Electron transfer in the membranes of endoplasmic reticulum. Participation of cytochrome b5 in the NADPH oxidation reaction. The evidence for two cytochromes b5 in liver microsomes.

The rate of the redox reactions of cytochromes b₅ and P-450 in the presence of NADPH and NADH has been studied. It has been shown that different factors: dimethylaniline, ferric pyrophosphate, carbon monoxide, and an increase in the ionic strength of the medium produce a similar effect on the rate of the redox reactions of cytochromes b₅ and P-450 reduced by NADPH. With NADH used as substrate, aerobic redox behavior of cytochrome b₅ was quite different. The data obtained gave grounds to suggest a scheme of electron transfer in the NADPH oxidation chain according to which one of the cytochrome b₅ subfractions (about 25% of the total pool of cytochrome b₅) functions between flavoprotein and cytochrome P-450.     

What information do inhibitors provide about the structure of the hydroquinone oxidation site of ubihydroquinone: Cytochromec oxidoreductase?

The Q cycle mechanism of thebc ₁ complex requires two quinone reaction centers, the hydroquinone oxidation (Q_P) and the quinone reduction (Q_N) center. These sites can be distinguished by the specific binding of inhibitors to either of them. A substantial body of information about the hydroquinone oxidation site has been provided by the analysis of the binding of Q_P site inhibitors to thebc ₁ complex in different redox states and to preparations depleted of lipid or protein components as well as by functional studies with mutantbc ₁ complexes selected for resistance toward the inhibitors. The reaction site is formed by at least five protein segments of cytochromeb and parts of the iron-sulfur protein. At least two different binding sites for Q_P site inhibitors could be detected, one for the methoxyacrylate-type inhibitors binding predominantly to cytochromeb, the other for the chromone-type inhibitors and hydroxyquinones binding predominantly to the iron-sulfur protein. The interactions with the protein segments, between different protein segments, and between protein and ligands (substrate, inhibitors) are discussed in detail and a working model of the Q_P pocket is proposed.     

Purification of characterization of a minor form of hepatic microsomal cytochrome P-450 from rats treated with polychlorinated biphenyls

A minor form of hepatic microsomal cytochrome P-450 has been purified to apparent homogeneity from rats treated with the polychlorinated biphenyl mixture, Aroclor 1254. This newly isolated hemoprotein, cytochrome P-450_e, is inducible in rat liver by Aroclor 1254 and phenobarbital, but not by 3-methylcholanthrene. Two other hemoproteins, cytochromes P-450_b and P-450_c, have also been highly purified during the isolation of cytochrome P-450_e based on chromatographic differences among these proteins. By Ouchterlony double-diffusion analysis with antibody to cytochrome P-450_b, highly purified cytochrome P-450_e is immunochemically identical to cytochrome P-450_b but does not cross-react with antibodies prepared against other rat liver cytochromes P-450 (P-450_a, P-450_c, P-450_d) or epoxide hydrolase. Purified cytochrome P-450_e is a single protein-staining band in sodium dodecyl sulfate-polyacrylamide gels with a minimum molecular weight (52,500) slightly greater than cytochromes P-450_b or P-450_d (52,000) but clearly distinct from cytochromes P-450_a (48,000) and P-450_c (56,000). The carbon monoxide-reduced difference spectral peak of cytochrome P-450_e is at 450.6 nm, whereas the peak of cytochrome P-450_b is at 450 nm. Ethyl isocyanide binds to ferrous cytochromes P-450_e and P-450_b to yield two spectral maxima at 455 and 430 nm. At pH 7.4, the 455:430 ratio is 0.7 and 1.4 for cytochromes P-450_b and P-450_e, respectively. Metyrapone binds to reduced cytochromes P-450_e and P-450_b (absorption maximum at 445–446 nm) but not cytochromes P-450_a, P-450_c, or P-450_d. Metabolism of several substrates catalyzed by cytochrome P-450_e or P-450_b reconstituted with NADPH-cytochrome c reductase and dilauroylphosphatidylcholine was compared. The substrate specificity of cytochrome P-450_e usually paralleled that of cytochrome P-450_b except that the rate of metabolism of benzphetamine, benzo[a]pyrene, 7-ethoxycoumarin, hexobarbital, and testosterone at the 16α-position catalyzed by cytochrome P-450_e was only 15–25% that of cytochrome P-450_b. In contrast, cytochrome P-450_e catalyzed the 2-hydroxylation of estradiol-17β more efficiently (threefold) than cytochrome P-450_b. Cytochrome P-450_d, however, catalyzed the metabolism of estradiol-17β at the greatest rate compared to cytochromes P-450_a, P-450_b, P-450_c, or P-450_e. The peptide fragments of cytochromes P-450_e and P-450_b, generated by either proteolytic or chemical digestion of the hemoproteins, were very similar but not identical, indicating that these two proteins show minor structural differences.     

