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Biomedical subjects

G Dooijewaard

Publications and source records attributed to G Dooijewaard.

53 records · Page 3Linked to original sources
PubMed1988

Renal and hepatic handling of endogenous tissue-type plasminogen activator (t-PA) and its inhibitor in man.

In eight male patients with normal liver and kidney function fibrinolytic components were measured in arterial blood and in renal and hepatic vein blood, obtained during catheterization for analysis of hypertension. Blood samples were collected simultaneously from veins und corresponding arteries before and 5 minutes after the completion of intravenous injection of desmopressin (DDAVP), 0.4 micrograms/kg body weight over a 10 minute period. DDAVP induced a rise in t-PA antigen and activity, and in von Willebrand factor, accompanied by a decrease in free PA-inhibitor level. We failed to detect a significant rise in plasma urokinase activity. The concentrations of fibrinogen, plasminogen, alpha 2-antiplasmin, antithrombin III and coeruloplasmin did not change either. Renal production of t-PA under basal conditions was inferred from a negative arterio-venous (A-V) difference in t-PA-activity and in t-PA-antigen levels but this could not be confirmed by orthogonal regression analysis of the same data. A-V differences of other fibrinolytic factors were negligible. In the hepatic vessels a significant positive A-V difference of t-PA-activity and of t-PA-antigen levels was a uniform finding. After DDAVP, when plasma levels were elevated, the mean A-V difference was proportionally higher, consistent with a constant fractional elimination rate. Free PA-inhibitor was virtually absent from arterial blood after DDAVP, but appeared in hepatic vein blood, indicating either production of the inhibitor by the liver or dissociation of a circulating complex of t-PA and its inhibitor in the liver. The blood levels of the other investigated components did not show any change upon passage through the liver.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1987

Increased plasminogen activator inhibition levels in malignancy.

Plasminogen activator(PA)-tissue and urokinase type-and PA-inhibition in plasma were investigated in 52 consecutive cancer patients with a variety of tumors. At first patients were analyzed as one group. Secondly patients were subdivided into two groups, one with (n = 42) and one without (n = 10) metastasis. Our results show that tissue-type-PA antigen (t-PA-antigen) and PA-inhibition were both significantly increased irrespective of the presence or absence of tumor metastasis (p less than 0.001 compared to age matched healthy controls. In the group without metastasis a significantly decreased level of t-PA activity was found (p less than 0.001) but in the group with metastasis t-PA activity was normal. These data seem to reject the hypothesis that decreased plasma fibrinolytic activity is one of the prerequisites for tumor metastasis.

Antigens↗
PubMed1987

Identification of two types of protein immunochemically related to urinary urokinase occurring in human plasma.

Plasma urokinase, a plasminogen activator immunochemically related to urinary urokinase (UK), was removed from human plasma (3.5 ng/ml) by immuno-depletion with antibodies raised against UK. The remaining plasminogen activator activity of the depleted plasma could not be inhibited by anti-UK antibodies and a sensitive ELISA for UK did not detect any UK levels that were higher than the background of the assay (0.1 ng/ml). However, when the depleted plasma was subjected to SDS-PAGE, substantial amounts of protein were found hereafter around 110 and 46 kD which now gave a positive reaction in the ELISA (35-350 ng/ml plasma). From these observations it is concluded that in human plasma two types of UK-related protein occur: Type I, among which the plasma urokinase, has antigenic determinants which are directly accessible to the anti-UK antibodies, Type II has determinants in a latent form. The function of the 110 kD type-II protein is that of a plasminogen activator; that of the 46 kD protein is not yet clear.

Electrophoresis, Polyacrylamide Gel↗
PubMed1987

alpha 2-Antiplasmin Enschede: dysfunctional alpha 2-antiplasmin molecule associated with an autosomal recessive hemorrhagic disorder.

