Neville Crawford

Isolation of pig platelet membranes and granules distribution and validity of marker enzymes

Abstract A method for the subcellular fractionation of pig platelet homogenates by sucrose density gradient centrifugation is described. The procedure is simple, highly reproducible and yields two major particulate fractions and a soluble phase. One particulate fraction consists almost entirely of membrane fragments and is relatively free from granule contamination. The other particulate zone contains the platelet granules and mitochondria. The distribution on the gradients of the enzymes lactate dehydrogenase, succinate dehydrogenase, 5′-nucleotidase, leucyl β-naphthylamidase and cholinesterase has been studied and organelle localisation further substantiated by electron microscopy. The degree of solubilisation of certain marker enzymes during homogenisation has been investigated and the parallel release of these enzymes with the soluble phase marker enzyme lactate dehydrogenase, suggests they have a true biphasic location between the soluble and particulate components of the cell. No significant difference was found in the molar ratios of cholesterol to phospholipid in the subcellular fractions but the content of each lipid was twice as high in the membrane fraction as in the granule fraction.

The isolation and characterisation of platelet microtubule proteins.

Abstract Tubulin, the microtubule subunit protein, has been isolated from a soluble extract of pig platelets, by an in vitro polymerisation process. Several physicochemical properties of platelet tubulin have been investigated and compared with those of mammalian brain tubulins. The molecular weight of the tubulin monomeric subunits was found to be 55 000 by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, and the platelet protein co-migrated with rat, rabbit, guinea-pig, mouse and calf brain tubulins. The [3H]colchicine-binding dimer form of platelet tubulin sedimented in a 5% to 20% linear sucrose gradient with a sedimentation coefficient of 5.7 S, compared with that of 5.9 S for rat brain tubulin. The amino acid compositions of platelet and mammalian brain tubulins were found to be very similar, with glutamate and asparate as the predominant residues. Moreover, the one and two dimensional fingerprints of 125I-labelled tryptic peptides of platelet and brain tubulins showed considerable similarity. Platelet tubulin could be separated into two non-identical (α and β) subunits by Tris/glycine discontinuous alkaline sodium dodecyl sulphate-polyacrylamide gel electrophoresis and the β-tubulin had two or three 125I labelled peptides not seen in the α-tubulin. Two, or perhaps three, high-molecular-weight proteins were always present in the platelet tubulin samples, prepared by in vitro polymerisation and these had molecular weights greater than 200 000 and were probably analogous to the high-molecular weight proteins reported to be present in brain microtubule preparations. Platelet tubulin with its associated high molecular weight proteins will assemble in vitro into tubular structures which in electron micrographs resemble brain microtubules and the microtubules seen in whole platelets. It is believed that this is one of the first mammalian microtubule systems other than that of nervous tissue which has been isolated and studied in detail at the subunit level.

Isolation of tubulin from pig platelets

Following the introduction of glutaraldehyde fixation [ 11, microtubules are now frequently seen in electron micrographs of blood platelets. They are present in the normal discoid-shaped cells as prominent circumferential rings lying subadjacent to the surface membrane. In the activated platelet, a condition which subsequently leads to increased adhesiveness and aggregation as part of the haemostatic process, the microtubules may then be seen extending into and throughout the length of the pseudopodia. Their morphology has been reviewed by Behnke [2], and by White [3]. Platelets also contain microfilaments which are smaller in diameter than the microtubules and these are now considered to be the components of an actomyosin-like contractile complex [4]. Whilst the microtubules are believed to have a cytoskeletal role and the microfilaments contractile potential [S], the exact functions and interrelationship of these two polymeric protein systems in platelet motile behaviour, such as shape change, aggregation and clot retraction, are by no means clear. The best studied mammalian microtubular system is that of brain where the subunit protein of the tubules, known as tubulin, has been isolated [6-81, and may accouni for between 1.5 and 40% of the soluble proteins of brain [9]. This subunit is a dimer, has a mol. wt of 110 000-l 20 000 and a sedimentation coefficient of about 6s [lo]. It can be split with denaturing agents to give two non-identical monomers

Effect of divalent cations and chelating agents on the ATPase activity of platelet contractile protein, "thrombosthenin".