On the function of cytochrome b5 in the cytochrome P-450-dependent oxygenase system

Complex formation between the phenobarbital-inducible form of rabbit liver microsomal cytochrome P-450 incorporated into phosphatidylcholine and detergent-solubilized cytochrome b₅ is associated with a low-to-high spin transition of the former pigment. It is concluded that the proteins combine in a 1:1 molar ratio. CD spectral analysis in the far uv region reveals that interaction of the cytochromes results in a conformational change of one or both hemoproteins. Such a cytochrome b₅-induced structural alteration of the reconstituted enzyme system is accompanied by an increase in affinity of 4-chloroaniline for cytochrome P-450, as measured in terms of cumene hydroperoxide-supported N-oxidation of the arylamine; the maximum velocity of the catalytic process remains unchanged. Similarly, incorporation into the assay media of cytochrome b₅ decreases the apparent K_d values of both the amine substrate and the oxygen donor, as determined by optical titration. Stopped-flow spectrophotometric studies on the influence of cytochrome b₅ on the kinetics of binding to cytochrome P-450 of 4-chloroaniline and/or cumene hydroperoxide show that the rates of formation and decay of the adducts change as the molar ratio of cytochrome b₅ to cytochrome P-450 varies. Moreover, cytochrome b₅ modifies the activation energies required for production of the substrate-bound oxy complex. These findings suggest that cytochrome b₅, apart from its well-known role as an electron carrier, might exert an effector function in the cytochrome P-450 system.     

The effects of sickling on ion transport. II. The effect of sickling on sodium and cesium transport

When the red cells from patients with sickle cell anemia (S-S) were kept in the disk shape by incubation in O₂, they maintained cell sodium in the steady state for at least 10 hours. The sodium flux in such cells at 37°C. was 6.0 ± 1.5 m.eq./ (liters RBC) x (hours). When S-S cells were sickled by incubation in N₂, sodium outflux increased two- to threefold, while influx increased four- to fivefold and the cells gained net sodium. A small but undetermined fraction of the sodium in disk and sickle shaped S-S cells exchanges at one or more rates which are substantially slower than those calculated here from the initial rate of transfer of tracer from cells to the medium. The penetration of tracer Cs into normal and both disk and sickled S-S cells was markedly inhibited by increasing the K concentration in the medium, indicating that Cs and K compete for an entrance pathway in all three cell types. The ratio of the inward rate constant for tracer Cs to that for K⁴² in normal and disk-shaped S-S cells increased only slightly when the K concentration in the medium was increased, indicating that almost all the Cs entered such cells in competition with K. Sickling accelerated the entrance of tracer cesium into S-S cells. Furthermore, the rate constant ratio increased with increasing external K concentration in sickled cells, suggesting the simultaneous presence of a non-competitive route for cesium influx in this cell type. The results are interpreted to support the view that sickling (a) accelerates inward transport of K and Cs and outward transport of Na by a non-diffusion, assumed carrier, process and (b) opens pathways for the diffusion of all three ions.     

