Plasma transport of 5-hydroxytryptamine-3H. II. Identification of the carrier protein of 5-hydroxyindoleacetic acid]

In the first note, we have demonstrated a 5-hydroxyindole acetic acid binding to plasma proteins. By gel filtration on Sephadex G 200 and polyacrylamide gel electrophoresis, the 5-hydroxyindole acetic acid carrier protein was identified to serum-albumin.     

Studies in vitro of the effects of adenosine 3'':5''-cyclic monophosphate on the phosphorylation of nuclear proteins in isolated rat heart nuclei.

Isolated rat heart nuclei were prepared by homogenization and sucrose-density-gradient centrifugation. The protein/DNA ratio of these nuclei was 3.1:1 (w/w), and the histones/non-histone proteins/DNA proportions were 1.4:1.6:1 (by wt.). Non-histone proteins were fractionated into six major groups by elution on a quaternized anion-exchanger (QAE-Sephadex A-50 column with increasing concentrations of NaCl in 5M-urea/0.01 M-Tris/HCl buffer (pH8.3). When isolated nuclei were incubated in a medium containing [gamma-32P]ATP, a differential distribution of 32P was observed in the six fractions of nonhistone proteins. The fractions eluted from the Sephadex column with 0.35M- and 0.6M-NaCl contained contained 80% of the total radioactivity incorporated into the non-histone proteins. This incorporation into the 0.35M- and 0.6M-NaCl fractions was increased by 66 and 112% respectively in the presence of cyclic AMP. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of these two particular fractions showed a selective increase in labelling of five protein bands in the presence of cyclic AMP.     

Uptake of H3-5-hydroxytryptophan by noradrenergic nerves in the mouse pancreas studied with electron microscopic autoradiography

Summary The uptake and distribution of radioactivity in vascular adrenergic nerves in the mouse pancreas following the injection of tritiated 5-hydroxytryptophan was studied by means of electron microscopic autoradiography. Autoradiographic silver grains were found selectively accumulated over axonal profiles. Quantitative analysis revealed a characteristic intraneuronal distribution of the silver grains, most of which probably represent 5-hydroxytryptamine formed by decarboxylation from the labeled precursor. Thus, the grain density over adrenergic nerve terminals, containing a mixed population of vesicles and granules, was about 5 times higher than the grain density recorded over non-terminal axonal parts and at least 20 times higher than the grain density found over surrounding adventitial tissue and smooth muscle cells. This was interpreted as an evidence that 5-hydroxytryptamine was taken up and stored in adrenergic terminals.     

Fast affinity purification coupled with mass spectrometry for identifying organophosphate labeled plasma butyrylcholinesterase

Classical plasma butyrylcholinesterase (BChE) purification involves dialysis and multiple steps of chromatography. We describe a procainamide affinity gel purification scheme that takes 15-30min to purify BChE from 1ml plasma. The method uses a microfuge spin column to build a 0.2ml procainamide affinity column. The eluted BChE contains 3-4mug of 500-fold purified BChE, free from 99% of contaminating plasma proteins. The BChE was further purified by gel electrophoresis. Tryptic peptides from the BChE containing gel electrophoresis band were prepared by in-gel digestion, separated by reverse phase liquid chromatography and identified by mass spectrometry. The 29 residue active site tryptic peptide labeled with the nerve agents soman or sarin was identified.     

Association of heparan sulfate proteoglycan and laminin with the cytoskeleton in rat liver

