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Biomedical subjects

J Visser

Publications and source records attributed to J Visser.

At least 235 records · Page 13Linked to original sources
PubMed1980

The pyruvate dehydrogenase complex of Pseudomonas aeruginosa PAO Purification, properties and characterization of mutants.

The pyruvate dehydrogenase complex of Pseudomonas aeruginosa PAO was purified by affinity chromatography on ethanol-Sepharose 2B followed by sucrose density gradient centrifugation. The overall purification was 130-fold based on enzyme activity. The purified complex contained three major and one minor polypeptide components when analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. These were identified by heat treatment, limited proteolysis and peptide mapping as pyruvate dehydrogenase (El; Mr 92500), acetyltransferases (E2; major component, Mr 76000, and minor component, Mr 77800) and lipoamide dehydrogenase (E3; Mr 58000). The purified complex had a sedimentation coefficient of 48S and the specific activity for the overall reaction of the complex was 6.5 micromol substrate transformed (mg protein)-1 min-1 at the optimum pH (7.8) and 25 degrees C. The lesions in four ace mutants lacking overall pyruvate dehydrogenase complex activity were identified after partial purification of the corresponding cell-free extracts. Three strains, designated ace A mutants, lacked pyruvate dehydrogenase activity (E1 component) and one strain, and ace B mutant, lacked the activity of the acetyltransferase (E2 component).

Antibodies↗
PubMed1980

Concentration of hemopoietic stem cells using a light-activated cell sorter.

Light-activated cell sorting although capable of highly specific selection of subpopulations of cells is rate-limited. The method as it now exists is insufficient to supply an adequate number of stem cells for bone marrow transplantation. However, other separation methods using electrophoresis, density gradients, and density centrifugation coupled with cell sorting can improve the yield for clinical utilization.

Animals↗
PubMed1979

The role of calcium, phosphate and citrate ions in the stabilization of casein micelles.

To obtain greater insight into the interaction of Ca, citrate and phosphate ions with casein, 31PNMR measurements were performed on combinations of these ions with alphas- and kappa-caseins. It was found that addition of alphas-casein to a Ca phosphate solution in D2O at 27 degrees C and pD 6.4 resulted in a downfield shift of the 31P singlet. An almost identical shift was observed with kappa-casein, but no shift was found when only phosphate ions were present or when Ca2+ were added to phosphate ions in the absence of casein. Separate experiments with poly-L-lysine, mol. wt approx. 35,000, resulted in similar downfield 31P chemical shifts of Ca phosphate as with both caseins, whereas no shift was observed when poly-glycine was added. From these results it can be concluded that Ca and phosphate ions associate with casein in a co-operative manner, probably in the way described by ter Horst (1963) as a complex with the NH3+-groups of lysine or arginine in a structure such as: (casein--NH3+)--PO43---Ca2+. The formation of this complex may be enhanced by citrate ions, since preliminary results have shown that addition of Na citrate to a solution of alphas-casein with added Ca phosphate produces a broadening of the 31P signal as well as a chemical shift.

Calcium↗
PubMed1979

Light scattering properties of pluripotent and committed haemopoietic stem cells.

Mouse bone marrow cells were separated according to their light scattering properties in a light-activated cell sorter. The intensity of the scattered light was measured in a forward direction (FLS) and perpendicular to the incident light (PLS). These two parameters were found to give an indication of cell size and shape. The fractions containing cells with different light scxattering properties were assayed for CFU-s (pluripotent stem cells) content and for the incidence of three types of CFU-c (cells committed to granulocyte/macrophage differentiation). These measurements confirm previous size calculations based on sedimentation rate and density separation. During maturation, the different colony-forming units (CFU) show a gradual increase in PLS intensity. This suggests that the transition from CFU-s through to CFU-c 3 is accompanied by a morphological differentiation.

Animals↗
PubMed1979

The physical separation of three subpopulations of granulocyte/macrophage progenitor cells from mouse bone marrow.

The heterogeneity among progenitor cells of granulocytes and macrophages capable of forming colonies in vitro (CFU-c) is analysed. If specific culture conditions and stimuli are used, it is possible to discriminate among three subpopulations of CFU-c. CFU-c directly responding to CSF prepared from pregnant mouse uteri are characterized by a density of 1.075 g.cm-3. The G1-phase cells of this CFU-c subpopulation have a sedimentation rate of 4.8 mm.h-1. CFU-c of two additional subpopulations are induced to proliferate by enhancing factors present in serum of mice injected with endotoxin 18 hours earlier and in lysate of rat erythrocytes. CFU-c responding to postendotoxin serum have a density of 1.070 g.cm-3 and a sedimentation rate of 4.3 mm.h-1 (G1 cells). The CFU-c responding to erythrocyte lysate have a density of 1.080 g.cm-3 and a sedimentation rate of 5.3 mm.h-1 (G1 cells). The calculated diameters of G1-phase cells of these three CFU-c subpopulations range from 7.4 to 7.6 micron and are not significantly different. It is proposed that the three CFU-c detected represent three sequential maturation stages of granulocyte/macrophage progenitor cells. These cells are similar in size but increase in density (1.070 g.cm-3 to 1.080 g.cm-3) as they mature.