Horseradish peroxidase as a label of injured cells

Synopsis The present study is concerned with artifacts likely to occur in a horseradish peroxidase exclusion test. Incubation of murine peritoneal macrophages and lymphocytes with the peroxidase showed a close relationship between the number of living cells and the percentage of cells excluding the tracer. The penetration of the cytoplasm by horseradish peroxidase is attributed to an increase in the permeability of the cell membrane during the incubation (ranging from 10 to 120 min). It was not increased by the presence of tracer throughout the incubation period. However, concomitant fixation of the cell in the presence of horseradish peroxidase caused an increase in the influx of the tracer. The horseradish peroxidase exclusion test applied to the guinea-pig organ of Corti has proved to be valid provided that: (a) mechanical lesions prior to the tracer incubation are avoided; (b) incubation is terminated by removal of the extracellular tracer; (c) fixation is carried out as soon as possible; (d) a low concentration of horseradish peroxidase is used; and (e) specimens are incubated in diaminobenzidine-H₂O₂ medium for the shortest possible period.Although fixation-induced cytoplasmic infiltration by horseradish peroxidase was not detected in cochlear specimens, the findings call attention to possible sources of error and define the level of significance of the test. Horseradish peroxidase does not appear to be a cytotoxic agent under the conditions used.     

Sow and piglet factors determining variation of colostrum intake between and within litters

Colostrum intake has a short- and long-term beneficial impact on piglet performance and mortality. Sows’ colostrum production and piglets’ colostrum intake are limited and highly variable. The present study investigated sow and piglet factors explaining the variation of colostrum intake between and within litters. The CV for colostrum intake and birth weight (BW_b) of all piglets within a litter was calculated to evaluate the variation of colostrum intake and BW_b within a litter (colostrum and litter BW_b heterogeneity, respectively). A total of 1937 live-born piglets from 135 litters from 10 commercial herds were included. Colostrum intake per piglet averaged 371±144 g and was affected by breed (P=0.02). It was lower when oxytocin was administered to the sow during parturition (P=0.001) and with increased litter size (P<0.001). It was higher when the interval between birth and first suckling decreased (t_FS, P<0.001). Colostrum intake was positively influenced by BW_b (P<0.001) and this association was more pronounced in piglets from Topigs (P=0.03) and Hypor (P=0.03) sows compared with piglets from Danbred sow breeds. The positive relationship between colostrum intake and BW_b was more pronounced when t_FS lasted longer (P=0.009). Heterogeneity in colostrum intake averaged 31±11%, it increased when oxytocin was applied during farrowing (P=0.004) and when stillbirth occurred (P=0.006). Colostrum heterogeneity was positively associated with litter size (P<0.001) and litter BW_b heterogeneity (P=0.01). The positive relationship between colostrum and litter BW_b heterogeneity was more pronounced when oxytocin was applied during farrowing (P=0.04). The present study demonstrated that oxytocin should be used cautiously in sows during farrowing. Farrowing and colostrum management should prevent or counteract the adverse influences of stillbirth, large and heterogeneous litters on colostrum intake and colostrum heterogeneity. The study also confirmed the expected association between BW_b and colostrum intake and indicated that the impact of BW_b on colostrum intake was different among breeds (Hypor v. Danbred) and dependent on piglets’ latency to first suckling. Hence, colostrum management should focus on low birth weight piglets, especially in some breeds, and low colostrum intake in low birth weight piglets can be counteracted by shortening the t_FS.     

