Showing papers in "Biochemical Journal in 1993"

New families in the classification of glycosyl hydrolases based on amino acid sequence similarities.

Abstract: 301 glycosyl hydrolases and related enzymes corresponding to 39 EC entries of the I.U.B. classification system have been classified into 35 families on the basis of amino-acid-sequence similarities [Henrissat (1991) Biochem. J. 280, 309-316]. Approximately half of the families were found to be monospecific (containing only one EC number), whereas the other half were found to be polyspecific (containing at least two EC numbers). A > 60% increase in sequence data for glycosyl hydrolases (181 additional enzymes or enzyme domains sequences have since become available) allowed us to update the classification not only by the addition of more members to already identified families, but also by the finding of ten new families. On the basis of a comparison of 482 sequences corresponding to 52 EC entries, 45 families, out of which 22 are polyspecific, can now be defined. This classification has been implemented in the SWISS-PROT protein sequence data bank.

Protein kinase C isoenzymes: divergence in signal transduction?

Abstract: The development and life-time of multicellular eukaryotic organisms represents a complex interplay of numerous proliferation and differentiation events that proceed in a highly ordered manner. As a prerequisite for those events, cells must respond to extracellular signals with a specific set of mechanisms that regulate or modulate gene expression. Between the signal and the gene, a system of rather different cellular components is assembled to guarantee a specific and successful process of signal transduction. Pathways of signal transduction, though differing remarkably in their complexity and in the use of cellular components, seem to obey certain principles which are evolutionarily conserved and ubiquitously distributed amongst living organisms. Extracellular signals, so-called ligands, either penetrate the cellular membrane or bind to the extracellular domain of receptors. Activated receptors as such, or in association with socalled transducers, are capable of activating effectors-either directly or by means of changing the amount or intracellular distribution of so-called second messengers. These second

Wortmannin is a potent phosphatidylinositol 3-kinase inhibitor: the role of phosphatidylinositol 3,4,5-trisphosphate in neutrophil responses

Abstract: Phosphatidylinositol 3,4,5-trisphosphate (PtdInsP3) is rapidly produced upon exposure of neutrophils to the chemoattractant N-formylmethionyl-leucylphenylalanine (fMLP), and has been proposed to act as a second messenger mediating actin polymerization and respiratory-burst activity. Here we present evidence that wortmannin, a known inhibitor of respiratory-burst activity, acts on PtdIns 3-kinase, the enzyme producing PtdInsP3 from PtdIns(4,5)P2. Pretreatment of 32P-labelled human neutrophils with 100 nM wortmannin totally abolished fMLP-mediated PtdInsP3 production, raised PtdInsP2 levels, and did not affect cellular PtdInsP and PtdIns contents. The inhibitory effect on PtdInsP3 formation in intact cells was dose-dependent, with an IC50 of approximately 5 nM. Similar results were obtained with PtdIns 3-kinase immunoprecipitated by antibodies against the p85 regulatory subunit: wortmannin totally inhibited PtdIns3P production in immunoprecipitates at concentrations of 10-100 nM (IC50 approximately 1 nM). These results illustrate the direct and specific inhibition of PtdIns 3-kinase by wortmannin. Since agonist-mediated respiratory-burst activation is most sensitive to wortmannin (IC50 = 12 nM), this suggests that agonist-mediated PtdInsP3 formation is indispensable for this cell response. Neutrophils pretreated with wortmannin develop oscillatory changes in F-actin content, but actin polymerization in response to fMLP is not inhibited. This, and the absence of PtdInsP3 under these conditions, are in agreement with a modulatory role for PtdInsP3 in cytoskeletal rearrangements, but imply that PtdInsP3 production is not a primary event triggering elongation of actin filaments in neutrophils.

Isoprenoid biosynthesis in bacteria: a novel pathway for the early steps leading to isopentenyl diphosphate.

Abstract: Incorporation of 13C-labelled glucose, acetate, pyruvate or erythrose allowed the determination of the origin of the carbon atoms of triterpenoids of the hopane series and/or of the ubiquinones from several bacteria (Zymomonas mobilis, Methylobacterium fujisawaense, Escherichia coli and Alicyclobacillus acidoterrestris) confirmed our earlier results obtained by incorporation of 13C-labelled acetate into the hopanoids of other bacteria and led to the identification of a novel biosynthetic route for the early steps of isoprenoid biosynthesis. The C5 framework of isoprenic units results most probably (i) from the condensation of a C2 unit derived from pyruvate decarboxylation (e.g. thiamine-activated acetaldehyde) on the C-2 carbonyl group of a triose phosphate derivative issued probably from dihydroxyacetone phosphate and not from pyruvate and (ii) from a transposition step. Although this hypothetical biosynthetic pathway resembles that of L-valine biosynthesis, this amino acid or its C5 precursors could be excluded as intermediates in the formation of isoprenic units.

