0.4 g/kg body weight on 5 consecutive days. The primary efficacy endpoint was an increase in platelet counts to ≧50 × 109/l. Secondary endpoints were time to and duration of platelet response and regression of bleeding. 34 PID patients with X-linked agammaglobulinemia, common variable immunodeficiency or IgG subclass deficiency were treated for 6 months with IVIG-F10 or Sandoglobulin at doses of 0.3–0.8 g/kg, infused at 3- or 4-week intervals. The primary efficacy endpoint was the number of days absent from school/work. Secondary endpoints were feeling of well-being, days of hospitalization, and use of antibiotics. Results: In ITP patients, the primary endpoint was met by 12/16 patients on IVIG-F10 and by 12/17 patients on Sandoglobulin (p = 1.000). Results of the secondary endpoints were comparable in the two study groups. In the PID study, 10/17 patients on IVIG-F10 and 9/17 patients on Sandoglobulin missed days at school/work, with monthly mean absences of 0.7 and 0.6 days (p = 0.746), respectively. There were no significant differences in the outcome of the secondary endpoints. Pharmacokinetics showed constant peak and trough serum IgG levels in PID patients. The median half-life (t1/2) of IgG was 33 days in the IVIG-F10 group and 41 days in the Sandoglobulin group. For anti-HBsAg, median t1/2 values were shorter, i.e. 17 and 19 days for IVIG-F10 and Sandoglobulin, respectively. In the ITP study, adverse events related to study drug were suspected in 9 and 14 patients treated with IVIG-F10 and Sandoglobulin, respectively; in the PID study adverse events occurred in 8 and 9 patients, respectively. Viral safety was ascertained in both studies by serology supplemented with nucleic acid amplification testing. Serum levels of the stabilizers transiently increased after infusion of IVIG-F10, but were back to baseline at the following day. Conclusions: The clinical studies in patients with chronic ITP and PID showed that the efficacy, tolerability, safety, and pharmacokinetics of IVIG-F10 were comparable to the properties of Sandoglobulin.

In this randomized, double-blind, parallel-group study, a commercially available human immunoglobulin-G product, IVIG, was compared with two test formulations: (1) IVIG-N, which is a nanofiltered formulation of IVIG, and (2) IVIG-L, which is a nanofiltered, liquid, ready-for-use IgG formulation containing nicotinamide, L-proline, and L-isoleucine as stabilizers. Three groups of 10 healthy subjects each received a single 0.6 g/kg dose of one of the formulations infused over 3.5 to 6.8 hours, depending on the total volume to be infused. Blood samples were obtained over a 6-week period to assess pharmacokinetics, immunogenicity, and the pharmacodynamic effects on leukocytes and TNF-alpha. A blood sample was taken at 6 months for a viral safety check. Administrations were generally well tolerated with only one reference IVIG infusion stopped prematurely due to headache. The IgG Cmax and AUC over the 6-week blood sampling period from both test formulations satisfied equivalence criteria compared with the reference formulation. In subjects receiving IVIG-L, peak concentrations of the stabilizer nicotinamide ranged from 0.34 to 0.47 mmol/L and of nicotinamide-N-oxide from 0.03 to 0.04 mmol/L, which are below those reported to cause adverse events. During the infusion of IVIG, leukocyte counts initially declined from a baseline of 5.7 +/- 1.1 x 10(9)/L to 3.7 +/- 0.8 x 10(9)/L at 2 to 4 hours and returned to baseline by 24 hours. TNF-alpha levels, reflecting activation of the monocyte-macrophage system by the infused IVIG, rose from a baseline of 13 +/- 4 pg/mL to a peak of 272 +/- 324 pg/mL at 2 to 4 hours and returned to baseline by 24 hours. These patterns were generally similar for the test formulations, with the exception that the increase in TNF-alpha levels was dampened for IVIG-N, although this was not statistically significant. There was no evidence of immunogenicity or viral transmission from any of the formulations. Hence, these three formulations were generally well tolerated, yielded similar systemic exposure to IgG over a 6-week period after administration, and did not give rise to immunogenicity or viral safety concerns.