Abstract 1. (1) the effect of various divalent cations and EDTA and EGTA on the pig platelet contractile protein “thrombosthenin” has been investigated. Under both low and high ionic strength conditions of assay the Ca 2+ ATPase activity greatly exceeds the Mg 2+ ATPase. With increasing ionic strength, Mg 2+ ATPase decreases and the Ca 2+ ATPase is greatly increased due to dissociation of the actomyosin-like component of thrombosthenin. Mg 2+ inhibits the Ca 2+ ATPase of thrombosthenin and inhibition can be detected at 25 μmolar concentrations of Mg 2+ in the presence of 3 m M Ca 2+ . 2. (2) At low chelate concentrations ( M ), EDTA enhances and EGTA inhibits thrombosthenin ATPase, measured in the absence of added divalent cations. At higher chelate concentrations (>0.5 m M ) both EDTA and EGTA were inhibitory towards the Ca 2+ ATPase activity measured in the presence of 3 m M Ca 2+ . The findings suggest that thrombosthenin preparations isolated by high ionic strength extraction and low ionic strength precipitation, contain both these divalent cations endogenously associated. 3. (3) ATPase activity profiles with various divalent cations (Ca 2+ , Mg 2+ , Sr 2+ , Mn 2+ , Be 2+ , Ba 2+ , Cd 2+ , Co 2+ and Zn 2+ ) revealed that only Ca 2+ and Mn 2+ significantly activated, Mg 2+ and Sr 2+ weakly activated and Cd 2+ , Co 2+ and Zn 2+ were all inhibitory (80–100%, at cation concentrations equimolar with substrate ATP-3 m M ). 4. (4) Although on the basis of the ATPase activities, thrombosthenin contains some actomyosin-like protein in the complex the findings suggest that it also includes a considerable excess of an unassociated myosin-like component, rich in Ca 2+ ATPase activity.

Subcellular distribution of 5 hydroxytryptamine in pig platelets.

Abstract The subcellular distribution of 5 hydroxytryptamine (5HT) in pig platelets before and after incubation of the cells with the amine has been investigated by density gradient fractionation. Conditions of homogenization and fractionation were designed to separate the platelet surface and intracellular membrane structures from the granular components. The major fractions have been partially characterized by electron microscopy and by certain marker enzymes. While a low density membrane zone separates well with minimal contamination the higher density granule fraction is a heterogeneous mixture of mitochondria and a -granules, small osmiophilic dense bodies and numerous unidentified organelles. Evidence has been presented for the existence of two separate storage compartments for 5HT. One site in the granule zone accounts for about 10 per cent of the total cell 5HT content stored as a relatively strongly bound complex. The other major storage compartment is within the extragranular cytoplasmic pool and this accounts for about 80 per cent of the cell 5HT. In this compartment the amine is either weakly associated or freely diffusible. The major proportion of the 5HT absorbed by the platelet during incubation in the presence of 5HT can be recovered from the low density fraction of the cell as a soluble component. Platelet granules exhibit a low capacity but have a high affinity for the absorbed amine.

The separation of platelet membranes and the various granular organelles by a simple density gradient procedure using the B14 zonal rotor

A new procedure is described for the subcellular fractionation of pig platelets, using the B14 titanium zonal rotor. By this procedure, it was possible to separate simultaneously, using a simple, sucrose linear gradient, the major platelet particulate components (i.e., membrane vesicles, hydrolase-containing organelles, mitochondria, and 5-HT storage granules), into clearly-resolved fractions. The evidence for this separation was provided by electron microscopy and the enrichment of various marker enzyme activities in different zonal fractions. The distribution of several hydrolase activities between the zonal fractions was also investigated. Fractions containing membrane vesicles showed five- to sixfold enrichment of phosphodiesterase activities towards bis-(p-nitrophenyl) phosphate and 5′-dTMP-p-nitrophenyl ester. The activities of various hydrolases (including acid phosphatases, glycosidases, and 3′-dTMP-p-nitrophenyl ester phosphodiesterase) were increased two- to fourfold in fractions containing granular organelles. These were clearly separated from fractions containing mitochondrial structures, in which mitochondrial enzyme activities were enriched 8- to 11-fold. Assays for 5-HT indicated that the amine storage granules were present as a further discrete fraction at the dense end of the zonal gradient.