Evolutionary families of peptidases

Abstract: The available amino acid sequences of peptidases have been examined, and the enzymes have been allocated to evolutionary families. Some of the families can be grouped together in 'clans' that show signs of distant relationship, but nevertheless, it appears that there may be as many as 60 evolutionary lines of peptidases with separate origins. Some of these contain members with quite diverse peptidase activities, and yet there are some striking examples of convergence. We suggest that the classification by families could be used as an extension of the current classification by catalytic type.

Inactivation of glycogen synthase kinase-3 beta by phosphorylation: new kinase connections in insulin and growth-factor signalling.

Abstract: The beta-isoform of glycogen synthase kinase-3 (GSK3 beta) isolated from rabbit skeletal muscle was inactivated 90-95% following incubation with MgATP and either MAP kinase-activated protein kinase-1 (MAPKAP kinase-1, also termed RSK-2) or p70 S6 kinase (p70S6K), and re-activated with protein phosphatase 2A. MAPKAP kinase-1 and p70S6K phosphorylated the same tryptic peptide on GSK3 beta, and the site of phosphorylation was identified as the serine located nine residues from the N-terminus of the protein. The inhibitory effect of Ser-9 phosphorylation on GSK3 beta activity was observed with three substrates, (inhibitor-2, c-jun and a synthetic peptide), and also with glycogen synthase provided that 0.15 M KCl was added to the assays. The results suggest that Ser-9 phosphorylation underlies the reported inhibition of GSK3 beta by insulin and that GSK3 may represent a point of convergence of two major growth-factor-stimulated protein kinase cascades.

The glucose transporter family: structure, function and tissue-specific expression.

Abstract: possess a transport system for glucose of the facilitative diffusion type; these transporters allow the movement of glucose across the plasma membrane down its chemical gradient either into or out of cells. These transporters are specific for the D-enantiomer of glucose and are not coupled to any energy-requiring components, such as ATP hydrolysis or a HI gradient [1]. The facilitative glucose transporters are distinct from the Na+dependent transporters, which actively accumulate glucose [2,3]. The importance of glucose as a cellular metabolite has led to

The bioactive phospholipid lysophosphatidic acid is released from activated platelets.

Abstract: Lysophosphatidic acid (LPA) is a water-soluble phospholipid with hormone-like and growth-factor-like activities. LPA activates a putative G-protein-coupled receptor in responsive cells, but the natural source of exogenous LPA is unknown. Here we show that LPA is present in mammalian serum in an active form (bound to albumin) at concentrations of 1-5 microM, but is not detectable in platelet-poor plasma, suggesting that LPA is produced during blood clotting. We find that thrombin activation of platelets prelabelled with [32P]Pi results in the rapid release of newly formed [32P]LPA into the extracellular environment. We conclude that LPA is a novel platelet-derived lipid mediator that may play a role in inflammatory and proliferative responses to injury.

Osteoblast-specific factor 2: cloning of a putative bone adhesion protein with homology with the insect protein fasciclin I.

Abstract: A cDNA library prepared from the mouse osteoblastic cell line MC3T3-E1 was screened for the presence of specifically expressed genes by employing a combined subtraction hybridization/differential screening approach. A cDNA was identified and sequenced which encodes a protein designated osteoblast-specific factor 2 (OSF-2) comprising 811 amino acids. OSF-2 has a typical signal sequence, followed by a cysteine-rich domain, a fourfold repeated domain and a C-terminal domain. The protein lacks a typical transmembrane region. The fourfold repeated domain of OSF-2 shows homology with the insect protein fasciclin I. RNA analyses revealed that OSF-2 is expressed in bone and to a lesser extent in lung, but not in other tissues. Mouse OSF-2 cDNA was subsequently used as a probe to clone the human counterpart. Mouse and human OSF-2 show a high amino acid sequence conservation except for the signal sequence and two regions in the C-terminal domain in which 'in-frame' insertions or deletions are observed, implying alternative splicing events. On the basis of the amino acid sequence homology with fasciclin I, we suggest that OSF-2 functions as a homophilic adhesion molecule in bone formation.

Studies on the inhibitory mechanism of iodonium compounds with special reference to neutrophil NADPH oxidase.