Rats were injected with 35SO4 and after 2 h their livers were removed and used to prepare a detergent-insoluble cytoskeleton fraction. Spectrin, cytokeratins, and actin were major protein components of the isolated cytoskeletons. The cytoskeleton fraction accounted for approximately 14% of the total trichloroacetic acid-insoluble 35SO4 radioactivity incorporated into the liver. The cytoskeleton-associated radioactivity was present in a single species of macromolecule. This molecule was not present to a significant extent in the detergent-soluble fraction containing the cell supernatant and dissolved membrane proteins. Further characterization revealed the cytoskeleton-associated molecule was a heparan sulfate proteoglycan: it was eluted from a Sepharose CL-4B column under denaturing conditions at Kav = 0.4; following mild alkaline hydrolysis the radioactivity was eluted at a Kav = 0.7; when this material was subjected to nitrous acid hydrolysis all of the radioactivity was eluted near the column included volume. The isolated cytoskeletons contained attached nuclei. Pure nuclei isolated without associated cytoskeletal elements contained less than 1% of the total liver trichloroacetic acid-insoluble 35SO4 radioactivity and no detectable heparan sulfate proteoglycan. These results suggested that other matrix proteins might be associated with the liver cytoskeleton. When the subcellular distribution of laminin was monitored by immunostaining proteins transferred to nitrocellulose, laminin was detected exclusively in the cytoskeleton fraction. These results provide evidence for an association between extracellular connective tissue proteins and intracellular structural proteins.     

5-Hydroxytryptamine transport in cells and secretory granules from a transplantable rat insulinoma.

Mechanisms of transport of 5-hydroxytryptamine in the pancreatic B-cell were investigated by using cell suspensions and secretory granules prepared from a transplantable rat insulinoma. (1) Cells incubated with 5-hydroxy[G-3H]tryptamine at concentrations ranging from 0.1 microM to 5 mM accumulated the radioisotope principally by a simple diffusion process. The incorporated radioactivity was recovered principally as the parent molecule and was recovered predominantly in soluble protein and secretory-granule fractions prepared from the tissue. (2) Isolated granules incubated in buffered iso-osmotic medium without ATP accumulated the amine to concentrations up to 38-fold that of the medium. This process was insensitive to reserpine and occurred over a wide range of 5-hydroxytryptamine concentrations (0.075 microM-25 mM). Above 5 mM, 5-hydroxytryptamine accumulation decreased in parallel with the breakdown of the delta pH across the granule membrane. Uptake was favoured by alkaline media and was reduced by the addition of (NH4)2SO4. In both cases a close correlation was observed between uptake and the transmembrane delta pH, a finding that suggested that 5-hydroxytryptamine permeated the membrane as the free base and equilibrated across the membrane with the delta pH. Binding of 5-hydroxytryptamine to granule constituents also played a part in this process. ATP caused a further doubling of granule 5-hydroxytryptamine uptake by a process that was sensitive to reserpine (0.5 microM). Inhibitor studies suggested that amine transport in this instance was linked to the activity of the granule membrane proton-translocating ATPase. (3) It was concluded that the uptake of amines driven by proton gradients across the insulin-granule membrane could account for the accumulation in vivo of amines in the B-cell.     

The uptake and intracellular distribution of [35S]trithiomolybdate in bovine liver in vivo

[35S] trithiomolybdate was administered intravenously to a group of four steer at two dose rates, 1 and 26 mg Mo per animal. Radioactivity appeared rapidly in the liver and was distributed in all the subcellular fractions examined. Examination by Sephadex G-100 gel-filtration of the cytosol fraction showed that distinct 35S-binding protein peaks were present. The protein-bound radioactivity was displaceable and was identified as [35S]thiomolybdates. No radioactivity eluted with metallothionein, but 35S was associated with the high molecular weight copper fraction, eluted in the void volume of the column, which increased transiently after the administration of the higher dose. It was suggested that the presence of protein-bound thiomolybdates in the liver gave rise to new ligands, which altered the equilibrium of copper between the different metal-binding proteins. This might be similar to the alteration in the copper-binding of albumin produced by the presence of thiomolybdates.     

Isolation and characterization of rat plasma fibronectin.

Rat plasma fibronectin has been isolated and characterized and monospecific antibodies were prepared to it. Two components of fresh rat plasma (in the presence of proteinase inhibitors) bound to a gelatin-Sepharose affinity column. One protein was eluted with 4.0 M-urea and was identified as fibronectin. Another protein was eluted from the gelatin-Sepharose column with 8.0 M-urea and was identified as a 70 000-Mr collagen-binding molecule. This 70 000-Mr fragment was found to be a normal constituent of blood plasma, and its presence did not represent a proteolytic degradation product formed during isolation. The antibodies prepared against rat fibronectin only weakly cross-reacted with plasma fibronectins of chicken, horse and human. These studies shed light on the metabolic interrelationships between fibronectin and other collagen-binding molecules.     

