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Biomedical subjects

N Crawford

Publications and source records attributed to N Crawford.

At least 73 records · Page 4Linked to original sources
PubMed1985

Electrophoretic analysis of red blood cells in polycythaemia.

In contrast with previously published observations, this study has found no increase in the net negative surface charge of the red blood cells (RBC) from patients with various forms of polycythaemia when compared with normal subjects. Two sub-populations of RBCs with different electrophoretic mobilities (EPM), termed fast and slow, were present in samples from both patients and normal individuals. Frequently, the slow cell population in RBCs from patients with polycythaemia, particularly the apparent polycythaemia sub-group, was of a lower EPM and contained more cells than the corresponding population in normal subjects. The membrane-bound sialic acid content of RBCs from patients with primary proliferative polycythaemia, associated with iron deficient RBC changes, was increased if the results were expressed per unit volume of RBCs and decreased if the sialic acid concentration was presented per number of RBCs. These results imply that differences in surface charge of RBCs are of no value in discriminating between primary and secondary polycythaemia.

Electrophoresis↗
PubMed1984

The inhibition of platelet cyclo-oxygenase by aspirin is associated with the acetylation of a 72kDa polypeptide in the intracellular membranes.

Previous studies by Roth & Majerus [J. Clin. Invest. (1975) 56, 624-632] showed that exposure of platelets to [acetyl-14C]aspirin resulted in the radioactive labelling of three polypeptides, two of which were in the cytosol and not saturable, whilst the third was located in particulate material, and was saturated at 30 microM-aspirin. By using high voltage free flow electrophoresis to separate a platelet mixed membrane fraction into highly purified surface and intracellular membrane subfractions, we have confirmed that the major polypeptide acetylated after exposing whole platelets to [acetyl-14C]aspirin is almost exclusively associated with intracellular membrane structures. We have shown previously that these intracellular membranes are the major site for prostanoid biosynthesis [Carey, Menashi & Crawford (1982) Biochem. J. 204, 847-851] and in the present study the extent of the radioactive labelling correlated well with inhibition of the cyclo-oxygenase activity in these intracellular membranes. In sodium dodecyl sulphate/polyacrylamide-gel electrophoresis the [14C]acetylated component, which appears to be a dimer, migrates with a mobility corresponding to 72kDa. Although cyclo-oxygenase is inhibited, there is no discernible radioactive labelling when the platelets are exposed to aromatic-ring-labelled [14C]aspirin. We suggest that the site or sites for aspirin acetylation and cyclo-oxygenase activity are structurally associated in the platelet's intracellular membranes referred to by electron microscopists as the dense tubular membrane system.

Acetylation↗
PubMed1984

Characterization of the calcium-sequestering process associated with human platelet intracellular membranes isolated by free-flow electrophoresis.

By using density-gradient fractionation and high-voltage free-flow electrophoresis, human platelet membranes were separated into highly purified subfractions of surface (SM) and intracellular (IM) origin. Associated exclusively with the IM fraction is an ATP-dependent Ca2+ uptake that, in the absence of oxalate, reaches steady-state levels in 5-10 min. When Ca2+-EGTA buffers were used to control the external Ca2+ concentrations (range 0.1-50 microM) there was an increase in the intravesicle steady-state level of Ca2+ up to 10 microM external Ca2+ concentration. Above this level the intravesicle space becomes saturated at a concentration between 10 and 20 nmol of Ca2+ X (mg of protein)-1. The ionophore A23187 promotes a rapid and almost total release of the sequestered Ca2+ (greater than 90%, t1/2 1-2 min). The presence of oxalate in the external medium greatly enhances the Ca2+ accumulation to levels as high as 200 nmol X (mg of protein)-1, but the uptake process is more variable and rarely reaches steady-state level even after 2 h incubation. Moreover, accumulation in the presence of oxalate effects ionophore release with less than 80% depletion in 45-60 min. These findings, taken together with the known presence in the platelet of a wide variety of functional and metabolic processes triggered by this cation, suggest that the platelet IM has a key role in controlling cytosolic Ca2+ concentrations.

Adenosine Triphosphate↗
PubMed1984

Subcellular localization and some properties of lipoxygenase activity in human blood platelets.

Lipoxygenase activity was measured in human platelet subcellular fractions. From a sonicated platelet preparation, a granule fraction, mixed membranes (surface and intracellular) and cytosol fractions were separated by differential centrifugation. With respect to activities in the sonicated preparation, the lipoxygenase was slightly enriched in both the cytosol and mixed-membrane fractions and consistently de-enriched in the granule fractions. Approx. 65% and 20% of the total cell enzyme activity were found in the cytosol and mixed membranes respectively, with only 8% present in the granule fraction. Additionally we measured the lipoxygenase activity in purified surface- and intracellular-membrane subfractions prepared from the mixed membranes by free-flow electrophoresis. There was a slight enrichment in activity in the intracellular membrane fraction compared with that in the mixed membranes, and a depletion of activity in the surface membranes. Characterization of the enzyme activity, i.e. time course, pH-dependence, Ca2+-dependence, Vmax. and Km for arachidonic acid, and the carbon-position specificity for this acid, failed to reveal any significant differences between the membrane-bound and soluble forms of the lipoxygenase. These findings suggest that in human platelets the same lipoxygenase is associated with the membranes as in the cytosol and that the membrane-bound activity predominates in intracellular membrane elements.