Abstract: Diphenyleneiodonium (DPI) and its analogues have been previously shown to react via a radical mechanism whereby an electron is abstracted from a nucleophile to form a radical, which then adds back to the nucleophile to form covalent adducts [Banks (1966) Chem. Rev. 66, 243-266]. We propose that the inhibition of neutrophil NADPH oxidase by DPI occurs via a similar mechanism. A reduced redox centre in the oxidase could serve as electron donor to DPI, and inhibition would occur after direct phenylation of the redox cofactor, or of adjacent amino acid groups by the DPI radical. In the absence of an activatory stimulus, human neutrophil NADPH-oxidase was not inhibited by DPI. The Ki for time-dependent inhibition by DPI of human neutrophil membrane NADPH oxidase was found to be 5.6 microM. Inhibitory potency of DPI was shown to be directly related to rate of enzyme turnover, indicating the need for a reduced redox centre. Adducts were formed between photoreduced flavin (FAD or FMN) and inhibitor (DPI or diphenyliodonium). These were separated by h.p.l.c. and characterized by absorbance spectroscopy, 1H-n.m.r. and fast-atom-bombardment m.s. and found to have properties consistent with substituted 4a,5-dihydroflavins. After incubation of pig neutrophil membranes with DPI, the quantity of recoverable intact flavin was greatly diminished when NADPH was present to initiate oxidase turnover, indicating that the flavin may be the site of DPI activation. These results may provide a common mechanism of action for iodonium compounds as inhibitors of other flavoenzymes.

Isoenzyme specificity of bisindolylmaleimides, selective inhibitors of protein kinase C

Abstract: The protein kinase C (PKC) family of isoenzymes is believed to mediate a wide range of signal-transduction pathways in many different cell types. A series of bisindolylmaleimides have been evaluated as inhibitors of members of the conventional PKC family (PKCs-alpha, -beta, -gamma) and of a representative of the new, Ca(2+)-independent, PKC family, PKC-epsilon. In contrast with the indolocarbazole staurosporine, all the bisindolylmaleimides investigated showed slight selectivity for PKC-alpha over the other isoenzymes examined. In addition, bisindolylmaleimides bearing a conformationally restricted side-chain were less active as inhibitors of PKC-epsilon. Most noticeable of these was Ro 32-0432, which showed a 10-fold selectivity for PKC-alpha and a 4-fold selectivity for PKC-beta I over PKC-epsilon.

Angiotensin I-converting enzyme in human circulating mononuclear cells: genetic polymorphism of expression in T-lymphocytes.

Abstract: The expression of angiotensin-I converting enzyme (ACE; EC 3.4.15.1) in human circulating mononuclear cells was studied. T-lymphocytes contained the highest level of enzyme, approx. 28 times more per cell than monocytes. No activity was detected in B-lymphocytes. ACE was present mainly in the microsomal fraction, where it was found to be the major membrane-bound bradykinin-inactivating enzyme. An mRNA for ACE was detected and characterized after reverse transcription and amplification by PCR in T-lymphocytes and several T-cell leukaemia cell lines. We have previously observed that the interindividual variability in the levels of ACE in plasma is, in part, genetically determined and influenced by an insertion/deletion polymorphism of the ACE gene. To investigate the mechanisms involved in the regulation of ACE biosynthesis, the ACE levels of T-lymphocytes from 35 healthy subjects having different ACE genotypes were studied. These levels varied widely between individuals but were highly reproducible and influenced by the polymorphism of the ACE gene. T-lymphocyte levels of ACE were significantly higher in subjects who were homozygote for the deletion than in the other subjects. These results show that ACE is expressed in T-lymphocytes and indicate that the level of ACE expression in cells synthesizing the enzyme is genetically determined.

Correlation of apoptosis with change in intracellular labile Zn(II) using zinquin [(2-methyl-8-p-toluenesulphonamido-6-quinolyloxy)acetic acid], a new specific fluorescent probe for Zn(II)

Abstract: Zinquin [(2-methyl-8-p-toluenesulphonamido-6-quinolyloxy)-acetic acid], a membrane-permeant fluorophore specific for Zn(II), was used with spectrofluorimetry and video image analysis to reveal and quantify labile intracellular Zn. Zinquin labelled human chronic-lymphocytic-leukaemia lymphocytes, rat splenocytes and thymocytes with a weak diffuse fluorescence that was quenched when intracellular Zn was chelated with NNN'N'-tetrakis-(2-pyridylmethyl)ethylenediamine (TPEN) and was greatly intensified by pretreatment of cells with the Zn ionophore pyrithione and exogenous Zn. There was substantial heterogeneity of labile Zn among ionophore-treated cells, and fluorescence was largely extranuclear. The average contents of labile Zn in human leukaemic lymphocytes, rat splenocytes and rat thymocytes were approx. 20, 31 and 14 pmol/10(6) cells respectively. Morphological changes and internucleosomal DNA fragmentation indicated substantial apoptosis in these cells when the level of intracellular labile Zn was decreased by treatment with TPEN. Conversely, increasing labile Zn by pretreatment with Zn plus pyrithione suppressed both spontaneous DNA fragmentation and that induced by the potent apoptosis-induced agents colchicine and dexamethasone. These results suggest that prevention of apoptosis is a function of labile Zn, and that a reduction below a threshold concentration in this Zn pool induces apoptosis.

Glycosaminoglycans and the regulation of blood coagulation.

