Ferdinando Capuano

Lipid peroxidation products and antioxidant enzymes in red blood cells during normal and diabetic pregnancy.

Oxygen free radicals produced during normal aerobic metabolism have been implicated in several pathophysiological mammalian processes. The importance of free radical-mediated fatty acid oxidation has received much attention. The generation of active oxygen species may lead to lipid peroxidation and formation of reactive products, which may be involved in severe damage of cell molecules and structures. Free radical metabolism in pregnancy and in diabetes mellitus is still unclear. To add new insights to the question, changes in lipid peroxidation products and activities of three antioxidant enzymes: catalase (CAT), glutathione peroxidase (GPX) and superoxide dismutase (SOD) in maternal red blood cells haemolysates were evaluated in pregnant women with insulin-dependent diabetes mellitus (IDDM-PW) and in healthy pregnant women (HPW). Healthy non-pregnant women were the control group for IDDM-PW and HPW, respectively. Pregnancy provoked an increase of lipoperoxidation products and an high SOD activity since early pregnancy, while CAT and GPX activities did not change during gestation. IDDM-PW showed higher content of lipoperoxidation breakdown products and lower SOD activity at each trimester, if compared with HPW; moreover, a slight increase of CAT and SOD activity is reported during late diabetic pregnancy. IDDM-PW were in very good metabolic control at time of sampling. The variations reported suggest an easier membrane lipoperoxidability and, consequently, an easier membrane damage during diabetic gestation.

Ultrastructural zonal heterogeneity of hepatocytes and mitochondria within the hepatic acinus during liver regeneration after partial hepatectomy.

Background information. Partial hepatectomy (70%) induces cell proliferation until the original mass of the liver is restored. In the first 24 h after partial hepatectomy, drastic changes in the metabolism of the remaining liver have been shown to occur. To evaluate changes in hepatocyte ultrastructure within the hepatic acinus during the liver regenerative process, we investigated, by light and electron microscopy observations on specimens taken 0 h, 24 h and 96 h after partial hepatectomy, the hepatocyte structure and ultrastructure in the periportal and pericentral area of the hepatic acinus, with a particular emphasis on mitochondria ultrastructure. Moreover, some biochemical events that could affect the mitochondria ultrastructure and function were investigated.

The H+/O stoicheiometry of mitochondrial respiration.

Mitochondrial respiration is compulsorily linked to proton ejection [ 1,2]. The mechanism by which transmembraneAcH+isgeneratedis, however,unknown [3]. Essential for exploring this issue is knowledge of the quantitative relationship between proton translocation and electron transport. Numerous determinations in mitochondria and bacteria produced a stoicheiometry of 2 H’ translocated for 2 etraversing an effective protonmotive redox loop, or energy conserving site of respiratory chain (reviewed [1,2,4]). Recently, however, the H’/2 eand H’/ATP stoicheiometries have been the subject of much controversy [2,5-91. Thus contrasting results have been reported, which indicate that in mitochondria the H+/O quotient for succinate or quinolrespiration(sites2 t 3) is either 4 [1,7,10-141, or 6 [6,8,15],or 8 [5,9,16,17]. We present here accurate spectrophotometric determination of the rate of respiration with hemoglobin and potentiometric determination of the acidification of the extramitochondrial space, which unequivocally show that the H’/O quotient for oxidation of succinate in mitochondria is, at neutral pH, 4.

The monocarboxylate carrier from rat liver mitochondria: Purification and kinetic characterization in a reconstituted system

The monocarboxylate (pyruvate) carrier was extracted from rat liver mitochondria with Triton X-100 in the presence of asolectin and partially purified by chromatography on HTP. The HTP eluate reconstituted in liposomes was shown to catalyze active pyruvatein/acetoacetateout, and acetoacetatein/pyruvateout counter-exchange. Kinetic characterization of the reconstituted pyruvate carrier was achieved by an original spectrophotometric method consisting of determination of substrate release from proteoliposomes with a coupled enzymatic assay.The monocarboxylate (pyruvate) carrier was extracted from rat liver mitochondria with Triton X‐100 in the presence of asolectin and partially purified by chromatography on HTP. The HTP eluate reconstituted in liposomes was shown to catalyze active pyruvatein/acetoacetateout, and acetoacetatein/pyruvateout counter‐exchange. Kinetic characterization of the reconstituted pyruvate carrier was achieved by an original spectrophotometric method consisting of determination of substrate release from proteoliposomes with a coupled enzymatic assay.

Spectrophotometric determination with hemoglobin of the rate of oxygen consumption in mitochondria

The study of respiratory processes and energy transduction in mitochondria or other respiring preparations often requires analysis of the kinetics of oxygen consulnption and of the quantitative relationship between the rates of oxygen utilization, ATP synthesis and ion transport [l-3]. Accurate determination of the rate of oxygen consurllption presents technical dif~culties when dealing with rapid, transient changes of the respiratory activity, as induced by addition to the respiring material of oxygen, reductants, ADP. Pi and cations. In fact the polarographic method which is generally used to measure oxygen consumption may be inadequate to measure accurately rapid changes of respiratory rates [2,4], as judged from its intrinsic response-time characteristics [S&j. This paper reports the application and suitability of a spectrophotometric method with hemoglobin [7,X] for accurate determination of initial rates of oxygen consunlption during rapid functional transitions of r-espiratory systems. Oxyhemoglobin is used both as oxygen donor and as indicator of respiration, taking advantage of the specific absorbance changes which it undergoes upon deoxygenation.