Arachidonate Lipoxygenases↗
PubMed1984

Two-dimensional polyacrylamide-gel electrophoresis of the proteins and glycoproteins of purified human platelet surface and intracellular membranes.

By using highly purified surface and intracellular membrane fractions prepared from human platelets by free-flow electrophoresis, the polypeptide and glycopeptides of these membranes have been characterized by high-resolution gel electrophoresis under reducing and non-reducing conditions. Silver staining and a variety of glycoprotein-staining procedures have been applied to identify the major components. The principal finding was the clear disparity between the distribution patterns for these two membrane fractions. There are proportionately more low-Mr acidic components present in the intracellular membrane than in the surface-derived membrane. Of the major platelet surface glycoproteins GPIb, IIb, IIIa and IIIb (or IV) well expressed in the surface membrane only, GPIIb and IIIa appear as trace components in the intracellular membrane. The cytoskeleton proteins, actin, myosin, tropomyosin, actin-binding protein and alpha-actinin are prominent features of the surface membrane and essentially absent from the intracellular membrane. Neuraminidase treatment at the whole-cell level, before homogenization, which is an essential requirement for good resolution of the two membrane subfractions, modifies a number of the glycoprotein subunits with respect to their pI characteristics, suggesting much molecular micro-heterogeneity with respect to sialic acid content. A comparison of the staining characteristics of the major glycoproteins with periodic acid/Schiff's reagent and concanavalin A/peroxidase detection and a combined procedure revealed significant differences in associated carbohydrate structures, and the major concanavalin A-binding component was shown to be GPIIIa. These observations are discussed in the context of functional activities of both membrane systems in the physiological behaviour of the platelet.

Blood Platelets↗
PubMed1983

Preparation of a highly purified surface membrane fraction from rabbit polymorphonuclear leucocytes by high-voltage free-flow electrophoresis.

A surface membrane fraction of high purity and good yield has been prepared from homogenates of rabbit peritoneal polymorphonuclear leucocytes, using a preliminary sorbitol density gradient sedimentation followed by preparative high voltage electrophoresis in a thin flowing buffer film. Enrichment values for the plasma membrane marker enzyme 5'-nucleotidase and 125I-labelled Lens culinaris lectin, after the latter had been applied at the whole cell level, were 18-fold and 6-fold, respectively. Contamination of the surface membrane fraction by other organelles was negligible and approximately 1 mg of surface membrane protein can be obtained from 2 . 10(9) leucocytes. A triacylglycerol-rich, protein-poor fraction that lacks any definable structure in electron microscopy separates discretely from the surface membrane vesicles during electrophoresis. It is considered that this may be a contaminant not previously recognized as present in membrane fractions prepared by more conventional procedures.

5'-Nucleotidase↗
PubMed1983

Redistribution of membrane 5'-nucleotidase in rabbit peritoneal polymorphonuclear leucocytes during phagocytosis.

Using a quantitative phagocytic model involving oil droplet internalisation rabbit polymorphonuclear leucocytes display selective segregation of membrane constituents during the phagocytic event. A resting cell surface membrane and fractions representing vesicle membranes and uninvolved surface membranes from the active cells have been purified by density gradient sedimentation and free flow electrophoresis. The specific activity of 5'-nucleotidase, a major neutrophil surface membrane glycoprotein, was 3-fold higher in the uninvolved membrane of phagocytosing cells than in the resting cell membrane. The activity in the vesicle membranes was substantially depleted. In contrast Lens culinaris receptors showed no redistribution during phagocytosis: the two surface domains showing essentially the same enrichment with respect to homogenate as the resting cell surface membrane.

5'-Nucleotidase↗
PubMed1982

Ecto-protein kinase activity in rabbit peritoneal polymorphonuclear leucocytes.

An ectoprotein kinase activity has been identified on intact rabbit peritoneal polymorphonuclear leucocytes and the time course of phosphate incorporation into proteins has been followed at different ATP levels. Saturation is reached at around 3 mM ATP and the activity is inhibited by p-chloromercuribenzoate. The possibility that the observed protein phosphorylation arises through the action of a membrane ATPase liberating phosphate for transfer into the cell, incorporation into ATP and its utilisation by endogenous kinases, has been excluded by studying both enzymes concomitantly and measuring the rate of [32P]orthophosphate uptake. Lactate dehydrogenase measurements in the extracellular media also exclude the possibility of kinase liberation from lysed cells. Moreover, the pattern of 32P-labelling of polypeptides when intact cells are exposed to [32P]ATP is quite different from that when homogenates are incubated with [32P]ATP or intact cells with [32P]-orthophosphate. We have been unable to demonstrate any cAMP dependency for this ectokinase activity.