Abstract: Blood coagulation involves the sequential activation of a series of serine proteinases, which culminates in the generation of thrombin and subsequent thrombin-catalysed conversion of fibrinogen into insoluble fibrin (Furie and Furie, 1988). Inhibitory modulation of this process, of paramount physiological importance, is primarily achieved by two principally different mechanisms (Figure 1). The enzymes may be inactivated by serine proteinase inhibitors (known as 'serpins'), which act by formation ofstable 1 :1 molar complexes with their target enzymes (Travis and Salvesen, 1983). Alternatively, the so-called protein C pathway leads to inactivation of auxiliary coagulation proteins (factors V. and VIII.) by cleavage at distinct sites (Esmon, 1989; Dahlbaick, 1991). The prime site of regulation is the surface of vascular endothelial cells, which have been known to possess anticoagulant properties (Colburn and Buonassisi, 1982). These properties are particularly conspicuous in the microcirculation, with its high wall surface to blood volume ratio (Busch, 1984). The ability of certain sulphated polysaccharides, glycosaminoglycans, to interfere with blood coagulation has a long-standing record, as illustrated by the extensive clinical use of heparin as an antithrombotic agent (see Roden, 1989). The main effect of heparin (and of its relative, heparan sulphate) is to accelerate the inactivation of coagulation enzymes by the serpin antithrombin (Rosenberg, 1977; Bjork and Lindahl, 1982). A more complex picture emerged with the finding of an additional serpin, heparin cofactor II, which is 'activated' not only by heparin, but also by another glycosaminoglycan, dermatan sulphate, and which selectively inactivates thrombin (Tollefsen et al., 1982; Tollefsen, 1989). Remarkably, also the other major regulatory mechanism, the protein C pathway, involves a glycosaminoglycan-containing molecular species, since the protein C activation cofactor, thrombomodulin, turned out to be a proteoglycan with a functionally important, covalently bound glycosaminoglycan chain (Bourin and Lindahl, 1990; Bourin et al., 1990). In this Review we attempt to summarize our current understanding of glycosaminoglycan involvement in the regulation of blood coagulation.

Platelet-activating factor: receptors and signal transduction

Abstract: During the past two decades, studies describing the chemistry and biology of PAF have been extensive. This potent phosphoacylglycerol exhibits a wide variety of physiological and pathophysiological effects in various cells and tissues. PAF acts, through specific receptors and a variety of signal transduction systems, to elicit diverse biochemical responses. Several important future directions can be enumerated for the characterization of PAF receptors and their attendant signalling mechanisms. The recent cloning and sequence analysis of the gene for the PAF receptor will allow a number of important experimental approaches for characterizing the structure and analysing the function of the various domains of the receptor. Using molecular genetic and immunological technologies, questions relating to whether there is receptor heterogeneity, the precise mechanism(s) for the regulation of the PAF receptor, and the molecular details of the signalling mechanisms in which the PAF receptor is involved can be explored. Another area of major significance is the examination of the relationship between the signalling response(s) evoked by PAF binding to its receptor and signalling mechanisms activated by a myriad of other mediators, cytokines and growth factors. A very exciting recent development in which PAF receptors undoubtedly play a role is in the regulation of the function of various cellular adhesion molecules. Finally, there remain many incompletely characterized physiological and pathophysiological situations in which PAF and its receptor play a crucial signalling role. Our laboratory has been active in the elucidation of several tissue responses in which PAF exhibits major autocoid signalling responses, e.g. hepatic injury and inflammation, acute and chronic pancreatitis, and cerebral stimulation and/or trauma. As new experimental strategies are developed for characterizing the fine structure of the molecular mechanisms involved in tissue injury and inflammation, the essential role of PAF as a primary signalling molecule will be affirmed. Doubtless the next 20 years of experimental activity will be even more interesting and productive than the past two decades.

Glycogen synthase kinase-3 is rapidly inactivated in response to insulin and phosphorylates eukaryotic initiation factor eIF-2B.

Abstract: We have studied the control of insulin-regulated protein kinases in Chinese hamster ovary cells transfected with the human insulin receptor (CHO.T cells). Among these enzymes is one that is obtained after chromatography of cell extracts on Mono-S, whose activity is decreased (7.3 +/- 1.9-fold) within 10 min of insulin treatment. This enzyme phosphorylates glycogen synthase and the largest subunit of protein synthesis eukaryotic initiation factor (eIF)-2B (the guanine nucleotide exchange factor). The kinase appears to be glycogen synthase kinase-3 (GSK-3), on the basis of: (1) its ability to phosphorylate a peptide based on the phosphorylation sites for GSK-3 in glycogen synthase, and (2) the finding that the fractions possessing this activity contain immunoreactive GSK-3, whose peak is coincident with that of kinase activity, as judged by immunoblotting using antibodies specific for the alpha- and beta-isoforms of GSK-3. The decrease in kinase activity induced by insulin was reversed by treatment of the column fractions with protein phosphatase-2A. These data indicate that insulin rapidly causes inactivation of GSK-3 and that this is due to phosphorylation of GSK-3. The implications of these findings for the control of glycogen and protein metabolism are discussed.