The fate of an N-formylated chemotactic peptide in stimulated human granulocytes. Subcellular fractionation studies

Experiments were performed to examine how human granulocytes process the chemotactic peptide N-formyl-Met-Leu-Phe after stimulation by the same peptide. Purified human granulocytes were stimulated with 50 nM N-formyl-Met-Leu-[3H]Phe at 37 degrees C for various times, washed, lysed by N2 cavitation, and fractionated by isopycnic sucrose density gradient sedimentation. The major subcellular fractions identified were plasma membrane, Golgi, granules, endoplasmic reticulum, and mitochondria. After 1 min of stimulation, radioactivity was found only in the plasma membrane (sedimentable) and cytosol (soluble) fraction. At 5, 10, and 25 min, radioactivity also appeared in a sedimentable, low density fraction (25-28% sucrose) enriched in galactosyl transferase activity and containing Golgi structures. The accumulation in the sedimentable fractions was maximal after 5 min but continued to increase linearly in the cytosol fraction. Incorporation of radioactivity into cells or membrane and soluble fractions was 60 to 85% specific and was inhibited if incubation with N-formyl-Met-Leu-[3H]Phe was performed at 4 degrees C. 80-90% of the radiolabel in the plasma membrane or Golgi-containing fractions remained sedimentable despite freeze thawing or sonication. Solubilization of these fractions in Triton X-100 followed by Sepharose 4B column chromatography revealed that the radiolabel eluted in the void volume. Our results are consistent with internalization which proceeds by passage of an occupied receptor in a high affinity, supramolecular complex from the plasma membrane to the Golgi followed by accumulation of peptide in the cytosol.     

Chondroitin sulphate proteoglycan in the substratum adhesion sites of Balb/c 3T3 cells. Fractionation on various ion-exchange and affinity columns.

Proteoglycans on the cell surface play critical roles in the adhesion of fibroblasts to a fibronectin-containing extracellular matrix, including the model mouse cell line Balb/c 3T3. In order to evaluate the biochemistry of these processes, long-term [35S]sulphate-labelled proteoglycans were extracted quantitatively from the adhesion sites of 3T3 cells, after their EGTA-mediated detachment from the substratum, by using an extractant containing 1% octyl glucoside, 1 M-NaCl and 0.5 M-guanidinium chloride (GdnHCl) in buffer with many proteinase inhibitors. Greater than 90% of the material was identified as a large chondroitin sulphate proteoglycan (Kav. = 0.4 on a Sepharose CL2B column), and the remainder was identified as a smaller heparan sulphate proteoglycan; only small amounts of free chains of glycosaminoglycan were observed in these sites. These extracts were fractionated on DEAE-Sepharose columns under two different sets of elution conditions: with acetate buffer (termed DEAE-I) or with acetate buffer supplemented with 8 M-urea (termed DEAE-II). Under DEAE-I conditions about one-half of the material was eluted as a single peak and the remainder required 4 M-GdnHCl in order to recover it from the column; in contrast, greater than 90% of the material was eluted as a single peak from DEAE-II columns. Comparison of the elution of [35S]sulphate-labelled proteoglycan with that of 3H-labelled proteins from these two columns, as well as mixing experiments, indicated that the GdnHCl-sensitive proteoglycans were trapped at the top of columns, partially as a consequence of their association with proteins in these adhesion-site extracts. Affinity chromatography of these proteoglycans on columns of either immobilized platelet factor 4 or immobilized plasma fibronectin revealed that most of the chondroitin sulphate proteoglycan and the heparan sulphate proteoglycan bound to platelet factor 4 but that only the heparan sulphate proteoglycan bound to fibronectin, providing a ready means of separating the two proteoglycan classes. Affinity chromatography on octyl-Sepharose columns to test for hydrophobic domains in their core proteins demonstrated that a high proportion of the heparan sulphate proteoglycan but none of the chondroitin sulphate proteoglycan bound to the hydrophobic matrix. These results are discussed in light of the possible functional importance of the chondroitin sulphate proteoglycan in the detachment of cells from extracellular matrix and in light of previous affinity fractionations of proteoglycans from the substratum-adhesion sites of simian-virus-40-transformed 3T3 cells.     