Animals↗
PubMed1978

Affinity chromatography studies with the pyruvate dehydrogenase complex of wild-type Escherichia coli.

1. The lifetime of thiamine pyrophosphate-Sepharose 2B affinity matrices synthesized according to Matsuura et al. (Matsuura, A., Iwashina, A. and Nose, Y. (1973) Biochem. Biophys. Res. Commun. 51, 241-246) has been improved. The matrix interacts with bacterial pyruvate dehydrogenase complexes. 2. The synthesis of a stable thiochrome-Sepharose 2B matrix is described. 3. Both matrices bind the pyruvate dehydrogenase complex of Escherichia coli in a 50 mM phosphate buffer, pH 7.0. Elution is possibly by an increase in ionic strength but not by the cofactor or metal-cofactor complexes. 4. The presence of Mg2+, reduces the capacity of the affinity matrices but leads to higher specificity for the multienzyme complex. 5. The pyruvate dehydrogenase complex of E. coli has been successfully purified by combining a classical purification step with these affinity chromatography systems. The method is less suitable for large scale operation.

Chromatography, Affinity↗
PubMed1977

Amino acid composition and sequence of kassinin, a tachykinin dodecapeptide from the skin of the African frog Kassina senegalensis.

Methanol extracts of the skin of the African amphibian Kassina senegalensis contain a dodecapeptide, kassinin, belonging to the family of tachykinins or physalaemin-like peptides. Kassinin, like all other natural tachykinins, possesses the characteristic C-terminal tripeptide Gly-Leu-Met-NH2 and a phenylalanine residue in position 5 from the C-terminus. However, the amino acid sequence in the N-moiety of the molecule differs sharply from that of the other tachykinins.

Amino Acid Sequence↗
PubMed1975

The pyruvate-dehydrogenase complex from Azotobacter vinelandii.

The pyruvate dehydrogenase complex from Axotobacter vinelandii was isolated in a five-step procedure. The minimum molecular weight of the pure complex is 600,000, as based on an FAD content of 1.6 nmol-mg protein-1. The molecular weight is 1.0-1.2 X 10(6), indicating 1 mole of lipoamide dehydrogenase dimer per complex molecule. Sodium dodecylsulphate gel electrophoretical patterns show that apart from pyruvate dehydrogenase (Mr89,000) and lipoamide dehydrogenase (Mrmonomer 56,000) two active transacetylase isoenzymes are present with molecular weight on the gel 82,000 and 59,000 but probably actually lower. The pure complex has a specific activity of the pyruvate-NAD+ reductase (overall) reaction of 10 units-mg protein-1 at 25 degrees C. The partial reactions have the following specific activities in units-mg protein-1 at 25 degrees C under standard conditions: pyruvate-K3Fe(CN)6 reductase 0.14, transacetylase 3.6 and lipoamide dehydrogenase 2.9. The properties of this complex are compared with those from other sources. NADPH reduced the FAD of lipoamide dehydrogenase as well in the complex as in the free form. NADP+ cannot be used as electron acceptor. Under aerobic conditios pyruvate oxidase reaction, dependent on Mg2+ and thiamine pyrophosphate, converts pyruvate into CO2 and acetate; V is 0.2 mumol 02-min-1-mg-1, Km(pyruvate)0.3 mM. The kinetics of this reaction shows a linear 1/velocity-1/[pyruvate] plot. K3Fe(CN)6 competes with the oxidase reaction. The oxidase activity is stimulated by AMP and sulphate and is inhibited by acetyl-CoA. The partially purified enzyme contains considerable phosphotransacetylase activity. The pure complex does not contain this activity. The physiological significance of this activity is discussed.

Acetyltransferases↗
PubMed1975

Separation of lipoamide dehydrogenase isoenzymes by affinity chromatography.

1. Lipoamide dehydrogenase NADH: lipoamide oxidoreductase, (EC 1.6.4.3) from pig heart has been separated into two sets of isoenzymes by chromatography on lipoyl- and NAD+-derivatized Sepharose-4B matrices. The first fraction is eluted at 30 mM sodium phosphate buffer (pH 7.2), the other requires a higher ionic strength. The two groups originate from the alpha-ketoglutarate and the pyruvate dehydrogenase complex respectively. 2, Hydrophobic chromatography on a homologous series of alkyl-Sepharoses lead to similar results. The first fraction is eluted with 30 mM phosphate buffer in the case of propyl- and butyl-Sepharose but a high ionic strength is required in the case of an increased chain length (C5--C6). The second fraction is reversibly bound on Sepharose-NC3 and -NC4 but binding becomes irreversible at higher chain lengths. 3. Aminoalkyl-Sepharose behave qualitatively as the alkyl derivatives although elution, particularly in the case of the second fraction, can be realized at lower ionic strength. 4. Matrices which are negatively charged (Sepharose-NCnCOOH, n equal 3--7) have no affinity at pH 7.2. 5. The influence of a neutral polar substituent has been studied by comparing the following matrices: Sepharose-NC6OH, Sepharose-NC6NH2 and Sepharose NC6. Binding of the various isoenzymes is completely reversible in the case of a Sepharose-NC6OH matrix and the elution behaviour is identical to that on propyl- and butyl matrices.

Alkanes↗