Regulation of matrix metalloproteinase expression in human vein and microvascular endothelial cells. Effects of tumour necrosis factor alpha, interleukin 1 and phorbol ester.

Abstract: Matrix metalloproteinases (MMPs) play a role in tissue remodelling and angiogenesis. We have investigated the expression and regulation of MMP-1 (interstitial collagenase), MMP-2 (gelatinase A), MMP-3 (stromelysin 1), MMP-7 (matrilysin), MMP-9 (gelatinase B) and their inhibitors TIMP-1 and TIMP-2 in human umbilical vein, femoral vein and microvascular endothelial cells, and compared these data with those obtained with human synovial fibroblasts. Non-stimulated vein endothelial cells expressed the mRNAs for MMP-1, MMP-2, TIMP-1 and TIMP-2. MMP-3 mRNA and protein were undetectable or only weakly expressed, but could be stimulated by the inflammatory mediator tumour necrosis factor alpha (TNF alpha). The expression of MMP-3 and MMP-1 was further enhanced by phorbol 12-myristate 13-acetate (PMA). Phorbol ester also induced TIMP-1 and MMP-9, the expression of the latter being further enhanced by TNF alpha or interleukin 1 alpha (IL-1 alpha). Similar stimulatory effects were observed in microvascular endothelial cells. Hence the inflammatory mediator TNF alpha induces/enhances the production of several matrix metalloproteinases in human endothelial cells. On the other hand, MMP-2 and TIMP-2 were not affected or were affected in a variable way by TNF alpha and/or phorbol ester, suggesting a dissimilar regulation of these proteins. The cyclic AMP-enhancing agent forskolin affected the production of MMPs in a cell-type-specific way. In human vein endothelial cells it enhanced the PMA-mediated induction of MMP-9, whereas it suppressed this induction in human microvascular endothelial cells and in synovial fibroblasts. On the other hand, forskolin suppressed the PMA-mediated induction of MMP-1 and MMP-3 in synovial fibroblasts, while it enhanced or did not affect this induction in various types of human endothelial cells. These observations may have implications for future pharmacological intervention in angiogenesis.

Oxidative stress and ageing in Caenorhabditis elegans.

Abstract: Mutations in the age-1 gene double both the mean and maximum life span of Caenorhabditis elegans. They also result in an age-specific increase of catalase and Cu/Zn superoxide dismutase activity levels. The higher superoxide dismutase activity levels in age-1 mutants confer hyperresistance to the superoxide-anion-generating drug paraquat. The rate of superoxide anion production by microsome fractions declines linearly with age in age-1(+) worms, but, after an initial decline, is stabilized at a higher level in senescent age-1 mutant nematodes. These results clearly show that oxidative stress resistance and potential life span are correlated in this organism, and they suggest that the natural product of age-1 either directly or indirectly downregulates the activities of several other genes as a function of age.

Proteasomes: multicatalytic proteinase complexes

Abstract: The multicatalytic proteinase complex (proteasome) is a highmolecular-mass (approximately 700 kDa) intracellular proteinase which has been isolated under a variety of different names from a wide variety of eukaryotic cells and tissues (reviewed, Rivett, 1989a; Orlowski, 1990). The proteinase complex is composed of at least 24 subunits which include many different polypeptides arranged in a cylindrical structure. Other multi-subunit complexes with cylindrical structures include 'prosomes', that are widely distributed 19 S ribonucleoprotein particles which were thought to be involved in the control of translation (Schmid et al., 1984; Martins de Sa et al., 1986), erythrocyte cylindrin (Harris, 1988), and a number of other partially characterized 16-22 S cylindrical particles. 'Prosomes' and certain related particles have been shown to have proteolytic activity and to share antigenic cross-reactivity with the multicatalytic proteinase (Falkenburg et al., 1988) and are therefore believed to be the same. The particles are most often referred to either as multicatalytic proteinase complexes (Dahlmann et al., 1988; Orlowski and Wilk, 1988) or, more recently, as proteasomes (Arrigo et al., 1988) and their properties are broadly similar irrespective of the

N.m.r. and molecular-modelling studies of the solution conformation of heparin

Abstract: The solution conformations of heparin and de-N-sulphated, re-N-acetylated heparin have been determined by a combination of n.m.r. spectroscopic and molecular-modelling techniques. The 1H- and 13C-n.m.r. spectra of these polysaccharides have been assigned. Observed 1H-1H nuclear Overhauser enhancements (n.O.e.s) have been simulated using the program NOEMOL [Forster, Jones and Mulloy (1989) J. Mol. Graph. 7, 196-201] for molecular models derived from conformational-energy calculations; correlation times for the simulations were chosen to fit experimentally determined 13C spin-lattice relaxation times. In order to achieve good agreement between calculated and observed 1H-1H n.O.e.s it was necessary to assume that the reorientational motion of the polysaccharide molecules was not isotropic, but was that of a symmetric top. The resulting model of heparin in solution is similar to that determined in the fibrous state by X-ray-diffraction techniques [Nieduszynski, Gardner and Atkins (1977) Am. Chem. Soc. Symp. Ser. 48, 73-80].