Adenosine Triphosphatases↗
PubMed1982

Localization of cyclo-oxygenase and thromboxane synthetase in human platelet intracellular membranes.

Platelet mixed membrane fractions can be separated into discrete vesicle subpopulations of surface and intracellular origin. Intracellular membrane vesicles are the predominant site of phospholipid-modifying enzymes that liberate arachidonic acid. We report the selective enrichment in intracellular membranes of cyclo-oxygenase and thromboxane synthetase activities. Surface membrane fractions show no such enrichment. These results suggest that a sequence of activities leading to the biosynthesis of thromboxane from arachidonate is associated with the intracellular membrane elements known as dense tubular membranes.

Arachidonic Acid↗
PubMed1982

The phospholipid and fatty acid composition of human platelet surface and intracellular membranes isolated by high voltage free flow electrophoresis.

Human platelets were labeled with tracer doses of [14C]arachidonic acid, then fractionated into mixed membranes which are separated into intracellular membranes and two different domains of surface membranes by high voltage free flow electrophoresis. Each subfraction was analyzed for its phospholipid content. Glycerophospholipids were separated by high performance liquid chromatography and their fatty acids analyzed by glass capillary gas chromatography. Intracellular membranes appeared substantially depleted in sphingomyelin, while enrichment of this phospholipid was seen in surface membranes. PC and PI were more enriched in intracellular membranes than in the surface membranes and the contrary was observed for PE. On the other hand, the pattern of the phospholipid labeling by [14C]arachidonate followed closely the glycerophospholipid profiles of the membrane subfractions, but the specific radioactivity of PI was higher than of PC, which itself was higher than that of PE. Moreover, the endogenous content of arachidonic acid accentuates these tendencies. The percentage of arachidonate in PE was higher in the surface membranes than in the intracellular membranes and the contrary was observed for arachidonyl-PC and PI. These differences were compensated for by certain saturated and monounsaturated fatty acids present in the composition profiles. These findings are discussed in relation to the membrane localization of lipases involved in the liberation of arachidonic acid for prostanoid synthesis.

Blood Platelets↗
PubMed1981

Characterization of human platelet surface and intracellular membranes isolated by free flow electrophoresis.

High voltage free flow electrophoresis has been applied to the separation of human platelet membranes. After short treatment with neuraminidase at the whole cell level, three membrane vesicle subpopulations have been isolated. Using a surface label (125I-labeled Lens culinaris lectin), the marker enzyme NADH-cytochrome c reductase, and lipid analysis, two of the fractions have been identified as of surface origin and the other consists of intracellular membrane elements. The distribution of adenylate cyclase, leucyl aminopeptidase, 5'-nucleotidase and Ca2+-ATPase has also been investigated, and their usefulness as markers for the different membrane fractions has been evaluated. All three fractions are vesicular but differ in size and character. Their phospholipid and cholesterol contents have been determined, and the cholesterol/phospholipid ratios of the two surface fractions are over twice that of the intracellular membrane, which also has a significantly lower microviscosity as determined by fluorescence polarization using diphenyl hexatriene. The polypeptide profiles from sodium dodecyl sulfate-polyacrylamide gel electrophoresis are particularly distinctive, with actin present in the two surface membrane fractions and absent from the intracellular membranes. Myosin, confirmed by its ATPase characteristics, is almost exclusively localized in one of the surface membrane fractions, and actin-binding protein is a prominent feature of the other.

5'-Nucleotidase↗
PubMed1981

Inhibition of transcription factor activity by poliovirus.

To study the poliovirus-induced inhibition of host-cell RNA synthesis, we prepared transcription extracts from mock-infected and poliovirus-infected HeLa cells. In contrast with the control extracts, poliovirus-infected cell extracts prepared 3 hr after infection were unable to transcribe specifically DNA templates recognized by RNA polymerase II. Accurate transcription by RNA polymerase III, however, was only slightly reduced. Supplementation of the infected cell extract with a crude preparation of transcription factors (S100) restored its ability to transcribe a polymerase II template specifically; supplementation with purified polymerase II had no effect. When the S100 was fractionated on a phosphocellulose column, the restoration activity eluted between 0.35 M and 1 M KCl. When we tested infected extracts for inhibitory activity by mixing uninfected and infected cell extracts, no in vitro inhibition of polymerase II transcription by the uninfected extract was evident. These results indicate that at least one factor required for specific transcription by polymerase II is deficient in extracts from poliovirus-infected cells.

HeLa Cells↗