Interaction of purified microsomal cytochrome P-450 with cytochrome b5

The interactions between purified microsomal cytochrome P-450 and cytochrome b₅ has been demonstrated by aqueous two-phase partition technique. Major forms of cytochrome P-450 induced by phenobarbital (P-450_LM2) and β-naphthoflavone (P-450_LM4) are almost exclusively distributed in the dextran-rich bottom phase (partition coefficient, K = 0.06), whereas NADPH-cytochrome P-450 reductase and cytochrome b₅ are mainly distributed in the polyethylene glycol-rich top phase (K = 3.5 and 2.5, respectively), when these enzymes were partitioned separately in the dextran-polyethylene glycol two-phase system. The mixing of P-450_LM with cytochrome b₅ changes the partition coefficients of both P-450_LM and cytochrome b₅ indicating that molecular interaction between P-450_LM and cytochrome b₅ occurred. Complex formation was also confirmed by optical absorbance difference spectral titration, and the stimulation of the P-450_LM-dependent 7-ethoxycoumarin and p-nitrophenetole O-deethylase activities by equal molar quantity of detergent-solubilized cytochrome b₅, but not trypsin-solubilized enzyme, in the reconstituted system. Cytochrome b₅ decreases the K_m's of both substrates for P-450_LM2-dependent O-deethylations and increases the V's of both reactions by two- to three-fold. This stimulatory effect requires the presence of phospholipid in the reconstituted enzyme system. These results suggest that cytochrome b₅ plays a role in some reconstituted drug oxidation enzyme systems and that molecular interactions among cytochrome P-450, reductase, and cytochrome b₅ are catalytically competent in the electron transport reactions.     

Domain conformational switch of the iron–sulfur protein in cytochrome bc1 complex is induced by the electron transfer from cytochrome bL to bH

Intensive biochemical, biophysical and structural studies of the cytochrome (cyt) bc₁ complex in the past have led to the formulation of the “protonmotive Q-cycle” mechanism for electron and proton transfer in this vitally important complex. The key step of this mechanism is the separation of electrons during the oxidation of a substrate quinol at the Q_P site with both electrons transferred simultaneously to ISP and cyt b_L when the extrinsic domain of ISP (ISP-ED) is located at the b-position. Pre-steady state fast kinetic analysis of bc₁ demonstrates that the reduced ISP-ED moves to the c₁-position to reduce cyt c₁ only after the reduced cyt b_L is oxidized by cyt b_H. However, the question of how the conformational switch of ISP-ED is initiated remains unanswered. The results obtained from analysis of inhibitory efficacy and binding affinity of two types of Q_P site inhibitors, Pm and Pf, under various redox states of the bc₁ complex, suggest that the electron transfer from heme b_L to b_H is the driving force for the releasing of the reduced ISP-ED from the b-position to c₁-position to reduce cyt c₁.     

膜上斑点洁显示人红细胞b5还原酶活性

人红细胞NADH-细胞色素b₅还原酶是使高铁血红蛋白还原的主要酶类, 其缺陷将导致遗传性高铁血红蛋白血症. 目前, 主要通过分光光度法测定b₅还原酶活性. 我们将b₅还原酶抗体点于硝酸纤维膜上, 以此捕获并富集红细胞胞浆b₅还原酶. 有b₅还原酶活性的斑点用噻唑蓝染色. 此法简单直观, 可用于b₅还原酶的定性和半定量测定, 为遗传性高铁血红蛋白血症的诊断提供了一种新的实验手段.     

Kinetic analysis of blood-brain barrier transport of amino acids

The Michaelis-Menten kinetics of blood-brain barrier transport of fourteen amino acids was investigated with a tissue-sampling, single-injection technique in the anesthetized rat. Tracer quantities of ¹⁴C-labelled amino acids and ³H₂O, used as a freely diffusible internal reference, were mixed in 0.2 ml of buffered Ringer's solution and injected rapidly into a common carotid artery. Circulation was terminated by decapitation at 15 s following injection. A brain uptake index (I_b) was determined from the ratio of ¹⁴C dpm in the brain tissue and the injection mixture divided by the same ratio for the ³H₂O reference. Brain clearance of tracer concentration of amino acid was saturable when various concentrations of unlabeled amino acid were added to the injection solution. Double reciprocal plots of the saturation data yielded K_m (mM) values that ranged from a low of 0.09 mM for arginine to a high of 0.75 mM for cycloleucine. Transport V values were determined from the relationship $\text{P =}\text{V}\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ where P is the blood-brain barrier permeability constant (ml/g per min): P was calculated from the I_b for each amino acid based on a cerebral blood flow of 0.56 ml/g per min and a fractional extraction of 0.75 for the ³H₂O reference 15 s following carotid injection. The V values ranged from a low of 6.2 nmol/g per min for lysine to a high of 64 nmol/g per min for l-DOPA. Efflux of the tracer amino acid during the 15-s period after injection was assumed to be slow, since the rate constant of cycloleucine from brain to blood was low, 0.11 min^?1.     

