Ligation, inhibition, and activation of cytochrome c oxidase by fatty acids.

Free fatty acids bind to beef heart cytochrome c oxidase and induce spectral shifts similar to those obtained with high spin ligands. Oleic (18:1(n-9)) and linoleic (18:2(n-6)) acids induce substantial blue shifts of the Soret peak of oxidized enzyme. Small saturated fatty acids (< 15 carbon atoms) shift the Soret peak to the red, and longer chain acids induce smaller blue shifts. Formate-induced spectral shifts are modified by short chain fatty acids but are unaffected by longer chain fatty acids for which effects are additive with those of formate. Inhibition by formate is partially relieved by all fatty acids tested. Palmitic and linoleic acids increase turnover at low cytochrome c levels and decrease the K(m) for cytochrome c at high cytochrome c levels. Oleic acid protects the enzyme against acid denaturation during turnover. Bovine serum albumin produces a red shift in the oxidase Soret peak and inhibits turnover of the isolated enzyme. Oleic acid and serum albumin modify the electron paramagnetic resonance spectrum of oxidized oxidase, oleic acid shifting the g = 3 (cytochrome a) peak towards low field and albumin towards higher field strengths. The oxidase may possess at least two fatty acid binding sites at one of which cytochrome c binding is modulated and at another spectral changes may be induced. One site is close enough to the binuclear centre to interact allosterically with ligand binding at that centre.

Cytochrome c oxidase in proteoliposomes visualised by platinum-carbon and by tungsten-tantalum shadowing: image analysis.

Beef heart cytochrome c oxidase complexes incorporated into phospholipid liposomes were examined by freeze-fracture electron microscopy. Enzyme molecules are inserted into the membrane asymmetrically, with larger projections on the 'C' side, where cytochrome c binding occurs, than on the 'M' (matrix-facing) side. Visualisation of the complexes was improved by: (i) image analysis, to determine details of size and shape, and (ii) tungsten-tantalum (W/Ta) rather than platinum-carbon (Pt/C) shadowing, which permits examination of smaller entities. Enzyme molecules are incorporated as dimers in the proteoliposomes. Some surface structural details of the embedded molecules can be discerned. Around each complex is seen a small area of modified lipid, the frozen annulus whose existence has been predicted with other methods.

Catalase HPII of Escherichia coli catalyzes the conversion of protoheme to cis-heme d.

Catalase HPII from aerobically grown Escherichia coli normally contains heme d but cultures grown with poor or no aeration produce HPII containing a mixture of heme d and protoheme IX. The protoheme component of HPII from anaerobically grown cells is converted into heme d during treatment of the purified enzyme with hydrogen peroxide. It is concluded that heme d found in catalase HPII is formed by the cis-hydroxylation of protoheme in a reaction catalyzed by catalase HPII using hydrogen peroxide as a substrate. The distal His128 residue of HPII is absolutely required for the protoheme to heme d conversion. Two mutant enzymes, Ala128 and Asn128, are catalytically inactive and contain only protoheme, which is unaffected by hydrogen peroxide treatment. The Asn201 residue is not an absolute requirement for heme conversion. The mutant enzyme Ala201 contains predominantly heme d and is partially active. However, insertion of a histidyl residue to give the His201 enzyme interferes with the heme conversion reaction. This mutant form is isolated as a protoheme enzyme with limited activity, and a reversible conversion to a heme d-like species occurs in vitro in the presence of continuously generated hydrogen peroxide.

The effect of antibodies to subunit V of cytochrome oxidase on cyanide inhibition of electron transfer.

Binding of antibodies raised against subunit V of mammalian cytochrome oxidase to the intact membranous enzyme is redox-sensitive, suggesting the existence of 'open' and 'closed' protein conformers (Freedman, J.A., Cooper, C.E., Leece, B., Nicholls, P. and Chan, S.H.P. (1988) Biochem. Cell Biol. 66, 1210-1217). Similar open and closed states for the oxygen-reacting site have been proposed to explain cyanide binding kinetics (Jensen, P., Wilson, M.T., Aasa, R. and Malmström, B.G. (1984) Biochem. J. 224, 829-837). We therefore examined cyanide inhibition of oxidase activity polarographically and spectrophotometrically using soluble oxidase preincubated with and without anti-subunit V or non-immune rabbit gamma-globulin. The subunit-specific antibody decreased the cyanide 'on' rate and essentially eliminated the rapid phase of cyanide binding. We conclude that (i), bound antibody blocks HCN binding; (ii), antibody and HCN probably bind to the same conformation of the oxidase and (iii), the 'open'-'closed' conformation change that modulates binding of HCN may be similar to that which modulates antibody binding. The results are consistent with some reciprocating models of electron transfer and energy transduction by the oxidase (cf., Wikström, M.K.F., Krab, K. and Saraste, M. (1981) Cytochrome Oxidase: A Synthesis).