alpha 2-Antiplasmin (alpha 2-AP) is a major fibrinolysis inhibitor, whose complete, congenital absence has been found to be associated with a distinct hemorrhagic diathesis. We studied a 15-yr-old male with a hemorrhagic diathesis after trauma from early childhood on. This bleeding tendency was associated with a minimal alpha 2-AP level recorded functionally in the immediate plasmin inhibition test: less than or equal to 4% of normal. However, a normal plasma concentration of alpha 2-AP antigen (83%) was found. His sister (5 yr old) showed similar results (2 and 92%). In their family, eight heterozygotes could be identified by half-normal activity results and normal antigen concentrations. The inheritance pattern is autosomal recessive. On analysis, the alpha 2-AP of the propositus was homogeneous in all respects tested, suggesting a homozygous defect. We designated the abnormal alpha 2-AP as alpha 2-AP Enschede. alpha 2-AP Enschede showed the following characteristics: (a) complete immunological identity with normal alpha 2-AP; (b) normal molecular weight (sodium dodecyl sulfate-polyacrylamide gel electrophoresis); (c) normal alpha-electrophoretic mobility; (d) presence in plasma of both molecular forms excluding an excessive conversion to the less reactive non-plasminogen-binding form; (e) quantitatively normal binding to lys-plasminogen and to immobilized plasminogen kringle 1-3; and (f) normal Factor XIII-mediated binding to fibrin. Functional abnormalities were found in: (i) no inhibition of amidolytic activities of plasmin and trypsin, even on prolonged incubation; (ii) no formation of plasmin-antiplasmin complexes in plasma with plasmin added in excess; and (iii) no inhibition of fibrinolysis by fibrin-bound alpha 2-AP. In the heterozygotes, the presence of abnormal alpha 2-AP did not interfere with several functions of the residual normal alpha 2-AP. One-dimensional peptide mapping showed an abnormal pattern of papain digestion. We conclude that in this family, abnormal antiplasmin molecules, defective in plasmin inhibition but with normal plasminogen-binding properties, have been inherited. The residual plasminogen-binding properties do not protect against a hemorrhagic diathesis.

Adolescent↗
PubMed1986

Quantitation of urokinase antigen in plasma and culture media by use of an ELISA.

An ELISA was set up using polyvinylchloride microtiter plates coated with rabbit anti-UK IgG's and affino-purified goat anti-UK IgG's as second antibody. Detection occurred with rabbit anti-goat IgG antibodies conjugated with alkaline phosphatase. The assay is specific for urokinase (UK) with a detection limit of 100 pg/ml sample. Tissue-type plasminogen activator, up to concentrations of 100 ng/ml, does not interfere. The assay measures the antigen of the inactive zymogen pro-UK, the active enzyme UK and the UK-inhibitor complex with equal efficiency and gives the total UK antigen present, irrespective of its molecular form. Culture media of fibroblasts, endothelial- and kidney cells showed, despite the absence of active UK, antigen levels of 1.2, 23 and 65 ng/ml, respectively. In human plasma the UK concentration was found to be 3.5 +/- 1.4 ng/ml (mean +/- SD, n = 54). The inter- and intra-assay variations were 20% and 6%, respectively.

Animals↗
PubMed1984

Stanozolol-induced changes in fibrinolysis and coagulation in healthy adults.

The effect of orally-administered stanozolol, 5 mg b.d. on fibrinolysis, coagulation and on various haematological and biochemical parameters have been studied in 16 healthy adults, 8 males and 8 females. Statistically significant enhancement of extrinsic (tissue-type) plasminogen activator activity was detected in all subjects studied. This was associated with significant increases in plasma plasminogen and a concomitant reduction in histidine-rich glycoprotein. There were no changes in plasma urokinase activity. Changes in the coagulation system included significant reduction in plasma fibrinogen and elevation of protein C and antithrombin III. Changes in plasma lipids included significant reduction of HDL cholesterol associated with an increase in LDL triglycerides. No change occurred in total cholesterol. There were no major differences between the sexes, nor were there serious side effects. The effects of stanozolol on extrinsic (tissue-type) plasminogen activator activity, "free" plasminogen, protein C and antithrombin III, argue strongly in favour of its therapeutic potential.

Adult↗
PubMed1979

The Escherichia coli phosphoenolpyruvate-dependent phosphotransferase system: observation of heterogeneity in the amino acid composition of HPr.

Resonances of the aromatic protons of tyrosine have been observed in the proton nuclear magnetic resonance (1H NMR) spectrum of purified HPr from Escherichia coli. Analysis of the NMR spectrum of native HPr suggests that the tyrosine is located in a single position in the secondary structure and that this position is on the interior of the molecule inaccessible to solvent. Previous reports suggested that E. coli HPr contained no tyrosine [Anderson, B., Weigel, N., Kundig, W., & Roseman, S. (1971) J. Biol. Chem. 246, 7023--7033]. In contrast, we find, by amino acid analysis and ultraviolet and NMR spectroscopy, that E. coli HPr does contain tyrosine but at a subintegral level of 0.5 +/- 0.1 mol of tyrosine per mol of HPr.

Amino Acids↗
PubMed1979

Escherichia coli phosphoenolpyruvate dependent phosphotransferase system. Copurification of HPr and alpha 1-6 glucan.

