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T Bruin

Publications and source records attributed to T Bruin.

33 records · Page 2Linked to original sources
PubMed1992

A missense mutation Pro157 Arg in lipoprotein lipase (LPLNijmegen) resulting in loss of catalytic activity.

Here we report on the molecular defect that leads to a deficiency of lipoprotein lipase (LPL) activity in a proband of Dutch descent. Southern-blot analysis of the LPL gene from the patient did not reveal any major DNA rearrangements. Sequencing of polymerase-chain-reaction-amplified DNA revealed that the proband is a homozygote for G725C, resulting in a substitution of Pro157 for Arg. This substitution alters a restriction site for PvuII, which allowed rapid identification of the mutant allele in family members. Site-directed mutagenesis and transient expression of the mutant LPL in COS cells produced an enzymatically inactive protein, establishing the functional significance of this mutation. This naturally occurring mutation which alters the Pro157 adjacent to Asp156 of the proposed catalytic triad, indicates that this region of the protein is indeed crucial for LPL catalytic activity.

Adult↗
PubMed1992

Two naturally occurring mutations at the first and second bases of codon aspartic acid 156 in the proposed catalytic triad of human lipoprotein lipase. In vivo evidence that aspartic acid 156 is essential for catalysis.

We are studying naturally occurring mutations in the gene for lipoprotein lipase (LPL) to advance our knowledge about the structure/function relationships for this enzyme. We and others have previously described 11 mutations in human LPL gene and until now none of these directly involves any of the residues in the proposed Asp156-His241-Ser132 catalytic triad. Here we report two separate probands who are deficient in LPL activity and have three different LPL gene haplotypes, suggesting three distinct mutations. Using polymerase chain reaction cloning and DNA sequencing we have identified that proband 1 is a compound heterozygote for a G----A transition at nucleotide 721, resulting in a substitution of asparagine for aspartic acid at residue 156, and a T----A transversion, resulting in a substitution of serine for cysteine at residues 216. Proband 2 is homozygous for an A----G base change at nucleotide 722, leading to a substitution of glycine for aspartic acid at residue 156. The presence of these mutations in the patients and available family members was confirmed by restriction analysis of polymerase chain reaction-amplified DNA. In vitro site-directed mutagenesis and subsequent expression in COS cells have confirmed that all three mutations result in catalytically defective LPL. The two naturally occurring mutations, which both alter the same aspartic acid residue in the proposed Asp156-His241-Ser132 catalytic triad of human LPL, indicate that Asp156 plays a significant role in LPL catalysis. The Cys216----Ser mutation destroys a conserved disulfide bridge that is apparently critical for maintaining LPL structure and function.

Amino Acid Sequence↗
PubMed1992

Regulation of adenylate cyclase in electropermeabilized Dictyostelium discoideum cells.

In Dictyostelium discoideum cells the enzyme adenylate cyclase is functionally coupled to cell surface receptors for cAMP. Coupling is known to involve one or more G-proteins. Receptor-mediated activation of adenylate cyclase is subject to adaptation. In this study we employ an electropermeabilized cell system to investigate regulation of D. discoideum adenylate cyclase. Conditions for selective permeabilization of the plasma membrane have been described by C.D. Schoen, J. C. Arents, T. Bruin, and R. Van Driel (1989, Exp. Cell Res. 181, 51-62). Only small pores are created in the membrane, allowing exchange of exclusively low molecular weight substances like nucleotides, and preventing the loss of macromolecules. Under these conditions functional protein-protein interactions are likely to remain intact. Adenylate cyclase in permeabilized cells was activated by the cAMP receptor agonist 2'-deoxy cAMP and by the nonhydrolyzable GTP-analogue GTP gamma S, which activates G-proteins. The time course of the adenylate cyclase reaction in permeabilized cells was similar to that of intact cells. Maximal adenylate cyclase activity was observed if cAMP receptor agonist or GTP-analogue was added just before cell permeabilization. If these activators were added after permeabilization adenylate cyclase was stimulated in a suboptimal way. The sensitivity of adenylate cyclase activity for receptor occupation was found to decay more rapidly than that for G-protein activation. Importantly, the adenylate cyclase reaction in permeabilized cells was subject to an adaptation-like process that was characterized by a time course similar to adaptation in vivo. In vitro adaptation was not affected by cAMP receptor agonists or by G-protein activation. Evidently electropermeabilized cells constitute an excellent system for investigating the positive and negative regulation of D. discoideum adenylate cyclase.

Adenosine Triphosphate↗
PubMed1991

Subcellular distribution and phosphorylation of vinculin isoforms in human blood platelets.