Peroxynitrite-induced luminol chemiluminescence

Abstract: Vascular endothelial cells, smooth muscle cells, macrophages, neutrophils, Kupffer cells and other diverse cell types generate superoxide (O2.-) and nitric oxide (.NO), which can react to form the potent oxidant peroxynitrite anion (ONOO-). Peroxynitrite reacted with luminol to yield chemiluminescence which was greatly enhanced by bicarbonate. The quantum chemiluminescence yield of the ONOO- reaction with luminol in bicarbonate was approx. 10(-3). Chemiluminescence was superoxide dismutase-inhibitable, indicating that O2.- was a key intermediate for chemiexcitation. O2.- appears to be formed secondarily to the reaction of a bicarbonate-peroxynitrite complex with luminol, yielding luminol radical and O2.-. Luminol radical reacts with O2.- to form the unstable luminol endoperoxide, which follows the light-emitting pathway. Neither .NO nor O2.- alone were capable of directly inducing significant luminol chemiluminescence in our assay systems. These results suggest that ONOO- can be a critical unrecognized mediator of cell-derived luminol chemiluminescence reported in previous studies. In addition, it is shown that bicarbonate can participate in secondary oxidation reactions after reacting with ONOO-.

Mammalian glucokinase and its gene.

Abstract: Mammalian glucokinase was identified 30 years ago as a distinct form of hexokinase in rat liver. The hexokinases (ATP: hexose 6phosphotransferases, EC 2.7.1.1) constitute a family of evolutionarily and stucturally related enzymes present in eukaryotic cells from yeast to mammals. In the cells of higher organisms, the physiologically significant substrate for these enzymes is Dglucose. The reaction catalysed by the hexokinases, ATP + Dglucose -+ ADP+ D-glucose 6-phosphate, is the first and obligatory step for glucose utilization after transport of the sugar into the cell. Mammalian tissues contain four different hexokinases which can be isolated by conventional protein separation techniques and for which cDNAs have been cloned. The isoenzymes of the rat have been designated hexokinases type I-IV or A-D in order of increasing negative net charge. The subject of this Review is hexokinase type IV or D, usually called glucokinase. Glucokinase stands apart from all the other hexokinases by a number of criteria. The first and most striking is its low affinity for glucose. The enzyme is half-saturated with glucose at 6 mM, compared with Km values in the micromolar range for the three other mammalian hexokinases. This feature led to the discovery of the enzyme and underlies its key role in the physiology of glucose homeostasis. The second hallmark ofmammalian glucokinase is its highly typical tissue distribution. The glucokinase gene is transcribed and the mRNA translated into active enzyme only in hepatocytes and insulin-secreting f-cells of the pancreatic islets of Langerhans, reflecting the great functional specialization of this isoenzyme. A third outstanding feature is the developmental and multihormonal regulation of the enzyme, illustrated most dramatically by the transcriptional induction of the glucokinase gene by insulin in the liver. The distinctive kinetics of glucokinase, its tissue-specific expression and its hormonal regulation were recognized within a few years of the discovery of the enzyme. However, our understanding of these particular characteristics has remained superficial until recently. The main reason for limited progress was the difficulty of purifying the enzyme, hence of raising specific antibodies or obtaining peptide sequence for the isolation of cDNA clones. Once this obstacle was surmounted, studies on glucokinase became a very fertile field of research. The most recent and medically rewarding outcome of this research has been the discovery of mutations of the glucokinase gene as the cause of one subtype of non-insulin-dependent diabetes mellitus (NIDDM). The purpose of this article is to review the recent developments on the structure and function of the glucokinase gene and its gene products, as they relate to our understanding of blood glucose homeostasis. The reader interested in historical perspectives and a complete background on the biochemistry of glucokinase should refer to the classical reviews of Walker [1], Weinhouse [2] and Colowick [3]. Recent commentaries on topical aspects are also available [4-8]. The glucokinase gene was first cloned from the rat and the structure of the gene in this species can serve as the standard of reference (Figure 1 a). The most remarkable feature is the presence of alternative promoters, responsible for the initiation of transcription at different sites on the DNA in hepatic and endocrine cells. The first clue to the existence of cell-type-specific promoters came from the sequences oftwo quasi-full-length cDNAs isolated from rat liver and insulinoma libraries [9,10]. The sequences were essentially identical for more than 2000 nucleotides starting from the 3' ends of the cDNAs, but segments of approximately 100 nucleotides at the 5' ends were found to differ entirely. The 5' specific sequences ofthe cDNAs were mapped to widely separated sites in genomic DNA by Magnuson and co-workers [10,11]. These investigators further identified nine exons, numbered 2-10 in the transcription unit, whose assembly gives rise to the common sequence found in the liver and insulinoma-derived cDNAs. The leader exon encoding the 5' end of the hepatic mRNA, termed exon 1L, was contained in a phage A clone which also carried the common exons 2-4. The leader exon for the 5' end of the insulinoma mRNA, termed exon 1lf in reference to the fl-cells of the islets of Langerhans, was localized in a different phage clone with non-overlapping genomic DNA. It was therefore concluded that the liver-specific exon IL was contiguous to the body of the structural gene, whereas the islet-type exon Ifi was located at an unspecified distance further upstream. The intervening sequence between the two leader exons has yet to be mapped accurately. Several attempts to isolate rat genomic DNA clones for the entire region have remained unsuccessful in my laboratory, perhaps suggesting unusual features of this DNA. In any event, more than 22 kb ofDNA separate the two leader exons in the rat gene (Figure la). The fact that the upstream exon lfl is used exclusively in isletderived cells, and the downstream exon 1L exclusively in liver, was established by primer extension and nuclease protection experiments and further confirmed by reverse transcription and PCR [10]. It should be noted that the two tissue-specific exons 1 of the glucokinase gene specify not only the 5' untranslated regions of the islet and liver mRNAs, but also their initial 45 nucleotides of protein coding sequence. It follows that the rat islet and liver glucokinase enzymes will differ in primary structure by 15 amino acids (including initiator Met) at the N-terminal ends of the molecules, for a total sequence of 465 amino acid residues. In addition to the differential splicing ofleader exons associated with the cell-specific control of transcription initiation, other modes of alternative splicing are known to affect glucokinase transcripts. An optional cassette exon has been identified in the rat gene between the originally described exons 1 L and 2 (Figure la). This cassette exon, termed exon 2A, is retained in a minor fraction of glucokinase mRNA in rat liver [12]. Alternative