A rapid, high-yield procedure has been developed for the purification of HPr from the Escherichia coli phosphoenolpyruvate dependent phosphotransferase system. During this procedure, the protein copurifies with a 2500-dalton homopolysaccharide which we have identified as alpha 1-6 glucan. The results of steady-state kinetic measurements of the phosphotransferase activity demonstrate that the polysaccharide works as an activator of the phosphotransferase system probably at the level of the HPr:P-E1 complex or the P-HPr:E11 complex.

Bacterial Proteins↗
PubMed1979

Escherichia coli phosphoenolpyruvate dependent phosphotransferase system. NMR studies of the conformation of HPr and P-HPr and the mechanism of energy coupling.

1H and 31P nuclear magnetic resonance investigations of the phosphoprotein intermediate P-HPr and the parent molecule HPr of the E. coli phosphoenolpyruvate dependent phosphotransferase system (PTS) show that HPr can exist in two conformations. These conformations influence the protonation state of the reactive histidine residue, thereby determining the reaction pathway in the phosphoryl group transfer step. A general mechanism is proposed for the energy-coupling process in the PTS.

Bacterial Proteins↗
PubMed1978

Chaotropic resolution of high molecular weight (type I) NADH dehydrogenase, and reassociation of flavin-rich (type II) and flavin-poor subunits.

1. Type-I NADH dehydrogenase (Complex I) was solubilized and dissociated into subunits by NaClO4. NADH slows the dissociation. On subsequent stepwise addition of (NH4)2SO4 the dissociation is partly reversed, as is to be expected from the opposing effects of ClO-4 and SO-24, which are on the salting-in and salting-out sides, respectively, of the lyotropic series. 2. In consequence, the aggregates of subunits that are separated by (NH4)2-SO4 fractionation consist of randomly associated subunits as well as fragments of Type I enzyme. The fraction precipitating at 27% satd. (NH4)2SO4 is flavin-poor, that remaining soluble at 55% satd. (NH4)2SO4 flavin-rich and those separating between 27 and 55% satd. (NH4)2SO4 intermediate in composition. 3. The fraction remaining soluble at 55% satd. (NH4)2SO4 contains the purified low-molecular-weight iron-sulphur flavoprotein (Type-II dehydrogenase). It is a dimer consisting of one molecule of FMN, one 28-kilodalton and one 56-kilodalton subunit per protomer. Work of others indicates that it contains 4 Fe and 4 acid-labile S atoms per molecule of FMN. Sometimes the fraction remaining soluble at 55% satd. (NH4)2SO4 contained an additional small subunit (12 kilodaltons) and four additional Fe and acid labile S atoms per protomer. The sedimentation coefficients (s020,w) of the two preparations were 5.3 and 6.6 S, respectively, with calculated frictional ratios of 1.5 and 1.24, respectively. 4. The intermediate fractions are mixtures of the various subunits present in Complex I. Specifically a fraction separating at 55% satd. (NH4)2SO4 was found to be a mixture of two fragments, the pure iron-sulphur flavoprotein and a 26-S fragment that contained per protomer four subunits of 12 kilodaltons, one each of 28, 32, 56 and 77 kilodaltons, one molecule of FMN and 20 Fe and acid-labile S atoms. It was probably tetrameric or even larger. 5. The oxidoreductase activity of the intermediate fractions is dependent on the protein concentration, the activity with ferricyanide increasing and that with ferricytochrome c decreasing with increasing protein concentration. This is interpreted as an increased association of subunits present in the intermediate fractions. Similar results are obtained when flavin-rich and flavin-poor fractions are mixed. The association is cooperative. NADH favours the association of the subunits. 6. Association of the subunits is accompanied by a 10-fold increase in k2 (rate constant for intramolecular electron flow), a 10-fold decrease of the accessibility of ferricyanide to the reduced enzyme and a 10(4)-fold decrease of the accessibility of ferricytochrome c. The Ks (NADH) is also decreased. Although the changes are in the direction to be expected from a conversion of Type II enzyme to Type I, the value of k2 is still much less than in the latter enzyme.

Flavin Mononucleotide↗
PubMed1977

EPR signals of NADH: Q oxidoreductase. Shape and intensity.

