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Biomedical subjects

N Crawford

Publications and source records attributed to N Crawford.

At least 91 records · Page 5Linked to original sources
PubMed1981

Platelet and leucocyte calmodulins: isolation and characterisation.

The calcium-dependent regulatory proteins, calmodulins, have been isolated from human blood platelets and guinea pig peritoneal polymorphonuclear leucocytes using the urea methanol procedure of Grand et al. [Biochem. J. 177, 521-529 (1978)]. The calmodulins were purified to homogeneity as indicated by polyacrylamide gel electrophoresis and both proteins comigrated with bovine brain calmodulin with mobilities corresponding to molecular weights of 16 000-17 000. The yield of calmodulin from platelets was higher on a wet weight basis than the yield from leucocytes but the former compared favourably with yields reported for brain and other tissues. Both calmodulin preparations significantly stimulated brain cyclic nucleotide phosphodiesterase, erythrocyte ghost Ca2+ ATPase and platelet phosphorylase kinase activities at the microgram level. Stimulation of Lubrol-solubilised brain adenylate cyclase was only marginally significant with platelet calmodulin and rarely demonstrable with the leucocyte preparations. Although biological activities of both proteins were retained during storage at -20 degrees C, higher-molecular-weight aggregates slowly formed which could not be dissociated during dodecylsulphate/mercaptoethanol denaturation.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗
PubMed1980

The isolation and characterisation of guinea-pig polymorphonuclear leucocyte actin and myosin.

The contractile proteins actin and myosin have been isolated from the soluble phase of guinea-pig polymorphonuclear leucocytes and partially characterised. Two forms of actin have been identified, designated 'Mg-actin' and 'KCl-actin'. They have different polymerising properties but their propensity to form synthetic homologous and heterologous actomyosins and to inhibit DNAase-1 does not significantly differ. Both show beta and gamma isoelectric forms in focusing gels and the Mg-actin accounts for about 5% of the soluble-phase protein and te KCl-actin around 2%. Leucocyte myosin has been isolated by affinity chromatography on N6-ADP-Sepharose with a good enrichment of both Ca2+-ATPase and the ATPase activity measured in the absence of Ca2+ or Mg2+ and in the presence of EDTA. This protein, too, has the capacity to form synthetic homologous and hybrid actomyosins with enhancement of the basal Mg2+-ATPase activity. The ratio of actin to myosin in the leucocyte calculated on a molar basis is well in excess of 100, a figure consistent with the findings from other non-muscle cells.

Actins↗
PubMed1980

Secondary structure model for bacterial 16S ribosomal RNA: phylogenetic, enzymatic and chemical evidence.

We have derived a secondary structure model for 16S ribosomal RNA on the basis of comparative sequence analysis, chemical modification studies and nuclease susceptibility data. Nucleotide sequences of the E. coli and B. brevis 16S rRNA chains, and of RNAse T1 oligomer catalogs from 16S rRNAs of over 100 species of eubacteria were used for phylogenetic comparison. Chemical modification of G by glyoxal, A by m-chloroperbenzoic acid and C by bisulfite in naked 16S rRNA, and G by kethoxal in active and inactive 30S ribosomal subunits was taken as an indication of single stranded structure. Further support for the structure was obtained from susceptibility to RNases A and T1. These three approaches are in excellent agreement. The structure contains fifty helical elements organized into four major domains, in which 46 percent of the nucleotides of 16S rRNA are involved in base pairing. Phylogenetic comparison shows that highly conserved sequences are found principally in unpaired regions of the molecule. No knots are created by the structure.

Bacillus↗
PubMed1980

Platelet phosphorylase kinase activity and its regulation by the calcium-dependent regulatory protein, calmodulin.

Platelet phosphorylase kinase (ATP:phosphorylase phosphotransferase, EC 2.7.1.38) was found to be a Ca2+-sensitive enzyme. It was two Ka values for Ca2+ viz. 0.25 and 2.6 microM, respectively. The "calcium-dependent regulator" or calmodulin can enhance the activity of phosphorylase kinase, increasing its affinity for Ca2+. In the presence of calmodulin phosphorylase kinase has only one, high affinity binding site for Ca2+ (Ka = 0.27 microM). Platelet phosphorylase kinase can be phosphorylated by endogenous cyclic AMP-dependent protein kinase increasing its catalytic activity and this activation process is reversed by dephosphorylation. The changing level of intracellular Ca2+ and cyclic AMP may control the activity of phosphorylase kinase, regulating the mobilization of glycogen.

Blood Platelets↗
PubMed1980

Platelet microtubule subunit proteins.