Eric Mohler, Arthur Nikkel, Richard Radek, Holly Robb, Michael Schrimpf, Jeffrey Waring, Chih-Hung Lee, Murali Gopalakrishnan, AbbVie Inc., North Chicago, Illinois, United States; AbbVie, North Chicago, Illinois, United States; AbbVie Inc., North Chicago, Illinois, United States; AbbVie Inc., North Chicago, Illinois, United States; AbbVie, North Chicago, Illinois, United States. Contact e-mail: robert.s.bitner@abbvie.com

Intravenous Immunoglobulin (IVIG) has been proposed as a potential therapeutic for Alzheimer's disease (AD) and its efficacy is currently being tested in mild-to-moderate AD. Earlier studies reported the presence of anti-amyloid beta (Aβ) antibodies in IVIG. These observations led to clinical studies investigating the potential role of IVIG as a therapeutic agent in AD. Also, IVIG is known to mediate beneficial effects in chronic inflammatory and autoimmune conditions by interfering with various pathological processes. Therefore, we investigated the effects of IVIG and purified polyclonal Aβ -specific antibodies (pAbs-Aβ) on aggregation, toxicity and phagocytosis of Aβ in vitro, thus elucidating some of the potential mechanisms of action of IVIG in AD patients. We report that both IVIG and pAbs-Aβ specifically bound to Aβ and inhibited its aggregation in a dose-dependent manner as measured by Thioflavin T assay. Additionally, IVIG and the purified pAbs-Aβ inhibited Aβ-induced neurotoxicity in the SH-SY5Y human neuroblastoma cell line and prevented Aβ binding to rat primary cortical neurons. Interestingly, IVIG and pAbs-Aβ also increased the number of phagocytosing cells as well as the amount of phagocytosed fibrillar Aβ by BV-2 microglia. Phagocytosis of Aβ depended on receptor-mediated endocytosis and was accompanied by upregulation of CD11b expression. Importantly, we could also show that Privigen dose-dependently reversed Aβ-mediated LTP inhibition in mouse hippocampal slices. Therefore, our in vitro results suggest that IVIG may have an impact on different processes involved in AD pathogenesis, thereby promoting further understanding of the effects of IVIG observed in clinical studies.

When the human body is invaded by foreign organisms it defends itself either through the propagation of a cellular response (cell-mediated immunity) or by the production of soluble proteins. This production of soluble proteins is known as humoral immunity, so-called because it refers to substances found within the humours, or body fluids. While there are a number of components that have been identified as important in the humoral immune response, including complement proteins [1], the key molecules necessary for mediating specific humoral immunity are immunoglobulins (Ig), also known as antibodies (Ab) (see also chapter A3).

Liquid intravenous immunoglobulin (IVIG) products offer improved convenience in preparation but often lack sufficient stability to allow room temperature storage. Furthermore, clinical tolerability may be affected due to formation of idiotype/anti-idiotype IgG dimers and/or aggregates. Here we report on the development of a 10% IVIG formulation with optimized stability achieved by the use of l-proline. The stability of concentrated liquid IVIG was strongly pH dependent. Aggregate formation, yellowish discoloration of the solution and loss of anti-hepatitis B surface antigen (HBs) antibody activity was minimal at intermediate pH (pH 4.8-5.3). Fragmentation of IgG was highest at low pH (pH 4.1). Idiotype/anti-idiotype IgG dimer formation was highest at neutral pH and was reduced with decreasing pH. The presence of L-proline further improved stability by inhibiting protein aggregation, reducing loss of anti-HBs antibody activity and decreasing coloring, particularly compared with glycine formulations. The IgG dimer content was up to 30% lower in solutions containing L-proline compared with those containing glycine or other stabilizers. In conclusion, a weakly acidic pH of approximately 5 and L-proline as stabilizer are optimal conditions for long-term stability of a liquid IVIG. L-proline, an amphiphilic, naturally occurring amino acid, is superior to glycine in restricting IgG dimer formation.