Subcellular distribution of reserpine and 5-hydroxytryptamine in blood platelets after treatment with reserpine in vitro and in vivo.

Abstract Using subcellular fractionation procedures with sucrose density gradients the distribution of reserpine and 5-HT in pig platelets has been investigated following exposure of the cells to the agents in vitro and in vivo . Pig platelets release about 50 per cent of their 5-HT content during 1–2 hr incubation at 37° with reserpine at levels to 0.2–1.0 μm/ml. The release is accompanied by a concomittant increase in the concentration of amine in the surrounding medium with little evidence of metabolic degradation. The platelets readily absorb 5-HT from the surrounding medium and will increase their content about 2-fold. This effect is completely blocked by the presence of reserpine at an incubation level of 1 μm/ml. Subcellular fractionation of platelets after exposure to reserpine revealed that the fraction containing granular organelles is most affected and total release from this store takes place even when the cells are preloaded with 5-HT before the reserpine exposure. Analysis of the reserpine content of the fractions showed binding to both the membranous and granular organelles and the proportion bound increases almost linearly with reserpine concentration, in the incubation media, in the range 1–4 μm/ml. Although reserpine levels as low as 0.2 μm/ml effect significant 5-HT release from the whole cell, concentrations as high as 15 μm/ml are required for significant release from isolated 5-HT granules. Rabbit platelets differ from the pig platelets in that they will absorb 5-HT both in vitro and in vivo after reserpinization in spite of the fact that reserpine induced 5-HT release is similar for both species. It seems that although the surface membrane of the platelet and the limiting membrane of the storage granule are both targets for reserpine and show similar and significant binding affinities for the alkaloid they differ in the magnitude of their response suggesting that high intracellular local concentrations of reserpine are required for granule release. However, since reserpine release from the whole cell primarily induces granule bound 5-HT depletion, the intracellular equilibrium between the tightly stored component and the more diffusable soluble phase pool is of considerable importance in the way in which the membrane responds to the agent by alterations in 5-HT flux.

The [3H]colchicine-binding properties of platelet tubulin

Abstract 1. 1. The [3H]colchicine-binding properties of platelet microtubule protein (tubulin) were studied in order to explore the nature of the interaction between platelet tubulin and the anti-mitotic alkaloid and to determine the optimum assay and storage conditions for the protein. 2. 2. A DEAE-cellulose filter disc assay was developed to estimate colchicine-binding activity. 3. 3. The colchicine-binding activity of platelet tubulin was quite sensitive to pH, with maximal binding occurring at pH 6.8 and was a fairly slow reaction, reaching a maximum after about 60 min at 37°C. 4. 4. The reaction was sensitive to incubation temperature (maximal binding at 37°C) and the ability of the protein to bind the drug was shown to decay at different rates at different temperatures. 5. 5. Sucrose, sodium glutamate and low temperature were all found to stabilize the colchicine-binding site of platelet tubulin. 6. 6. The association of colchicine and platelet tubulin was shown to be non-covalent and work with colchicine analogues suggested that a methoxy or equivalent group on the tropolone ring of the colchicine molecule may be a requirement for platelet tubulin binding.

Evidence against tubulin--actin homology.