Hepatic clearance of adenosine 3:5-cyclic monophosphate from plasma in the rat.

Rats were given intravenous injections of cyclic [3H]AMP and the disappearance of radioactivity from plasma and its appearance in bile were followed. Livers were removed and the cyclic [3H]AMP content was measured. The binding of radioactivity to soluble proteins was measured after preparations of a cytoplasmic fraction. Experiments in vitro to determine the ability of hepatic cytoplasmic proteins to bind cyclic [3H]AMP were also carried out. A role for cytoplasmic proteins in the clearance of cyclic AMP from plasma is discounted.     

A general method for fractionation of plasma proteins. Dye-ligand affinity chromatography on immobilized Cibacron blue F3-GA.

The chromatographic behaviour of 27 different plasma proteins on fractionation of human plasma on immobilized Cibacron Blue F3-GA was studied. The column was eluted by using a three-step procedure. First, a low-molarity buffer (30 mM-H3PO4/Na3PO4, pH 7.0, I0.053) was used, then a linear salt gradient (0-1 M-NaCl in the buffer above) was applied, followed by a wash with two bed volumes of 1.0 M-NaCl. Finally, bound proteins were 'stripped' with 0.5 M-NaSCN. Up to 1 ml of whole plasma could be loaded per 5 ml bed volume. No denaturation of proteinase inhibitors or complement fractions was observed. The recovery of individual proteins ranged between 52 and greater than 95%. Enrichment of four individual plasma components (alpha 1-antitrypsin, caeruloplasmin, antithrombin III and haemopexin) was between 10-fold and 75-fold. These results indicate that chromatography on immobilized Cibacron Blue F3-GA can be a useful initial step in the purification of plasma proteins.     

Histamine and 5-hydroxytryptamine-inhibition of transmitter release mediated by H2- and 5-hydroxytryptamine receptors.

The vasodilatation caused by histamine and 5-hydroxytryptamine may be due, at least in part, to their inhibitory action on adrenergic neurotransmission. The evidence for this is as follows: 1) contractions of isolated canine vascular strips caused by sympathetic nerve stimulation are depressed by these substances whereas contractions caused by norepinephrine are either unchanged or augmented; 2) histamine and 5-hydroxytryptamine inhibit the release of norepinephrine evoked by sympathetic nerve stimulation of isolated vascular strips previously incubated with the labeled transmitter. This inhibitory effect can be demonstrated using concentrations of the substinces less than those required to affect the smooth muscle cells directly. By contrast, neither histamine nor 5-hydroxytryptamine inhibits the displacement of neuronal norepinephrine by tyramine, suggesting that these substances interfere with the exocytotic process. Additional studies have identified the histamine-H2 receptor as the probable mediator of this prejunctional action of histamine, whereas the nature of the receptor for 5-hydroxytryptamine remains to be clarified.     

The chemical characterization of the radioactive products derived from [[3H]PheB1]insulin in the circulation of the rat.

1. Gel filtration of rat plasma taken 1 h after subcutaneous injection of [[3H]PheB1]insulin gives three peaks of radioactivity. 2. The material in these peaks was characterized by electrophoresis and chromatography. 3. We conclude that [[3H]PheB1]insulin is rapidly degraded to free tritiated phenylalanine. The phenylalanine is subsequently used for synthesis of plasma proteins de novo.     

Chemoattractant and guanosine 5''-[gamma-thio]triphosphate induce the accumulation of inositol 1,4,5-trisphosphate in Dictyostelium cells that are labelled with [3H]inositol by electroporation.