S U N D A Y 311 Selective Exclusion of High Titer Donor Plasma and Immunoadsorption Chromatography As Steps to Reduce Isoagglutinin Titers in IVIG Liane Hoefferer, Ibrahim El Menyawi, Brigitte Siani, Martin Imboden, Annette Gaida, Isabelle Glauser, Reinhard Bolli, Katharina Willimann, Sandra Wymann, A. Adriano Marques, Eleanora Widmer; CSL Behring, Berne, Switzerland. RATIONALE: Hemolysis is a rare but potentially serious complication of high-dose IVIG therapy. Isoagglutinins originating in donor plasma are believed to play a major role in these reactions, but host factors are also important. We determined the effects of excluding plasma from donors with high isoagglutinin titers from the pools used to prepare IVIG, and of specific anti-A/B immunoaffinity chromatography (IAC), on the anti-A and anti-B titers of IVIG products prepared using the process for Privigen/ Hizentra. METHODS: A high-throughput assay was used to identify plasma donors with the highest anti-A titers. The effects of addition of IAC with A/Btrisaccharide-coupled resins into the manufacturing process were also studied. Anti-A/B isoagglutinin titers in the resulting IVIG preparations were measured by indirect agglutination and flow cytometry. RESULTS: Exclusion of the 5% of plasma donors with the highest anti-A titers reduced both anti-A and anti-B isoagglutinins in the final product by approx. 50% (a single 2-fold dilution step, i.e. reduction of the titer from 1:16 to 1:8, and 1:8 to 1:4, respectively. N530 lots). Specific IAC reduced anti-A and anti-B titers by at least two 2-fold dilution steps in the final product (>80% reduction shown by flow cytometry on standard red cells), while the content of antibodies against common microbial antigens remained unchanged. CONCLUSIONS: Anti-A/B isoagglutinin reduction in IVIG products is feasible using screening and exclusion of a small percentage of high anti-A titer donors andwith specific IAC. These approachesmay reduce the risk of hemolysis in IgG therapy.

The autoimmune disease pemphigus vulgaris (PV) manifests as loss of keratinocyte cohesion triggered by autoantibody binding to desmoglein (Dsg)3, an intercellular adhesion molecule of mucous membranes, epidermis, and epidermal stem cells. Here we describe a so far unknown signaling cascade activated by PV antibodies. It extends from a transient enhanced turn over of cell surface-exposed, nonkeratin-anchored Dsg3 and associated plakoglobin (PG), through to depletion of nuclear PG, and as one of the consequences, abrogation of PG-mediated c-Myc suppression. In PV patients (6/6), this results in pathogenic c-Myc overexpression in all targeted tissues, including the stem cell compartments. In summary, these results show that PV antibodies act via PG to abolish the c-Myc suppression required for both maintenance of epidermal stem cells in their niche and controlled differentiation along the epidermal lineage. Besides a completely novel insight into PV pathogenesis, these data identify PG as a potent modulator of epithelial homeostasis via its role as a key suppressor of c-Myc.

Intravenous immunoglobulin (IVIG) preparations are derived from the pooled plasma of thousands of healthy donors and contain a complex mix of antibodies. Depending on the formulation, IVIG preparations contain variable amounts of monomeric and dimeric IgG. The biological and therapeutic significance of these IVIG fractions is still ill defined. Kinetic analysis of monomeric and dimeric IgG isolated by size-exclusion chromatography revealed a stable monomeric versus an unstable dimeric IgG fraction tending to dissociation. Biochemical analysis by 2D gel electrophoresis and isotype analysis showed no significant differences between the fractions. In contrast, comparative analysis by immunodot, ELISA, FACS, and immunohistology of monomeric and dimeric IgG fractions showed a preferential reactivity of the dimeric IgG on a variety of both self-antigens and exoantigens.

A method is described for the large scale preparation of reconstituted high density lipoproteins (rHDL) suitable for therapeutic use. Apolipoprotein A-I (apoA-I was isolated from precipitates obtained by cold ethanol fractionation of human plasma. This process includes several steps for virus removal and virus inactivation, among them pasteurization. Reconstitution of lipoprotein particles was performed by cholate dialysis using soybean phosphatidylcholine as the lipid source. An apoA-I:lipid ratio of 1:150 (mol:mol) was obtained. Redissolved rHDLs were disc-shaped particles resembling nascent HDL, as assessed by electron microscopy. The method was optimized for low content of free apoA-I protein as well as the low concentration of free lipid. The product was stabilized by lyophilization in the presence of sucrose. In vitro studies show potential effects it the prevention of gram-negative septic shock and in the inhibition of atherosclerosis.