Electron microscopy of cytochrome c oxidase-containing proteoliposomes: imaging analysis of protein orientation and monomer-dimer behaviour.

1. Cytochrome c oxidase-containing vesicles were prepared by cholate dialysis using bovine heart cytochrome c oxidase with egg and dioleoylphosphatidylcholine/dioleoylphosphatidylethanolamines (1:1, w/w) at two ratios of phospholipid to protein (25 mg/mg and 10 mg/mg). With each mixture, one or two (FII, FIII) fractions with mostly outward-facing cytochrome aa3 were separated from a fraction (FI) containing mostly inward-facing enzyme and protein-free liposomes by DEAE-Sephacel chromatography. 2. FII and FIII fractions from egg phospholipid mixtures had 60-80% outward-facing enzyme; FII and FIII fractions from dioleoyl phospholipids showed 50-70% outward-facing enzyme. Egg and dioleoyl phospholipid mixtures maintained good respiratory control ratios (8-13) only at the higher lipid/protein ratios. 3. Platinum/carbon replicas of freeze-fractured vesicle surfaces were subjected to image analysis. The results showed two types of membrane projection with average heights of 7.5 nm and 3.5 nm from the fracture plane. The former were more numerous on the convex faces. Calculated areas of the projections indicated the probable presence of both enzyme dimers and higher aggregates. Oxidase dimers may have membrane areas of 70-80 nm2 at the high (7.5 nm) side and 40-50 nm2 on the low (3.5 nm) side. 4. Proteoliposomes prepared with enzyme depleted of subunit III contained predominantly much smaller projecting areas. These probably represent monomers with high side areas of 35-40 nm2 and low side areas of 20-25 nm2. Electron microscopy thus directly confirms the predicted change of aggregation state resulting from subunit depletion. 5. The results are compared with those from two-dimensional crystals. Assuming that the high and low projections are two sides of one family of transmembrane molecules, a total length of 11 nm matches 11-12 nm lengths obtained by crystallography. Our membrane areas match the areas obtained in earlier 'crystal' studies better than the small areas obtained recently by electron cryomicroscopy.

Analytical problems encountered during high-performance liquid chromatographic separation and coulometric detection of bopindolol metabolites in human plasma.

Good clinical control of hypertension is achieved with low doses of the potent beta-blocker bopindolol. Pharmacokinetic evaluations therefore require an analytical technique of high sensitivity. Analysis of plasma by high-performance liquid chromatography (HPLC) using electrochemical detection provides this sensitivity. This article describes the development of an analytical procedure and presents a reversed-phase HPLC method with coulometric detection suitable for plasma analyses during pharmacokinetic investigations of bopindolol therapy.

The renal handling of human urinary ribonuclease by rat kidneys.

The purpose of the study was to find out how poly(C)-avid human urinary ribonuclease is handled by the kidney. Purified human urinary ribonuclease (molecular weight 33 000) was radiolabelled with 125I. The enzyme was injected intravenously into dogs and monkeys with and without kidneys. The disappearance rate from the animals without kidneys was markedly prolonged. In the dog and monkey with kidneys, the radiolabelled enzyme which was infused was recovered in the urine unchanged. No large molecular weight fragments were found. When 125I-labelled ribonuclease was infused into rats the material recovered in the urine was primarily identical with the material infused. A very small fraction of the material recovered was found to contain some fragments which had chromatographic characteristics of monoiodotyrosine and diiodotyrosine. Two other fragments were detected but could not be identified. Autoradiographic studies of the rat kidneys also showed some reabsorption of the radiolabelled ribonuclease, particularly in the proximal tubules. With electron microscopy the radiolabelled material could be seen in the lysosomes. These observations corroborate findings discovered for other low molecular enzyme such as lysozyme (molecular weight 14 000) and suggest that the human ribonuclease is mainly excreted by the kidneys unchanged and that a minor amount may be reabsorbed by the proximal tubules and metabolized in the lysosomes.

A highly sensitive method for identification of amino termini of proteins: application to multiple forms of poly(C)-avid ribonuclease and 17 beta-hydroxysteroid dehydrogenase.

