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Protein Structure and Function
Protein & Cell Aggregation
protein & cell aggregation
absorbance
Glycosylation at Asn-184 inhibits the conversion of single-chain to two-chain tissue-type plasminogen activator by plasmin
Biochemistry 29: 4175–4180 (1990).
Arthur J. Wittwer and Susan C. Howard.
Department of Cell Culture and Biochemistry, Central Research Laboratories, Monsanto Company, St. Louis, MO 63167 USA.
Summary. Tissue-type plasminogen activator (tPA) a glycosylated serine protease is an effective thrombolytic agent. The native single-chain tPA (sc-tPA) is converted by plasmin to a two-chain tPA (tc-tPA). Two glycoforms of native sc- tPA are known. Type I sc-tPA is fully glycosylated, while type II lacks glycosylation at Asn-184. The rates at which type I and type II human melanoma sc-tPA were converted to type I and type II tc-tPA by plasmin were determined by observing the amidolytic activity using a nitroanilide chromogenic substrate and following the rate of change at 405 nm in a Molecular Devices VMax kinetic microplate reader. Results indicate that glycosylation of tPA at Asn-184 hinders conversion of sc-tPA to tc-tPA. Glycosylation of tPA may serve to modulate activity permitting type I sc-tPA to persist in the single-chain form longer than type II sc-tPA under physiological conditions.
Measuring platelet aggregation with a microplate reader. A new technical approach to platelet aggregation studies
Am. J. Clin. Pathol. 94: 613–617 (1990).
Joseph C. Fratantoni and Betty J. Poindexter.
Division of Blood and Blood Products, Center for Biologics Evaluation and Research, Bethesda, MD 20892 USA.
Summary. Platelet aggregation measurements were performed with the use of a VMax microplate reader (Molecular Devices Corp.) Authors report that many wavelengths were evaluated and found to yield equivalent results. The largest change in optical density is observed at lower wavelengths. The 96-well microplate permits all test and control platelet samples including replicates to be observed simultaneously over the course of a 10 to 15 minute assay. The technique was validated by demonstrating the similarity of dose-response curves to those obtained with a standard aggregometer for the stimulants adenosine diphosphate, thrombin, and arachidonic acid.
Dramatic aggregation of Alzheimer Ab by Cu(II) is induced by conditions representing physiological acidosis
J. Biol. Chem. 273: 12817–26 (1998).
Craig S. Atwood1, Robert D. Moir2, Xudong Huang1, Richard C. Scarpa1, N. Michael E. Bacarra1, Donna M. Romano2 , Mariana A. Hartshorn1, Rudolph E. Tanzi2, and Ashley I. Bush3.
1 Department of Psychiatry and Genetics and Aging Unit.
2 Department of Neurology and Genetics and Aging Unit, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts 02114.
Methods: protein aggregation. Turbidity measurements, as an assay for aggregation, were performed in a flat-bottomed 96-well microtiter plate (Corning Costar Corp.), and absorbances (405 nm) were measured using a SpectraMax Plus spectrophotometric microplate reader (Molecular Devices, Corp.) Automatic 30-second plate agitation mode was selected for the plate reader to evenly suspend the aggregates in the wells before all readings.
Protein C inhibitor acts as a procoagulant by inhibiting the thrombomodulin-induced activation of protein C in human plasma
Blood 91: 1542–47 (1998).
Marc G.L.M. Elisen, Peter A.Kr. von dem Borne, Bonno N. Bouma, and Joost C.M. Meijers.
Department of Haematology, University Hospital and Institute of Biomembranes, Utrecht University, Utrecht, The Netherlands.
Methods: gel assembly assay. Turbidimetry was used to monitor the TM-mediated thrombin inhibition by PCI. The change in turbidity during fibrin formation was monitored at 405 nm in a microplate reader. An increase in turbidity indicated gel assembly. All experiments were performed in citrated plasma recalcified with CaCl2 (final concentration of 17 mmol/L) resulting in a free Ca2+ concentration of 2.3 mmol/L. A mixture of recombinant tissue factor (Innovin, final dilution 3 x 104) and calcium necessary for recalcification was added to 67.5 µL of plasma to initiate clotting. The volume was adjusted to 125 µL with HEPES buffer, resulting in a final plasma concentration of 54%. After mixing, 100 µL of the reaction mixture was transferred to a microplate, and turbidity at 405 nm was monitored at 37 °C using a SpectraMax 340 microplate reader (Molecular Devices Corp.)
Clusterin has chaperone-like activity similar to that of small heat shock proteins
J. Biol. Chem. 274: 6875–81 (1999).
David T. Humphreys, John A. Carver, Simon B. Easterbrook-Smith, and Mark R. Wilson.
Methods: protein precipitation assay. Solutions of clusterin were prepared in 0.3 mL of 50 mM sodium phosphate containing 0.1 M NaCl and incubated at 37 °C with or without 20 mM DTT. During this period absorbance readings at 360 nm (measures light scattering) were acquired every 5 minutes for a total of 5 hours in a SpectraMax 250 plate reader (Molecular Devices Corp.)
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