In this study we investigated human blood platelet vinculin microheterogeneity, the subcellular localization and phosphorylation of the different isoforms before and after platelet stimulation. At least 5 vinculin isoforms could be detected, as well as meta-vinculin. These isoforms did not demonstrate a specific subcellular localization, i.e. their relative content was similar in cytoskeleton, membrane skeleton and cytosol. Upon platelet stimulation with thrombin a small increase in alpha-vinculin was noted in all platelet subfractions. The cytoskeleton of non-stimulated platelets contained a minor quantity of vinculin. Upon thrombin stimulation of the platelets the cytoskeletal vinculin content increased significantly; previously we already reported a maximal 10% incorporation of the total platelet vinculin content into the cytoskeleton upon stimulation. A phosphorylation of a minor vinculin-isoform, i.e. at the alpha'/alpha location was mainly detected in the cytoskeleton. This phosphorylation was observable in the non-stimulated platelet cytoskeletal vinculin. These findings argue against a regulatory role for vinculin phosphorylation in the uptake of the main isoforms of this protein in the platelet cytoskeleton upon thrombin stimulation. The function of the phosphorylated cytoskeletal vinculin remains to be established.

Blood Platelets↗
PubMed1990

Vinculin is a permanent component of the membrane skeleton and is incorporated into the (re)organising cytoskeleton upon platelet activation.

Vinculin, a 130-kDa protein discovered in chicken gizzard smooth-muscle cells and subsequently also described in platelets, is believed to be involved in membrane-cytoskeleton interactions. In this study we investigated vinculin distribution in human blood platelets. Two skeletal fractions and a remaining cytosolic fraction were prepared with a recently described Triton X-100 lysis buffer causing minimal post-lysis breakdown by proteolysis. The presence of vinculin was demonstrated in the membrane skeleton and cytosol of resting and thrombin-activated human platelets. Upon thrombin stimulation vinculin also appeared in the cytoskeleton. this cytoskeletal incorporation was completed during the early stages of platelet aggregation and secretion, when the uptake of myosin, actin-binding protein and talin was still not maximal. We conclude therefore, that vinculin may play an important role in the structural (re)organisation of the human platelet cytoskeleton upon platelet activation.

Blood Platelets↗
PubMed1990

A missense mutation at codon 188 of the human lipoprotein lipase gene is a frequent cause of lipoprotein lipase deficiency in persons of different ancestries.

Lipoprotein lipase (LPL) plays a crucial role in the regulation of lipoprotein metabolism by hydrolysing the core triglycerides of circulating chylomicrons and VLDL. Human, bovine, mouse, and guinea pig complementary DNA clones have recently been isolated and the organization of the human LPL gene is now known to comprise 10 exons spanning approximately 30 kb. Here we report a similar mutation on 21 alleles from 13 unrelated affected probands with LPL deficiency of French Canadian, English, Polish, German, Dutch, and East Indian ancestry. We show that an identical missense mutation within exon 5, resulting in an amino acid substitution of glutamic acid for glycine at position 188, is responsible for LPL deficiency in 21 of 88 LPL alleles assessed. This mutation alters an Ava II restriction site in exon 5 and will allow a rapid screening test for this mutation in patients with LPL deficiency. This mutation has occurred on the same haplotype in all the unrelated affected persons suggesting a common origin. The amino acid substitution lies within the longest segment of homology for LPL in different species and results in a protein that is catalytically defective.

Alleles↗
PubMed1989

Intracellular localization of secretable cAMP in relaying Dictyostelium discoideum cells.

Dictyostelium discoideum cells synthesize and secrete the chemoattractant cAMP within minutes after chemotactic stimulation. During development, this signal-relay process is instrumental in cell aggregation, pattern formation, and differentiation. Cyclic AMP is known to accumulate inside the cell before secretion. In this study we investigated the subcellular localization of the nascent cAMP. After chemotactic stimulation at 0 degrees C and subsequent accumulation of intracellular cAMP, the newly synthesized chemoattractant could be released by gently opening cells in two different ways. Both methods make the cytosolic compartment accessible, whereas intracellular compartments surrounded by a membrane remain largely intact. The first method involved rapid lysis by forced passage through a 5-micron pore-size Nuclepore filter. The second technique was electropermeabilization under carefully controlled conditions that ensured the formation of small, stable pores in the plasma membrane. These pores allowed the passage of small molecules, such as cAMP, but not of macromolecules. To confirm the selectivity for the plasma membrane of both methods, we showed that a typical vesicular cell compartment, the lysosome, remained intact. Both procedures immediately released all intracellularly accumulated cAMP. We interpret our results as strong evidence for accumulation of nascent cAMP in the cytosolic compartment rather than in a vesicular compartment before it is secreted. This implies that cAMP secretion takes place via a trans-membrane transport mechanism, rather than by exocytosis.