Surface Potentials and the Calculated Selectivity of Ion Channels

Ion channels catalyze the transport of ions across biological membranes. A proper understanding of ion-channel functioning is essential to our knowledge of cell physiology, and, in this context, ion-channel selectivity is a key concept. The extent to which a channel permeates two ion species, a and b, is expressed by the permeability ratio, P_a/P_b. This paper addresses a complication in the calculation of P_a/P_b that is related to the existence of surface potentials (ψ) and that so far has not been fully appreciated. This paper shows the rather surprising effect of ψ on the calculated P_a/P_b of a channel that is permeable to two ion species of different valence. If we ignore ψ, we conclude, for instance, P_a > P_b. If we implement ψ in the calculation of P_a/P_b, we may, however, conclude exactly the reverse, i.e., P_a < P_b. Because electrostatic potentials arise at the surface of essentially all biological membranes, this paper argues for a more critical evaluation of ion channel selectivity measurements.     

Chlorophyllase in citrus leaves. Kinetic aspects of the reaction

The reaction: Chlorophyll + Enzyme → Chlorophyllide + Phytol follows a first order kinetics with regard to the quantity of enzyme, when it is saturated by substrate. K_m and V_m were determined from the average reaction rates for the substrates: Chlorophylla andb, pheophytina andb, methylchlorophyllidea and methylpheophorbide a: The lowest K_m corresponded to chlorophylla and the highest V_m to methylpheophorbidea. For a substrate of chlorophyll (a + b), K_m and V_m were determined also using the initial reaction rates. “Enzyme efficiency” was calculated using both methods of determination. The reaction products partially inhibit the hydrolytic process.     

Oxidation of ruthenium red for use as an intercellular tracer

Summary When ruthenium red (RR) is combined with OsO₄, an electronopaque complex forms which readily binds to the cell surface coat. However, the RR-OsO₄ complex is often excluded from intercellular spaces in many cell types, and thus is not dependable as a tracer of regions continuous with the extracellular space. Postfixation of erythrocytes agglutinated by the lectin helix (Helix promatia) and intact carotid artery endothelium with a freshly prepared mixture of 1% OsO₄ containing 0.1% ruthenium red (RR) resulted in a dense surface deposit of these cells, but intercellular regions were penetrated to a minimal degree by the stain. When a similar mixture of RR-OsO₄ was allowed to stand 3 h before use, RR is oxidized by OsO₄ to yield a ruthenium compound that has a spectrophotometric absorbance maximum at 365 nm. This RR molecule has a reduced number of cationic sites due to binding with osmium dioxide OsO ₂ ⁼ . Postfixation of agglutinated RBCs and carotid artery endothelium with this oxidized ruthenium-OsO₄ mixture resulted in a 50–80% decrease in surface deposition but markedly enhanced penetration into intercellular regions. The enhanced penetration is attributed to decreased binding affinity of the oxidized ruthenium for anionic surface membrane components, permitting effective stain penetration into regions of cell-to-cell contact rather than extensive surface deposition. These studies indicate that the ruthenium compound formed by OsO₄ oxidation of ruthenium red may be a useful tracer for ultrastructural visualization of intercellular spaces and junctions.     