(1) The EPR spectrum of Center 1 of NADH dehydrogenase in isolated Complex I or submitochondrial particles from beef heart consists of two overlapping nearly axial signals of the same intensity. They are defined as Center 1a (gll = 0.021, gl = 1.938) and Center 1b (gll = 2.021, gl = 1.928). (2) The line shape of the EPR spectrum of the Center 3+4 can be interpreted as an overlap of two rhombic signals of the same intensity. We define Center 3 by the g-values: gz=2.103, gy = 1.93-1.94, gx=1.884, and Center 4 by the values gz=2.04, gy=1.92-1.93, gx=1.863. (3) Direct quantitation of the individuals signals as well as computer stimulation suggests that the amount of the Centers 1a and 1b is only 25% of that of the other individuals centers and FMN. As EPR spectra of beef-heart submitochondrial particles at 10-20 K are nearly identical to those of Complex I, the same relative concentrations of the Fe-S centers are also present in the particles. (4) The signals either observed by us in EPR spectra of Complex I and submitochondrial particles at 4.2 K and high microwave powers can now be explained without assuming more than 5 paramagnetic centers in NADH dehydrogenase.

Animals↗
PubMed1976

Steady-state kinetics of high molecular weight (type-I) NADH dehydrogenase.

(1) Studies of the steady-state kinetics of the NADH dehydrogenase activity of Complex I (NADH: Q oxidoreductase) revealed that the reaction mechanism with the one-electron acceptor ferricyanide or the two-electron acceptor 2,6-dichloro-indophenol is ping pong bi bi, with double substrate inhibition. NADH inhibits the reaction of the reduced form of the flavoprotein with the acceptors, and the acceptors prevent NADH from reacting with the oxidized form. This implies that both NADH and acceptors react with the same site on NADH dehydrogenase. (2) The velocity at infinite NADH and acceptor concentrations (corrected for the double substrate inhibition) is much larger with ferricyanide than with the indophenol. It is concluded that the latter binds to the reduced enzyme. Thus, with ferricyanide the rate constant measured refers to the dissociation of bound NAD+ from the reduced enzyme (k2) and with the indophenol to the rate constant of oxidation of reduced enzyme by bound acceptor (k4). The latter value is not an estimate for the situation in vivo, where ubiquinone is the acceptor. (3) The rate constant of the dissociation of bound NAD+ from the reduced enzyme (k2) increases with pH. It is suggested that an ionizing group on the enzyme is involved in the dissociation. (4) After extraction of ubiquinone from Complex I with pentane curve relating activity at infinite ferricyanide concentration to NADH concentration changes from hyperbolic to sigmoidal. The hyperbolic curve is restored by reincorporating ubiquinone. It is concluded that ubiquinone is an effector for NADH dehydrogenase.

2,6-Dichloroindophenol↗
PubMed1976

Steady-state kinetics of low molecular weight (type-II) NADH dehydrogenase.

(1) The steady-state kinetics of the NADH dehydrogenase activity of Type-II (low molecular weight) NADH dehydrogenase with the acceptors ferricyanide, cytochrome c and 2,6-dichloroindophenol are consistent with the simultaneous operation of an ordered and a ping-pong mechanism. Thus, depending on the acceptor concentration, the reduced enzyme is preferentially oxidized before or after NAD+ disociates from it. (2) The acceptors are able to oxidize the reduced enzyme and its NAD+ complex equally well. In contrast to the kinetics of the Type-I (high molecular weight) enzyme, double substrate inhibition is not found, implying that the site of oxidation of the reduced enzyme by acceptors and the NADH-binding site are remote. (3) With the indophenol, in the concentration range measured, the ordered mechanism is mainly operative. At infinite NADH and acceptor concentrations the rate constant of the reduction of enzyme by bound NADH is measured. (4) With ferricyanide and cytochrome c, in the concentration range measured, erroneous conclusions may be drawn from extrapolations owing to the fact that extrapolated lines in double-reciprocal plots of turnover number against acceptor concentration, at different NADH concentrations, intersect in the third quadrant. A method is described that allows the extrapolation of these data to zero acceptor concentrations. (5) The relation between activity and NADH concentration is sigmoidal (h = 2.0) with ferricyanide or cytochrome c as acceptor, but hyperbolic with 2,6-dichloroindophenol. The latter is also an inhibitor, competitive with respect to NADH. It is concluded that this two-electron acceptor, like ubiquinone, acts as an allosteric effector. (6) Type II is isolated from Type I without gross changes in tertiary structure, as judged by the unaltered rate constants of dissociation of NADH (k-1) and NAD+ (k4) and association of NADH (k1). (7) Type II differs from Type I in two respects, (a) The accessibility of the acceptors is greater by at least two orders of magnitude (k3). (b) The redox potential of the prosthetic group FMN is 120 mV less, as judged by a drop in the value of k2 by four orders of magnitude. It is suggested that one or more of the iron-sulphur proteins present in Type-I but lacking in Type-II dehydrogenase functions as an effector, regulating the redox potential of the FMN.

2,6-Dichloroindophenol↗