A radiochemical method for the determination of the amino terminus on very small amounts (0.5-5 nmol) of protein is described. The high sensitivity of the method is achieved by using undiluted 1-fluoro-2,4-dinitro-[3,5-3H]benzene [( 3H]Dnp-F) as the labelling reagent under conditions in which a maximum amount of radioactive label is incorporated. Chemical homogeneity is achieved by reacting with excess unlabelled Dnp-F. High recovery is obtained by adding Dnp-albumin as carrier protein. A mixture of Dnp 14C-labelled amino acids is added prior to hydrolysis and identification of the amino terminus is made on the basis of the 3H/14C ratios of the separated Dnp-amino acids. The method was tested on insulin, pancreatic ribonuclease, and lysozyme which gave high 3H/14C ratios only in the expected amino-terminal amino acids. Application to multiple forms of poly(C)-avid ribonuclease gave only amino-terminal lysine. Two of four putative isozymes of 17 beta-hydroxysteroid dehydrogenase had serine as the amino terminus while the other two had aspartic acid or asparagine.

The isolation, purification, and properties of a ribonuclease (Mr 18 000) from human uremic serum, and its relation to the human urinary ribonuclease (Mr 33 000). II. Properties of the enzymes.

A low molecular weight ribonuclease (Mr 18 000) isolated and purified from human uremic serum was found to have similar properties to the high molecular weight ribonuclease (Mr 33 000) isolated from human urine. A detailed comparative study of both enzymes was undertaken to investigate the relationship between them. It is suggested that poly(C)-avid human ribonucleases have similar amino acid compositions but variable carbohydrate contents and that variations in the sugar content are responsible for variations in the molecular weight.

Ribonuclease activity in renal failure. Evidence for toxicity.

Ribonuclease isolated from human urine is a glucoprotein of molecular weight 33,000. The purified enzyme inhibits: (1) the stimulation of 3H-thymidine uptake into lymphocytes by phytohemagglutinin, pokeweed, and concanavalin A; (2) the growth of pancreatic fibroblastoid cells in in vitro cell culture, and (3) the growth of colonies in bone marrow cell cultures. Ribonuclease levels in the uremic patient vary from 9,500 to 35,000 U/ml (normal 1,041 +/- 247). Serum ribonuclease levels are unaffected by dialytic procedures. It is suggested that the ribonuclease glycoprotein may represent a large number of nondialyzable high molecular weight uremic 'toxins'.

Ribonuclease activity in human serum, cerebrospinal fluid, and urine.

Poly(C)-avid ribonucleases of molecular weight 33 000 are present in the serum, cerebrospinal fluid and urine of humans. Purified human urinary ribonuclease was used to produce a monospecific antibody in rabbits. The antibody was capable of: (i) inhibiting the enzyme activities in the serum, CSF, and urine; (ii) reacting with antigens in the serum and CSF. The antigens in the serum, CSF and urine were found to be immunologically identical. Immunoelectrophoresis data suggested that the urinary and CSF RNAase are chemically identical. Succesful renal transplantation reduced elevated serum RNAase to normal levels. The data suggest that the most likely source of both urinary and CSF ribonuclease activity is the blood stream.

Ribonuclease activity of human cerebrospinal fluid.

The ribonuclease activity of cerebrospinal fluid of 219 patients was studied. The normal level was 269 +/- 95 units/ml. Consistent elevations above 550 units/ml were found in: 1. Chronic cerebrovascular disease; 2. Spinal cord compression; 3. Tumors. The molecular weights of the ribonucleases in the cerebrospinal fluid are approximately 33,000; 21,000 and 15,000; the major species is the one with m.w. 33,000. Although the increase in the CSF ribonuclease activity is not disease specific, the measurement has provided corroborative help in cases when the CSF protein is normal. The increase in CSF RNAase is not due to red or white blood cells and the immunologic data suggest that the CSF enzyme activity is derived from the blood stream. Further studies are necessary to rule out a nerve cell origin of the CSF ribonuclease activity.

Ribonuclease activity in renal failure: evidence for toxicity.

The normal level of serum or plasma poly C-avid ribonuclease activity is 1047 +/- 247 U/mL. Serum levels increase proportionately with elevations in serum creatinine, reaching levels of 9,500-35,000 in patients undergoing dialysis. The levels can be normalised by successful renal transplantation but not by dialysis. Purified human urinary ribonuclease, a glycoprotein enzyme similar to the serum ribonuclease, was capable of: 1) inhibiting the incorporation of 3H-thymidine into mitogen-stimulated lymphocytes; 2) inhibiting the proliferation and growth of bone marrow red cell colonies; and 3) adversely affecting the growth and viability of precursor fat cells.