Starting from isolated chloroplasts of the Chlamydomonas reinhardii cw 15 mutant, several mRNA-containing chloroplast subfractions, i.e. thylakoid-bound polysomes, detached polysomes or isolated RNA, were prepared and incubated in homologous and heterologous translation systems. In the reticulocyte lysate these fractions gave rise to strikingly different product patterns. A most prominent difference concerned the in-vivo rapidly labelled 32,000-dalton thylakoid polypeptide. Neither this membrane protein nor its 34,000-dalton precursor was formed when membrane-containing or free polysomes were translated, while the 34,000-dalton precursor was a main product of the RNA isolated from the same membranes. The influence of thylakoid membranes during translation was also observed in homologous translation systems with lysed chloroplasts supplemented with ATP. Membrane and soluble fractions, when translated separately, yielded product patterns which differed from each other, although the RNAs extracted from the respective fractions gave the same product patterns when translated in reticulocyte lysate; the latter included a soluble protein, the large subunit of ribulose-1,5-bisphosphate carboxylase, and a membrane protein, the 34,000-dalton precursor of the 32,000-dalton membrane protein, as major labelled translation products. These results point to a regulatory role of thylakoid membranes in the expression of chloroplast mRNA and argue against compartmentation of the chloroplast mRNAs between the soluble and membrane fractions.

The monomeric and dimeric forms of bovine cytochrome c oxidase (EC 1.9.3.1) were obtained from gel filtration chromatography on Ultrogel AcA 34 and analyzed. Both species contained all 12–13 subunits described for this enzyme. In the dimer 320 molecules [3H]dodecyl-β- d -maltoside were bound per heme aa3 and in the monomer 360 molecules per heme aa3. The monomers contained 10 mol of tightly bound phospholipid/mol heme aa3 and the dimers 14. Sedimentation coefficients of 15.5–18 S for the dimer and 9.6 S for the monomer were calculated from sucrose density centrifugation analysis and analytical centrifugation. By the laser beam light-scattering technique a Stokes radius of 70 A for the dimeric detergent-lipid-protein complex was measured. From those parameters and the densitometric determined partial specific volumes of the detergent and the enzyme, the molecular weights of 400,000 for the protein moiety of the dimer and 170,000–200,000 for the monomer were calculated. Under very low ionic strength conditions the monomer/dimer equilibrium was found to be dependent on the protein concentration. At low enzyme concentrations (10−9 m ) monomers were predominant, whereas at concentrations above 5 × 10−6 m the amounts of dimers and higher aggregates were more represented. The cytochrome c oxidase activity, measured spectrophotometrically and analyzed by Eadie-Hofstee plot, was biphasic as a function of cytochrome c concentration for the dimeric enzyme. Pure monomers gave monophasic kinetics. The data, fitting with a homotropic negative cooperative mechanism for the dimer of cytochrome c oxidase, are discussed and compared with other described mechanisms.

Bovine cytochrome c oxidase in 0.1% dodecylmaltoside, 50 mM KCl and 10 mM Tris-HCl, pH 7.4 is monodisperse with an apparent Mr 360,000 (dimer) as estimated by filtration on Ultrogel AcA 34. In the absence of added KCl the apparent Mr is 160,000 (monomer). The dimeric enzyme has a high and a low affinity site for cytochrome c; the monomeric, only the high affinity site. The results are consistent with the existence of one active site per monomer, having high affinity for cytochrome c. Since in a dimer the two sites are in close proximity, the binding of the first molecule of cytochrome c to the first site hinders the binding of the second molecule to the second site. The kinetic data fit with a model of homotropic negative cooperativity. The effect of salts on the cytochrome c oxidase kinetics is also present in isolated bovine heart mitochondria.

Cytochromec oxidase fromParacoccus denitrificans was homogenously dispersed in Triton X-100. Using gel exclusion chromatography and sucrose gradient centrifugation analysis a molecular weight of the detergent-protein complex of 155,000 was determined. After subtraction of the bound detergent (111 mol/mol hemeaa3) a molecular weight of 85,000 resulted, which agreed well with the model of a monomer containing two subunits. This monomer showed high cytochromec oxidase activity when measured spectrophotometrically in the presence of Triton X-100 (Vmax=85 s−1). The molecular activity, plotted according to Eadie-Hofstee, was monophasic as a function of the cytochromec concentration. AKm of 3.6×10−6 M was evaluated, similar to theKm observed in the presence of dodecyl maltoside [Naleczet al. (1985).Biochim. Biophys. Acta808, 259–272].