Cell Membrane↗
PubMed1987

The function of the human factor V carbohydrate moiety in blood coagulation.

Human factor V was subjected to desialation and deglycosylation to investigate the function of the molecular carbohydrate moiety. Removal of 90% of the sialic acid residues resulted in a 1.5-2-fold increase in clotting activity, and up to 70% deglycosylation in a concurrent decrease in clotting activity. Desialation had no effect on thrombin-induced activation, whereas deglycosylated factor V activation was impaired. Lectin-blot experiments with sialic-acid-specific Limax flavus agglutinin (LFA), galactose-specific Ricinus communis agglutinin (RCA-II) and mannose-specific concanavalin A on thrombin-induced factor V fragments revealed the presence of carbohydrate residues in fragments B, C1, D and F1F2. Interestingly, sialic acid was present in C1 whilst galactose was not detectable. Fragment F1F2 contained terminal galactose residues. LFA and RCA-II inhibited the procoagulant activity of native factor V and of desialated factor V respectively. These investigations distinctly indicate the important role of the human factor V carbohydrate moiety in the process of blood coagulation.

Blood Coagulation↗
PubMed1986

Purified radiolabeled antithrombin III metabolism in three families with hereditary AT III deficiency: application of a three-compartment model.

Purified human radioiodinated antithrombin III (125I-AT III) was used to study its metabolism in six members from three different families with a known hereditary AT III deficiency. Six healthy volunteers served as a control group. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and crossed immunoelectrophoresis (CIE) showed the purified AT III to be homogeneous. Amino acid analysis of the protein revealed a composition identical to a highly purified internal standard. The specific activity was 5.6 U/mg. Analysis of plasma radioactivity data was performed, using a three-compartment model. Neither plasma disappearance half-times nor fractional catabolic rate constants differed significantly between patients and control subjects. The mean absolute catabolic rate in the patient group was significantly lower than that of the control group at 2.57 +/- 0.44 and 4.46 +/- 0.80 mg/kg/day, respectively. In addition, the mean patient alpha 1-phase, flux ratio (k1,2 and k2,1) of the second compartment alpha 2-phase and influx (k3,1) of the third compartment were significantly reduced as compared with control values. It has been tentatively concluded that the observed reduction in the second compartment may be caused by a decrease in endothelial cell surface binding.

Adult↗
PubMed1985

Antithrombin III metabolism in two colitis patients with acquired antithrombin III deficiency.

125I-Antithrombin III metabolism studies were performed in 2 patients with ischemic and ulcerative colitis, respectively. Both patients had acquired antithrombin III deficiency and objectively diagnosed deep venous thrombosis. A decreased 125I-antithrombin III plasma disappearance halflife and an increased fractional catabolic rate was found in both patients. The transcapillary flux ratio was elevated in the patient with ischemic colitis. A follow-up study of the first patient during a period when no signs of an ischemic colitis were present and no medication was taken showed completely normal tracer data. The data are consistent with both gastrointestinal loss and intravascular consumption of antithrombin III. The antithrombin III deficiency could not be explained by other causes such as proteinuria, liver dysfunction, or obvious disseminated intravascular coagulation. Reduced antithrombin III plasma levels were considered to have contributed to the development of deep venous thrombosis in both patients.

Aged↗
PubMed1983

Identification of a contaminant protein in the purification of human factor V. Comparison of two purification methods.

Coagulation factor V has been isolated from human plasma by the methods of Bolhuis and of Kane to investigate reported differences in purity and specific activity. The specific activities of the preparations are 10 and 40 units/mg, respectively. The Kane preparation shows a single band (Mr 330 000) in polyacrylamide gel electrophoresis in the presence and absence of dithiothreitol, but the Bolhuis preparation shows an additional band at Mr 190 000 upon reduction with dithiothreitol. This suggests the presence of a contaminant which could account for the lower specific activity. Modification of the Bolhuis technique to include ion-exchange chromatography on DEAE-Sepharose resulted in separation of the two isolated proteins. The proteins were identified as factor V, with characteristics indistinguishable from Kane factor V, and a dimeric subunit of alpha 2-macroglobulin (Mr 350 000). Contamination by alpha 2-macroglobulin may explain the I2-component described in some preparations of bovine factor V, which is the basis of speculation that bovine factor V has a second polypeptide chain modulating stability and activity.

Blood Proteins↗