Plastid development in primary leaves of Phaseolus vulgaris: The light-induced development of the chloroplast cytochromes

Summary Etioplasts obtained from the primary leaves of dark-grown bean plants contained cytochromes f, b-559_LP and b-563 in a molar ratio of approximately 1.0:2.0:1.5. On illumination of the plants there was a lag of between 10 and 15 h before these cytochromes increased in amount, but after 48 h they had increased from 6- to 10-fold on a per plastid basis. The presence of cytochrome b-559_HP in the plastids was first detected after 15 h of illumination, which coincided with the commencement of grana formation and the onset of a number of photosynthetic reactions in the greening leaves. After 48 h of illumination the molar ratio for cytochromes f, b-559_HP, b-559_LP and b-563 was 1.0:1.2:2.8:2.6.Agranal chloroplasts formed by the exposure of dark-grown plants to intense light flashes contained high amounts of cytochromes f, b-559_LP and b-563 but cytochrome b-559_HP could not be detected.As the light-induced formation of cytochromes f, b-559_LP and b-563 was substantially inhibited by D-threo chloramphenicol, but not by the L-threo isomer, it seems likely that their formation was dependent on 70S ribosomes. Both chloramphenicol isomers gave plastids which lacked cytochrome b-559_HP.     

A study of human furin specificity using synthetic peptides derived from natural substrates, and effects of potassium ions

We explored furin substrate requirements in addition to the motif R-X-K/R-R using synthetic fluorescent resonance energy transfer (FRET) decapeptides. These decapeptides were derived from furin cleavage sites in viral coat glycoproteins and human and bacterial protein precursors. The hydrolysis by furin of most substrate was activated by K⁺ ion, whereas kosmotropic anions of the Hofmeister series were inhibitors. The analysis of furin hydrolytic activity showed that its efficiency is highly dependent on the particular combinations of amino acids at different substrate positions. There is a clear interdependence of furin subsites that must be taken in account in determining its specificity and also for the design of inhibitors. However, clear preferences were detected for substrates with S at P₁′, and V at P₂′, at P₃′ the amino acids D, S, L and A are almost equally frequent. In the non-prime subsites the best substrates presented S and H at P₆; basic amino acids at P₅; and no clear tendency at P₃. Interestingly, two amino acid substitutions on the prime side of the peptide derived from H5N1 influenza hemagglutinin furin processing site highly improved its hydrolysis. These modifications are possible by single point mutations, suggesting a potential yield of a more infectious virus.     

Simultaneous purification of a cytochrome P-450 and cytochrome b5 from the house fly,Musca domestica L.

Cytochrome P-450, cytochrome b₅ and cytochrome P-450 reductase were purified from house fly abdomens using high performance liquid chromatography (HPLC). Using a new technique, cytochrome P-450 was separated from the bulk of other proteins after polyethylene glycol fractionation and hydrophobic interaction chromatography (HIC) using a phenyl-5PW column. This technique resulted in 91% recovery of the cytochrome P-450s in a single concentrated fraction that also contained the remaining cytochrome b₅ and cytochrome P-450 reductase activity. Further purification by anion exchange on a DEAE-5SW column resolved the cytochrome P-450s, cytochrome b₅ and cytochrome P-450 reductase into individual fractions. The ion exchange step yielded one fraction that contained a high specific content of P-450 (14.4 nmol/mg protein). This cytochrome P-450 fraction ran as a single band at 54.3 kDa in sodium dodecyl sulfate polyacrylamide (SDS-PAGE) gel electrophoresis and had a carboxy ferrocytochrome absorbance maximum at 447 nm.Further purification of the anion exchange cytochrome b₅ fraction, by C8 reverse phase HPLC, resulted in a cytochrome b₅ fraction with a specific content of 51.8 nmol/mg protein and an apparent molecular mass of 19.7 kDa by SDS-PAGE. The anion exchange HPLC fraction containing the cytochrome P-450 reductase activity was further purified by NADP-agarose affinity chromatography. This step yielded cytochrome P-450 reductase with an apparent molecular mass of 72 kDa.     