2-Cyano-4-hydroxycinnamate was covalently linked, through a diazo bond, to Sepharose 4B, which had been elongated with a hydrophobic spacer. A Triton X-100 extract from bovine heart mitochondria was pre-purified by hydroxylapatite chromatography and passed through the 2-cyano-4-hydroxycinnamate affinity resin in the presence of 0.7% deoxycholate. At pH 6 and in the presence of 0.2 M sodium chloride, a single polypeptide with an Mr of 34,000 was eluted. Subsequently, at pH 8 and in the presence of 2-cyano-4-hydroxycinnamate, another single protein with an Mr of 31,500 was released. Both proteins were reconstituted into phospholipid vesicles and their transport activities were measured. High, delta pH-dependent, 2-cyanocinnamate-sensitive pyruvate uptake was measured in vesicles containing only the 34-kDa protein. alpha-Ketobutyrate and other alpha-ketomonocarboxylic acids were competitive inhibitors of the pyruvate uptake, whereas di- and tricarboxylates had only small effects. alpha-Ketoglutarate-alpha-ketoglutarate exchange could only be measured in vesicles containing the 31.5-kDa protein. The molecular weight of this protein and its functional properties were similar to those of the alpha-ketoglutarate carrier isolated by a different method (Bisaccia, Indiveri, C., and Palmieri, F. (1985) Biochim. Biophys. Acta 810, 362-369). 2-Cyano-4-hydroxycinnamate inhibited the alpha-ketoglutarate exchange in a noncompetitive manner with an apparent Ki of 0.7 mM. It is concluded that by the described affinity chromatography procedure, two mitochondrial carriers transporting alpha-ketoacids, i.e. the monocarboxylate and the alpha-ketoglutarate carrier, could be purified in a functionally active state.

The monocarboxylate (pyruvate) carrier from bovine heart mitochondria was extracted from submitochondrial particles with Triton X-114 in the presence of cardiolipin. By a single hydroxylapatite chromatography step a 125-fold purification of the carrier protein could be achieved. High pyruvate/pyruvate-exchange activity was recovered, when the protein was reconstituted into phospholipid vesicles. No transport activity was observed, when the isolation occurred in the absence of phospholipids. The 2-cyano-4-hydroxycinnamate sensitive pyruvate exchange reaction was strongly temperature sensitive and dependent on the amount of protein reconstituted. Other 2-ketoacids caused competitive inhibition of the pyruvate uptake. Inhibitors of other mitochondrial carries, however, had very low or no effect on the monocarboxylate exchange. The influence of different -SH group reagents on the measured pyruvate/pyruvate-exchange in the reconstituted system was similar to the one observed with intact mitochondria. It is concluded that the described procedures for extraction, purification and reconstitution of the mitochondrial monocarboxylate carrier conserved the functional properties of the protein.

Glycosyl-phosphatidylinositol-specific phospholipase D (GPI-PLD) is an amphiphilic protein which, in serum, is associated with high-density lipoproteins (HDL). It is shown that the major component of the HDL fraction, apolipoprotein A-I (apo A-I), is responsible for this association. In the absence of apo A-I, purified GPI-PLD occurred as virtually inactive aggregates which became disaggregated by apo A-I. The enzyme/apo A-I complex efficiently hydrolyzed the solubilized GPI-anchored substrate, acetylcholinesterase. Triton X-100 was also able to dissociate aggregated GPI-PLD, however, it strongly inhibited enzyme activity at detergent concentrations above the critical micellar concentration.

Cytochrome c reductase and oxidase have traditionally been purified from a large number of species and tissues by techniques based on detergent solubilization of these membrane enzymes and subsequent fractionation by ammonium sulfate precipitation [ Nelson (1978), Yu et al. (1979); for a review on the enzymes see Azzi et al. (1985) and Rich (1984)]. These procedures are useful only for large-scale preparations and they have to be adapted for the isolation of the enzymes from different sources.