The analysis of the inositol cycle in Dictyostelium discoideum cells is complicated by the limited uptake of [3H]inositol (0.2% of the applied radioactivity in 6 h), and by the conversion of [3H]inositol into water-soluble inositol metabolites that are eluted near the position of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] on anion-exchange h.p.l.c. columns. The uptake was improved to 2.5% by electroporation of cells in the presence of [3H]inositol; electroporation was optimal at two 210 microseconds pulses of 7 kV. Cells remained viable and responsive to chemotactic signals after electroporation. The intracellular [3H]inositol was rapidly metabolized to phosphatidylinositol and more slowly to phosphatidylinositol phosphate and phosphatidylinositol bisphosphate. More than 85% of the radioactivity in the water-soluble extract that was eluted on Dowex columns as Ins(1,4,5)P3 did not co-elute with authentic [32P]Ins(1,4,5)P3 on h.p.l.c. columns. Chromatography of the extract by ion-pair reversed-phase h.p.l.c. provided a good separation of the polar inositol polyphosphates. Cellular [3H]Ins(1,4,5)P3 was identified by (a) co-elution with authentic [32P]Ins(1,4,5)P3 and (b) degradation by a partially purified Ins(1,4,5)P3 5-phosphatase from rat brain. The chemoattractant cyclic AMP and the non-hydrolysable analogue guanosine 5'-[gamma-thio]triphosphate induced a transient accumulation of radioactivity in Ins(1,4,5)P3; we did not detect radioactivity in inositol 1,3,4-trisphosphate or inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4]. In vitro, Ins(1,4,5)P3 was metabolized to inositol 1,4- and 4,5-bisphosphate, but not to Ins(1,3,4,5)P4 or another tetrakisphosphate isomer. We conclude that Dictyostelium has a receptor- and G-protein-stimulated inositol cycle which is basically identical with that in mammalian cells, but the metabolism of Ins(1,4,5)P3 is probably different.     

Assay of 3-hydroxy-3-methylglutaryl CoA reductase activity using anionic-exchange column chromatography.

A rapid, easy, and sensitive method is described in this paper for the assay of 3-hydroxy-3-methylglutaryl CoA (HMG CoA) reductase, a key enzyme in cholesterol biosynthesis. [14C]HMG CoA was used as the substrate and the product formed, i.e., [14C]mevalonate, was allowed to be converted to its lactone form (mevalonolactone) in the presence of HCl. The reaction mixture was applied to a column containing an anionic exchanger. The column was made up of QAE-Sephadex (A25, formate form) packed to a height of 4 cm in Pasteur pipets. Under these conditions, mevalonolactone was not retained by the column and was eluted with ammonium formate solution while HMG CoA, being negatively charged, was retained by the gel and eluted by HCl above 0.05 M. Determination of the amount of radioactivity in mevalonolactone was then used to quantitate the activity of HMG CoA reductase. This assay has been successfully used for determining the activity of this enzyme in a microsomal fraction prepared from the liver of the rat.     

Studies on the bioactivity of radiolabeled,highly-purified bovine thyrotropin

Bovine thyrotropin radiolabeled stoichiometrically with chloramine T was subjected to high pressure liquid chromatography on a Waters' Protein I-125 column. More than 80% of the radioactivity eluted after the Na¹²⁵I (“salt”) peak. In contrast, thyrotropin bioactivity eluted before the salt peak. Radiolabeled thyrotropin affinity-purified with thyroid plasma membranes eluted after the salt peak. Discordance between the thyrotropin bioactivity and radioactivity elution profiles was confirmed by labeling thyrotropin with ¹²⁵I by lactoperoxidase and then measuring both bioactivity and radioactivity in each chromatographic fraction. These data suggest that the bioactivity in radiolabeled thyrotropin may not be inherent in the radiolabeled molecules.     