Ursodeoxycholate promotes protein phosphorylation in the cytosol of rat hepatocytes.

A study is presented of the effect of the bile salt ursodeoxycholate (UDC) on protein phosphorylation by [γ‐32P]ATP in the cytosol from ra hepatocytes. Gel electrophoresis and corresponding autoradiograms of cytosolic proteins show that UDC promotes phosphorylation of at least eight different protein bands. Four of them (the 36, 60, 64 and 76 kDa) are phosphorylated by Ca2+ and phospholipid‐dependent protein kinase (PKC); three (the 31, 51 and 71 kDa) are phosphorylated by cAMP‐dependent protein kinase (PKA) and one protein band, with molecular weight of 34 kDa, apparently contains substrates of both PKC and PKA. Data are reported indicating that UDC can directly affect the intrinsic activity of protein kinases.

The H+‐ATP Synthase of Mitochondria in Tissue Regeneration and Neoplasia

The mitochondrial H+-ATPase complex, H+-ATP synthase (EC 3.6.1.34), is a membrane-associated enzyme that utilizes the electrochemical proton gradient generated by the respiratory chain to produce most of the ATP necessary to the cell. The ATP synthase is structurally and functionally organized in three parts: the catalytic sector, or F,,’ which catalyzes ATP synthesis or ATP hydrolysis; the membrane sector, or F,: which functions as a transmembrane proton translocator during the catalytic cycle of the enzyme; and a stalk, which connects F, and F, and is involved in the coupling between chemical catalysis and proton translocation. The subunit composition of the ATP synthase is complex, the Escherichia coli enzyme being the simplest defined so far. This prokaryotic complex appears to be composed of eight polypeptide subunit^.^ Five of them, a, 8, y, 6 and c subunits, in the F, sector and three, a, b and c subunits, in the F, sector. On the other hand, the subunit composition of mammalian ATP synthase is not yet established. It has been reported, in fact, that the bovine heart enzyme contains between 14 and 15 different polypept ide~ .~ , ’ The F, moiety of mammalian ATP synthase has an overall composition and organization similar to that of prokaryotic F,. The subunit composition of F, remains unclear, however. The mammalian F, contains subunits that have no counterparts in Escherichia coli. Among these, there are the inhibitor protein that binds to &FI6 and Factor 6, which is involved in binding of F, to F,.’ The genes for the Escherichia coli enzyme are located in the unc or atp operon,* whereas the subunits of the mammalian enzyme are encoded partly by nuclear and partly by mitochondrial genes. Thus, normal biosynthesis of ATP synthase in eukaryotes requires a concerted expression of genes located in two different genomes. The F, subunits: OSCP, Factor 6, the inhibitor protein,” subunit c, and a subunit of 25-kDa of F, (F,I)” (see also Ref. 12) are encoded by nuclear DNA, whereas two subunits of F,, ATPase 6 and A6L, are the products of mitochondrial genes.I3 There appear to be two genes for subunit c ,~~*‘’ and more pseudogenes for OSCP,’o which may be differently expressed in various mammalian tissues. This study concerns the alteration of structure and function of the mitochondrial H+-ATPase complex that can be observed in liver regeneration and in Morris hepatoma 3924% a rapidly growing tumor, poorly differentiated and characterized by a high rate of aerobic glycolysis.

The mitochondrial ATP synthase in normal and neoplastic cell growth

Rapidly growing cancer cells have a reduced number of mitochondria, increased glycolytic activity and a shift from respiratory to fermentative ATP supply which can cover 50% or more of their energy requirement (Nakashima et al., 1984). This phenotype of cancer cells raises a number of questions. Which are the characteristics and the causes of the changes in the energy metabolism? Do they provide cancer cells with an advantage for their growth? If so, is there a possibility to control or suppress neoplastic cell growth by affecting their energy metabolism?

Bile acids, cell proliferation and protein phosphorylation

Bile acids are endogenous compounds with amphiphilic properties normally present in human body fluid. Cholic and chenodeoxycholic acids, the so-called “primary” bile acids, are synthesised in the liver from cholesterol. Under normal physiological conditions, 90–95% of these bile acids are then conjugated in the liver with glycine or taurine and secreted to the duodenum. Here, they facilitate lipid absorption and participate in a complex enterohepatic circulation. Cholic and chenodeoxycholic acids can also reach the colon where they are converted by the bacterial enzyme 7α-dehydroxylase, to the “secondary” bile acids, deoxycholic and lithocholic acids respectively.

Structural and Functional Alterations of Mitochondrial FoF1-ATPsynthase in Various Pathophysiological States

The structure and function of mitochondrial FOF1 ATPsynthase in rat liver regeneration, heart of senescent rats and in rapidly growing Morris hepatoma 3924A have been studied. In the Morris hepatoma 3924A, the ATPase activity exhibited a Km for ATP considerably higher than in normal rat liver. In rat liver 24 h after partial hepatectomy and in heart of aged rats (24 month old rats) the Vmax for ATP hydrolase activity decreased and the oligomycin sensitive proton conductivity, in vesicles of the inner mitochondrial membrane, increased.