Oxidation of low-density lipoprotein with hypochlorite causes transformation of the lipoprotein into a high-uptake form for macrophages

Abstract: Oxidation of low-density lipoprotein (LDL) lipid is thought to represent the initial step in a series of oxidative modification reactions that ultimately transform this lipoprotein into an atherogenic high-uptake form that can cause lipid accumulation in cells. We have studied the effects of hypochlorite, a powerful oxidant released by activated monocytes and neutrophils, on isolated LDL. Exposure of LDL to reagent hypochlorite (NaOCl) at 4 degrees C resulted in immediate and preferential oxidation of amino acid residues of apoprotein B-100, the single protein associated with LDL. Neither lipoprotein lipid nor LDL-associated antioxidants, except ubiquinol-10, represented major targets for this oxidant. Even when high concentrations of NaOCl were used, only low levels of lipid hydroperoxides could be detected with the highly sensitive h.p.l.c. post-column chemiluminescence detection method. Lysine residues of apoprotein B-100 quantitatively represented the major target, scavenging some 68% of the NaOCl added, with tryptophan and cysteine together accounting for an additional 10% of the oxidant. Concomitant with the loss of LDL's amino groups, chloramines were formed and the anionic surface charge of the lipoprotein particle increased, indicated by a 3-4-fold increase in electrophoretic mobility above that of native LDL on agarose gels. While both these changes could be initially reversed by physiological reductants such as ascorbic acid and methionine, incubation of the NaOCl-modified LDL at 37 degrees C resulted in increasing resistance of the modified lysine residues against reductive reversal. Exposure of mouse peritoneal macrophages to NaOCl-oxidized LDL resulted in increased intracellular concentrations of cholesterol and cholesteryl esters. These findings suggest that lipid-soluble antioxidants associated with LDL do not efficiently protect the lipoprotein against oxidative damage mediated by hypochlorite, and that extensive lipid oxidation is not a necessary requirement for oxidative LDL modification that leads to a high-uptake form of the lipoprotein.

The role of high-density lipoprotein and lipid-soluble antioxidant vitamins in inhibiting low-density lipoprotein oxidation.

Abstract: 1. The oxidation of low-density lipoprotein (LDL) is believed to play a central role in atherogenesis. We have compared the effect of antioxidant vitamins and high-density lipoprotein (HDL) on the Cu(2+)-catalysed oxidation of LDL. 2. Antioxidant vitamin supplementation significantly reduced conjugated diene formation but did not affect the formation of lipid peroxides. 3. Conversely, HDL did not affect conjugated diene formation but inhibited the formation of lipid peroxides by up to 90%. 4. The inhibition by HDL of lipid peroxide formation in oxidized LDL was dependent on the concentration of HDL and was not due to HDL chelating Cu2+. 5. Large interindividual variations in the inhibition of lipid peroxide formation by autologous HDL were evident, which were related to the rate of lipid peroxide generation in the LDL. 6. We conclude that HDL is a powerful antioxidant or more probably inhibitor of LDL oxidation in vitro and may play an important role in vivo in preventing atherosclerosis by inhibiting LDL oxidation in the artery wall.