Manganese substrate complexes of phosphofructokinase studies by pulsed magnetic resonance

The formation of binary, ternary, and quaternary complexes between phosphofructokinase, manganese, and substrates has been demonstrated by use of pulsed nuclear magnetic resonance techniques. A Scatchard plot of the interaction of manganese with phosphofructokinase as determined by electron paramagnetic resonance shows two types of manganese binding sites. Phosphofructokinase seems to contain one or two of the metal binding sites with K_d = 20 μm and ?_b ≦ 4, and perhaps, as many as 14 binding sites with K_d ~ 0.8 mm and ?_b ≦ 12 ± 2 per enzyme. Addition of ATP or ADP results in a further enhancement of the relaxation rate indicating ternary complex formation. The concentration of ATP and ADP which results in half maximal change of enhancement is 30–100 μm and 80 μm, respectively. No change in the water proton relaxation rate was detected upon addition of fructose-6-P or fructose-1,6-bisphosphate. A quaternary complex was detected by proton relaxation measurements upon addition of fructose-6-P to a reaction mixture containing β, γ-methylene ATP, manganese, and enzyme with 50 μm fructose-6-P required to obtain the half maximal observed effect. This evidence for a quaternary complex is consistent with a sequential reaction mechanism for phosphofructokinase.     

Crystal structure of a bacterial protein proteinase inhibitor (Streptomyces subtilisin inhibitor) at 2.6 A resolution

The crystal structure of a bacterial protein proteinase inhibitor (Streptomyces subtilisin inhibitor) was solved at 2·6 Å resolution. Each subunit of the dimeric inhibitor has a five-stranded antiparallel β-sheet and two short α-helices. The subunit-subunit interface formed by a stack of two β-sheets provided by the two subunits resembles the dimer-dimer interface of concanavalin A. Conformation of the reactive site around the scissible bond Met73-Val74 seems very rigid. Between bovine pancreatic trypsin inhibitor (Kunitz) and the Streptomyces inhibitor, the reactive site conformations are almost identical with each other from the P₂ to P₂′ residues, while between the soybean trypsin inhibitor (Kunitz) and the Streptomyces inhibitor they are similar from the P₂ to P₁′ residues. There are overall similarities in conformation extending from the P₃ to P₂′ residues between the Streptomyces inhibitor and a hypothetical substrate presumed (Robertus et al., 1972b) to be bound to subtilisin BPN′ in a productive binding mode. Apart from the reactive site, there seems to be no structural relationship among the Streptomyces, bovine pancreatic and soybean inhibitors, suggesting their convergent evolution from separate ancestral proteins.     

Limitations of Augmentation Index in the Assessment of Wave Reflection in Normotensive Healthy Individuals

Objectives

Augmentation index (AIx) is widely used as a measure of wave reflection. We compared the relationship between AIx and age, height and sex with ‘gold standard’ measures of wave reflection derived from measurements of pressure and flow to establish how well AIx measures wave reflection.

Materials and Methods

Measurements of carotid pressure and flow velocity were made in the carotid artery of 65 healthy normotensive individuals (age 21–78 yr; 43 male) and pulse wave analysis, wave intensity analysis and wave separation was performed; waveforms were classified into type A, B or C. AIx, the time of the first shoulder (T_s), wave reflection index (WRI) and the ratio of backward to forward pressure (P_b/P_f) were calculated.

Results

AIx did not correlate with log WRI or P_b/P_f. When AIx was restricted to positive values AIx and log WRI were positively correlated (r = 0.33; p = 0.04). In contrast log WRI and P_b/P_f were closely correlated (r = 0.66; p<0.001). There was no correlation between the T_s and the timing of P_b or the reflected wave identified by wave intensity analysis. Wave intensity analysis showed that the morphology of type C waveforms (negative AIx) was principally due to a forward travelling (re-reflected) decompression wave in mid-systole. AIx correlated positively with age, inversely with height and was higher in women. In contrast log WRI and P_b/P_f showed negative associations with age, were unrelated to height and did not differ significantly by gender.

Conclusions

AIx has serious limitations as a measure of wave reflection. Negative AIx values derived from Type C waves should not be used as estimates of wave reflection magnitude.