The microtubule protein, tubulin, from brain, blood platelets, cilia and flagella and the microfilament protein, actin, from muscle and cytoplasmic sources all have similar amino acid compositions [ 1,2] and the involvement of the polymeric forms of these two subunit proteins in a variety of cellular motile activities has raised the question of the possible presence of regions of homology in their structure. This has become a topic of some considerable controversy. Puszkin and her colleagues [3,4] reported that colchicine-binding proteins (or presumptive tubulins) isolated from brain and blood platelets enhanced over 20-fold the Mg2+-dependent ATPase activity of muscle myosin, a property generally regarded as specific for actin [5]. Stephens, however, in a fingerprint study of tryptic and chymotryptic peptides of adductor muscle actin and ciliary tubu1i.r from the scallop Pecten irradians clearly demonstrated that these two proteins had very dissimilar twodimensional peptide patterns [6]. In another report Mohri and Shimomura [7] compared the properties of tubulin and actin isolated respectively from seaurchin sperm flagella and sea-urchin latern muscle and showed that although both proteins formed superprecipitates with rabbit skeletal muscle myosin in the presence of ATP, only the latern muscle actin increased the Mg”-dependent ATPase activity of muscle myosin at low ionic strength. Furthermore, only the synthetic actomyosin and not the tubulinmyosin mixture showed an ATP-inducible viscosity decrease under high ionic strength conditions. In a more recent study however, _Alicea and Renaud [8] examined the effect of Tetrahymena ciliary tubulin on the Mg’Tdependent ATPase of chicken breast muscle myosin and recorded an almost 4-fold increase in Mg”-ATPase activity. They concluded that this was strong evidence for actin tubulin homology. In our studies [9,10] of the properties of actins and tubulins isolated from pig platelets and mammalian brains we showed that although there are some similarities in certain properties the two subunit proteins differ significantly in their molecular weights, drug binding affinities, subcellular localisation and the polymeric structures which they form. Although the amino acid compositions of platelet and brain tubulin are virtually identical, they differ significantly from those of platelet and muscle actins in at least 6 residues [2] . Furthermore, fluorescently-labelled anti-actin and anti-tubulin antibodies stained morphologically quite different structures in bovine lens epithelial cells [lo] . In the light of these findings and the controversy in the literature, we felt it pertinent to re-examine the homology question, and in this paper we present experimental results which we consider do not support the concept of tubulin-actin homology. The studies have been based largely upon actin-myosin and tubulin-myosin combination experiments but since it has also recently been shown that G-actin inhibits the enzyme DNase I and forms a stoichiometric complex with it [ 1 l] , we have also compared the effect of muscle actin and brain tubulins on DNase I activity.

The subcellular distribution and characterisation of ATPase activity in pig platelets

Abstract The distribution of ATPase activity has been studied in pig platelets subcellular fractions prepared by sucrose density gradient centrifugation. ATPase activities in the presence of Ca 2+ have been measured and the effect of monovalent cations, of ouabain, 2,4-dinitrophenol and Salyrgan investigated. ATPase activity was present in the soluble phase and in the membrane and granule fractions of the cell. The mean ratios for Ca 2+ /Mg 2+ activity being 2.6, 0.62 and 0.60 in these fractions. A large proportion of this divalent cation-activated ATPase can be accounted for by the contractile proteins by the platelet. The Mg 2+ -ATPase of the granular fraction could be stimulated by 2,4-dinitrophenol. The effects of additions of Na + and K + and ouabain over a range of concentrations were most variable and the presence of a Mg 2+ -activated (Na + + K + )-ATPase in the cell could not be unequivocally demonstrated. High monovalent cation concentration (approx. 200 mM) showed a more consistent effect. Homogenate Mg 2+ -ATPase was stimulated by high levels of Na + or K + , Ca 2+ -ATPase, however, was only slightly increased by 100 mM Na + and very markedly by 200 mM K + . A similar effect with 200 mM K + was observed with all the subcellular fractions. The Ca 2+ -ATPase of the membrane fraction could be increased for example, 2–3-fold by the addition of 200 mM K + . Salyrgan was found to inhibit the ATPase activities of all fractions and the soluble phase activity was almost completely inhibited by this agent in the presence of either bivalent cation. The granule fraction was the least sensitive to Salyrgan. The findings well illustrate the difficulties in identifying other minor ATPase enzymes in the presence of a large contractile protein ATPase component of wider distribution in the cell.