In order to obtain information on the role of subunit III in the function and aggregation state of cytochrome c oxidase, the kinetics of ferrocytochrome c oxidation by the bovine cytochrome c oxidase depleted of its subunit III were studied and compared with those of the oxidase isolated from P. denitrificans which contains only two subunits. The aggregation state of both enzymes dispersed in dodecyl maltoside was also compared. The two-subunit oxidase from P. denitrificans gave linear Eadie-Hofstee plots and the enzyme resulted to be monomeric (Mr = 82000) both, in gel filtration and sucrose gradient centrifugation studies. The bovine heart subunit III depleted enzyme, under conditions when the P. denitrificans cytochrome c oxidase was in the form of monomers, was found to be dimeric by sucrose gradient centrifugation analysis. At lower enzyme concentrations monomers were, however, detected by gel filtration. Depletion of subunit III was accompanied by the loss of small polypeptides (VIa, VIb and VIIa) and of almost all phospholipid (1–2 molecules were left per molecule of enzyme). The electron-transfer activity of the subunit III-depleted enzyme showed a monophasic Eadie-Hofstee plot, which upon addition of phospholipids became non-linear, similar to that of the control bovine cytochrome c oxidase. One of the roles of subunit III may be that of stabilising the dimers of cytochrome c oxidase. Lack of this subunit and loss of phospholipid is accompanied by a change in the kinetics of electron transfer, which might be the consequence of enzyme monomerisation.

Bovine heart submitochondrial particles were treated with a medium containing Triton X-114 and cardiolipin. The extract was subjected to hydroxyapatite chromatography. Only a few major polypeptides of similar molecular masses were found in the eluate, as shown by electrophoresis in an SDS-polyacrylamide gel stained with silver. The eluate was reconstituted into liposomes and was shown to catalyse two different transport activities: 2-oxoglutarate-2-oxoglutarate exchange sensitive to phthalonate and phenylsuccinate and pyruvate-pyruvate exchange sensitive to 2-cyano-4-hydroxycinnamate. Since both activities were found to have characteristics similar to those described for intact mitochondria, it was concluded that at least two of the polypeptides found in the hydroxyapatite eluate correspond to the two mitochondrial carriers.

Bovine heart cytochrome b-c1 complex dispersed in 0.1% dodecylmaltoside, 10 mM Tris-HCl (pH 7.4), was subjected to filtration on Ultrogel AcA 34 columns. Apparent Mr values of about 400,000 and 170,000 were estimated for the enzyme-detergent complex in the presence and absence of 50 mM KCl, respectively. Similar Mr values (about 390,000 and 160,000) were obtained after sucrose gradient centrifugation of the b-c1 complex species isolated using Ultrogel filtration. Both species contained eight polypeptides, as in the original cytochrome b-c1 complex. The experiments suggest that the two species represent a dimer and a monomer of the b-c1 complex. The molecular conversion between the monomeric and dimeric state of the enzyme was found to be reversible. Both monomers and dimers of the b-c1 complex were competent to catalyze QH2:cytochrome c reductase activity with approximately the same maximal velocity. The finding that both molecular forms of the enzyme appear equally active does not support functional models based exclusively on a dimeric b-c1 complex.

Structural and functional research on cytochrome c oxidase has allowed in the last eight years to achieve some results in our laboratory which will be discussed in the lines below. This represents a contribution to the understanding of its mechanism of action, but, by no means, a balanced review article on cytochrome c oxidase.