Blood platelets contain microtubule proteins (tubulin and HMWs) which can be polymerised "in vitro" to form structure which resemble the microtubules seen in the intact platelet. Platelet tubulin is composed of two non-identical subunits alpha and beta tubulin which have molecular weights around 55,000 but can be resolved in alkaline SDS-polyacrylamide gel electrophoresis. These subunits associate as dimers with sedimentation coefficients of about 5.7 S although it is not known whether the dimer protein is a homo- or hetero-dimer. The dimer tubulin binds the anti-mitotic drug colchicine and the kinetics of this binding are similar to those reported for neurotubulins. Platelet microtubules also contain two HMW proteins which appear to be essential and integral components of the fully assembled microtubule. These proteins have molecular weights greater than 200,000 daltons. Fluorescent labelled antibodies to platelet and brain tubulins stain long filamentous microtubular structures in bovine lens epithelial cells and this pattern of staining is prevented by exposing the cells to conditions known to cause depolymerization of cell microtubules.

Animals↗
PubMed1980

Platelet phosphoprotein phosphatase activity. Its subcellular distribution and regulation.

Phosphoprotein phosphatase (EC 3.1.3.-) activity was found in human platelet homogenates and this activity was stimulated up to 20-fold by preincubation with trypsin. Both Mg2+ and Mn2+ greatly decreased the activity of trypsin-activated phosphatase but the activity of the untreated phosphatase was not affected by increasing the concentration of these divalent cations. It was also shown that the activity of the phosphatase underwent a transient inhibition upon addition of ATP and a permanent one with ATP-gamma-S.

Adenosine Triphosphate↗
PubMed1978

The subcellular distribution of [3H]-colchicine-binding activity and tubulin in pig blood platelets.

The subcellular distribution of the [3H]-colchicine-binding protein, believed to be tubulin, the subunit protein of microtubules, has been investigated in mammalian blood platelets. Studies on a soluble extract from pig platelets and two particulate fractions (viz. membrane-rich and granule-rich fractions) have shown that about 98% of the colchicine-binding activity in a platelet homogenate is located in the soluble phase. This result is in agreement with polyacrylamide gel electrophoresis experiments which show that the soluble fraction contains a substantial amount of 55,000 MW tubulin, whereas the membrane and granule-rich fractions contain very little of this component. The [3H]-colchicine-binding activity of the platelet soluble phase is largely precipitated by 40-50% ammonium sulphate and also by vinblastine sulphate in millimolar concentrations. Moreover the colchicine-binding protein in the platelet soluble fraction has a sedimentation coefficient of 5.9 S, is eluted in the void volume of a Sephadex G-100 column, and binds to DEAE-Sephadex at low ionic strength and is eluted from this ion-exchanger at an ionic strength of 0.47 M-KCl. In addition, most of the colchicine-binding activity of the platelet soluble phase is associated with protein which will undergo temperature-dependent polymerization in vitro and which has a molecular weight on SDS-polyacrylamide gels of 55,000. All these experimental findings suggest that the colchicine-binding activity of pig platelet homogenates is due to the presence of the microtubule protein, tubulin, which is largely found in the soluble compartment of the cells.

Actins↗
PubMed1977

Bis-(4-methylumbelliferyl) phosphate as a substrate for the surface membrane-associated phosphodiesterase activity of pig platelets.

It was found that the newly-available compound, bis-(4-methylumbelliferyl) phosphate, could be used as a substrate for the pig platelet surface membrane-associated phosphodiesterase activity, usually assayed with bis-(p-nitrophenyl) phosphate. This enzyme activity is distinct from the phosphodiesterase activity towards 5'-dTMP-P-nitrophenyl ester, which is probably associated with intracellular membrane structures in platelets. Consequently, the use of the 4-methylumbelliferyl derivative as substrate for the phosphodiesterase activity provides a sensitive, fluorimetric assay for this marker enzyme of the platelet surface membrane.

Animals↗
PubMed1977

Localization in platelets of sodium 51Cr-chromate, 125I-antibody to whole membrane, and 3H-diisopropylfluorophosphate using electron microscopic autoradiography.

Platelets from the rabbit and rat, respectively, were incubated with sodium 51Cr-chromate and 3H-diisopropylfluorophosphate (3H-DFP) in the same manner as for platelet survival studies. A third isotope, 125I, was incorporated into a labeled antibody raised against a partially purified pig platelet membrane fraction. The 51Cr label was found to be concentrated predominantly in the cytoplasm, with a marked degree of association with mitochondria, whereas the tritium label appeared to be largely confined to the periphery of the platelet, particularly in association with the open canalicular system. The 125I-labeled antibody showed a very marked association with the plasma membrane and the open canalicular system, suggesting a membrane-bound affinity.

Animals↗