The cerebrospinal-fluid soluble form of Alzheimer's amyloid beta is complexed to SP-40,40 (apolipoprotein J), an inhibitor of the complement membrane-attack complex.

Abstract: The amyloid fibrils deposited in Alzheimer's neuritic plaque cores and cerebral blood vessels are mainly composed of aggregated forms of a unique peptide, 39-42 amino acids long, named amyloid beta (A beta). A similar, although soluble, A beta ('sA beta') has been identified in cerebrospinal fluid, plasma and cell supernatants, indicating that it is normally produced by proteolytic processing of its precursor protein, amyloid precursor protein (APP). Using direct binding experiments we have isolated and characterized an 80 kDa circulating protein that specifically interacts with a synthetic peptide identical with A beta. The protein was unmistakably identified as SP-40,40 or ApoJ, a cytolytic inhibitor and lipid carrier, by means of amino acid sequence and immunoreactivity with specific antibodies. Immunoprecipitation with anti-SP-40,40 retrieved soluble A beta from cerebrospinal fluid, indicating that the interaction occurs in vivo.

p40phox, a third cytosolic component of the activation complex of the NADPH oxidase to contain src homology 3 domains.

Abstract: The NADPH oxidase generates superoxide in phagocytic cells. It is important for immunity and its deficiency leads to chronic granulomatous disease (CGD). It consists of a membrane-bound flavocytochrome b that lies dormant until activated by the translocation to the plasma membrane of cytosolic proteins, p47phox (phox for phagocyte oxidase), p67phox and p21rac, a small GTP-binding protein. We show here that a novel component, p40phox, forms an activation complex with p47phox and p67phox with which it translocates to the membrane to associate with the flavocytochrome b. cDNA cloning and amino acid analysis revealed that p40phox has an src homology 3 (SH3) domain and a large region of sequence similarity with the N-terminus of p47phox. The primary association of p40phox appears to be with p67phox, and it is present in reduced amounts in patients with CGD lacking p67phox.

Hydrolysis of human and pig brain natriuretic peptides, urodilatin, C-type natriuretic peptide and some C-receptor ligands by endopeptidase-24.11

Abstract: Endopeptidase-24.11 (E-24.11, EC 3.4.24.11) is widely believed to play a physiological role in metabolizing atrial natriuretic peptide (ANP). Since the discovery of ANP, new natriuretic peptides have been isolated and other peptides synthesized as receptor ligands. The hydrolysis in vitro of six related peptides by the endopeptidase has been studied, mainly by h.p.l.c. The initial attack on the 32-residue form of pig brain natriuretic peptide (pBNP-32) was shown to be at the Ser20-Leu21 bond, as had been previously shown for the 26-residue form. In contrast, human brain natriuretic peptide-32 (hBNP-32), which differs in ten residues from pBNP-32, was attacked first at the Met4-Val5 bond, releasing the N-terminal tetrapeptide, and only later at bonds within the ring: at Arg17-Ile18 and subsequently at four other sites. Urodilatin, which has a four-residue extension at the N-terminus compared with alpha-human atrial natriuretic peptide-28 (alpha-hANP), was degraded at about half the rate of the latter, though the C-terminal Phe-Arg-Tyr was released at the same rate. The 22-residue C-type natriuretic peptide was hydrolysed more rapidly than alpha-hANP, as were two C-receptor ligands (peptides with deletions within the ring): C-ANP4-23 (rANP4-23 des-Gln18,Ser19,Gly20,Leu21,Gly22) and SC 46542 (hANP5-28 des-Phe8,Gly9,Ala17,Gln18). Angiotensin-converting enzyme failed to hydrolyse pBNP-32, hBNP-32 or 125I-rat (r) ANP, even after prolonged incubation. Km and kcat values were determined for the hydrolysis of alpha-hANP, porcine BNP-26, porcine BNP-32 and 125I-rANP by E-24.11. Ki values were determined for six peptides, alpha-hANP, urodilatin, hBNP-32, C-type natriuretic peptide (CNP), SC 46542 and C-type natriuretic peptide (C-ANP4-23), in radiometric assays of E-24.11 with either [125I] insulin B chain or [125I] rANP as substrate. The Ki values (2.5-13 microM) for CNP were the lowest of any of the group, whereas those for hBNP-32 (151-172 microM) were the highest. The physiological significance of these results is discussed, especially in regard to the relative resistance of hBNP-32 to attack and the ability of the C-receptor ligands to compete with natriuretic peptides for hydrolysis by E-24.11.