Purification and partial characterization of ceruloplasmin receptors from rat liver endothelium

Ceruloplasmin (CP), a circulating glycoprotein, is known for its copper transport. Recently the spectrum of its activity has been increased to include numerous enzymatic functions. CP binds to the liver endothelium and is transported across the cell via a mechanism involving receptor-mediated endocytosis. To isolate CP receptors, we obtained purified preparations of liver endothelium in rats. The membrane was then isolated by ultracentrifugation and solubilized in Triton X-100. Membrane proteins were labeled with 125I and passed through an affinity column in which CP was covalently linked to Sepharose 4B. Most of the radioactivity was eluted with buffer during the first 5 days. When no more radioactivity was eluted with buffer, elution was done either competitively with cold excess CP or 1 M NaCl. By this technique, a sharp single peak of radioactivity was obtained and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing and nonreducing conditions. Under both conditions receptors appeared as a single band with Mr of 35,000 containing 3% carbohydrate and an isoelectric point of 5.2.     

Solubilization and photoaffinity labeling of renal membrane cyclic AMP receptors.

Renal cortical plasma membranes were solubilized with sodium deoxycholate. The membrane-bound cyclic AMP receptors retained biologic activity in the detergent-dispersed state exhibiting the properties of high affinity for cyclic AMP, saturability and specificity. Half-maximal binding of cycle [3H]-AMP to these receptors was found to occur at 0.06 muM and 1.5 pmol of cyclic [3H]AMP was bound per mg membrane protein at saturation (0.5 muM cyclic [3H]AMP). Sodium deoxycholate-solubilized membrane proteins were chromatographed on Biogel A-5m. Cyclic [3H]AMP receptors eluted in the internal volume at positions equivalent to molecular sizes of 50 000 and 20 000 daltons and in the void volume at molecular size greater than 450 000. After photoaffinity labeling the renal membrane receptors with cyclic [3H]AMP, we found peaks of tritium radioactivity which eluted at similar molecular size positions on this Bogel A-5m column. Further treatment of photoaffinity labeled membranes with sodium dodecyl sulfate, mercaptoethanol and urea, followed by polyacrylamide gel electrophoresis, showed bands of tritium-labeled receptor protein with relative mobilities corresponding to molecular sizes of 26 000 and 21 000 daltons. This study shows that porcine renal cortical membranes contain at least two molecular species of cyclic AMP receptors which may be associated with regulation of the membrane-bound cyclic AMP-dependent protein kinase.     

Isolation and characterization of the major proteins of ram seminal plasma

Mammalian seminal plasma contains among others, two major families of proteins, namely spermadhesins and those proteins that contain fibronectin type II domains. Spermadhesins are the major proteins of boar and stallion seminal plasma and homologous proteins have been identified in the bull. These proteins appear to be involved in capacitation and sperm-egg interaction. In bovine seminal plasma, proteins containing fibronectin type II domains are the major proteins and are designated BSP proteins. These proteins play a role in sperm capacitation. In this study, we present the isolation and characterization of the major proteins of ram seminal plasma. Precipitated proteins from Suffolk ram seminal plasma were loaded onto a gelatin-Agarose column. The unadsorbed (fraction A) and retarded proteins (fraction B) were removed by washing the column with phosphate buffered-saline and the adsorbed proteins (fraction C) were eluted with 5 M urea. SDS-PAGE of fraction B indicated the presence of a 15.5 kDa protein, which is the major protein of ram seminal plasma (approximately 45% of total protein by weight) and was identified as a spermadhesin by N-terminal sequencing. SDS-PAGE analysis of fraction C revealed the presence of four proteins, which represented approximately 20% of total ram seminal plasma proteins by weight, and were identified as proteins of the BSP family and named RSP proteins. These RSP proteins were designated RSP-15 kDa, RSP-16 kDa, RSP-22 kDa, and RSP-24 kDa. Only RSP-15 kDa and -16 kDa proteins cross-reacted with antibodies against BSP proteins. Ram spermadhesin and RSP proteins interact with heparin but only RSP proteins bind to hen's egg yolk low-density lipoprotein. In conclusion, spermadhesin is the major protein of ram seminal plasma and other major proteins belong to the BSP protein family.