Resume Les monomeres et les dimeres de la cytochrome c oxidase bovine (EC 1.9.3.1.) ont ete separes par filtration moleculaire sur Ultrogel AcA 34 ou par centrifugation en gradient de saccharose. Les facteurs qui influencent l'interconversion moleculaire ont ete analyses. En presence du detergent dodecyl-maltoside et a force ionique tres faible, le monomere de l'oxidase est la forme principale. La presence de cations semble stabiliser la forme dimere, les cations divalents etant plus efficaces que les ions monovalents. Des concentrations en enzymes elevees favorisent la formation des dimeres, aussi a force ionique tres faible. Le choix du detergent est tres important : en presence de Triton X-100 ou dodecyl-maltoside (a force ionique forte) l'enzyme est homogene dans sa forme dimere tandis qu'en presence de Tween-80 la filtration moleculaire ne montre que de grandes particules, qui sont eluees dans le volume d'exclusion. En presence de cholate des monomeres et des agregats sont observes mais pas de dimeres. L'etat moleculaire de l'enzyme influence la cinetique en etat stationnaire de l'oxidation du ferrocytochrome c. Les monomeres montrent une fonction lineaire dans la presentation d'Eadie-Hofstee tandis que les dimeres montrent une cinetique biphasique ou courbe. La force ionique, la concentration en enzyme et le detergent influencent la cinetique de l'enzyme d'une facon qui correspond a la forme moleculaire determinee par la filtration ou par l'analyse de sedimentation. Ces resultats confirment un mecanisme de cooperation negative dans l'interaction du cytochrome c avec la forme dimere mais non avec le monomere de la cytochrome c oxidase. Ce mecanisme a ete deja propose dans une etude precedente

The existence of a system transporting pyruvic acid through the inner mitochondrial membrane has been for a long time a matter of debate. In 1971, when such a system was described for the first time (Paradies et al, 1971) and since then it has been characterized in terms of its substrate specificity. Both other 2-oxo acids and their derivatives substituted in position 2 with halogens turned out to be good and competitive substrates in the reactions of either net pyruvate uptake according to the transmembrane pH difference or in the exchange reaction (Halestrap, 1975; Paradies & Papa, 1977). Halestrap described a series of chemical compounds which exhibited an inhibitory effect on the pyruvate transport measured in intact mitochondria, among them the most widely applied being α-cyanocinnamate and its 4-hydroxy derivative (Halestrap, 1975).

Chloroplast polysomes that were originally bound to thylakoid membranes were isolated from the cell wall mutant CW-15 from Chlamydomonas reinhardii . Polysomes were isolated from synchronously gorwn cells harvested in the middle of the third light period, when the ratio of chloroplast to cytoplasmic polysomes was maximal. Thylakoid membranes were isolated from a chloroplast fraction and polysomes were released by Triton X-100. Analyses of subunits on sucrose gradients showed that the polysomes consisted predominantly of the 70 S-type ribosomes. The detached polysomes as well as polysomes still bound to the thylakoid membrane were active in in vitro protein synthesis when supplemented with Escherichia coli -soluble factors. The in vitro activity was inhibited by chloramphenicol and aurintricarboxylic acid, but not by cycloheximide.

The determination of the overall state of association of a protein requires the measurement of its molecular weight. In the case of a globular, water-soluble protein, this can be done easily and accurately by a gel filtration expe ntn, in which the permeation of the protein into the pores of a gel matrix is compared with that of proteins of known size and molecular weight. A solubilized membrane protein, however, is a detergent-protein-complex, which often contains a considerable amount of bound lipid. To estimate the molecular weight of the protein moiety in such a complex, one has first to determine the amounts of each of the components bound, and one has to know also their partial specific volumes. Gel permeation chromatography and centrifugation techniques may then be applied to determine size, molecular weight, and aggregation state of the protein complex. The obtained result has to be interpreted carefully, considering especially the asymmetry of the protein complex, the possible interaction of the detergent with the gel matrix, and the influence of other components of the medium (e.g., pH, ionic strength).

Publisher Summary his chapter discusses the techniques for the study of bovine cytochrome-c oxidase monomer-dimer association. At neutral pH and in the presence of nonionic detergents, the bovine heart oxidase is in the form of dimers. In crystals prepared in deoxycholate, the enzyme exists as a monomer in detergent-rich sheets and in crystals obtained in Triton X-100, the oxidase is a dimer inserted across the lipid bilayer. Several factors regulate interconversion of the bovine enzyme from the monomeric to the dimeric form. An increase in ionic strength facilitated. dimerization of the enzyme in dodecyl-β-D-maltoside. Lowering pH and detergent concentration reversed the monomerization of oxidase, while monomers were obtained at high pH and high Triton X-100 concentration. The composition of the protein-lipid-detergent complex can also influence the molecular form of the enzyme. The monomerization of the enzyme can be correlated with lower lipid content. Gel filtration and centrifugation studies (equilibrium centrifugation, sedimentation velocity measurements, and density gradient centrifugation) are applied most often to the study of the molecular form of cytochrome-c oxidase.