Guy C. McLeod

Accumulation of vanadium by tunicate blood cells occurs via a specific anion transport system

Tunicates, or sea squirts, are known to sequester vanadium to very high concentrations within specialized blood cells. They selectively accumulate the element from seawater against a 106- to 107-fold concentration gradient, and store it mainly as V(III). The mechanism for this selective accumulation involves the facilitated diffusion of vanadate across the blood cell plasma membrane followed by intracellular reduction to a non-transportable cation. Evidence for this mechanism was obtained by studying vanadate and [48V]vanadate influx into living blood cells (vanadocytes). Influx of [48V]vanadate into the cells is a rapid (t12 = 57 s at 0°C) process which can be saturated (Km = 1.4 (±2%) mM). Net vanadate accumulation is equal to isotopic influx, and accumulated vanadate is not released by washing cells with EDTA. Uncouplers of oxidative phosphorylation and glycolytic inhibitors have no effect on the rate of influx. Phosphate competes with vanadate for transport, and is itself taken up by the cell. The similar anions, sulfate and chromate, neither inhibit transport, nor are they taken up by the vanadocyte. Influx is inhibited by those stilbene disulfonate derivatives known to bind specifically to the external transport site of the anion exchange protein in the human erythrocyte membrane. During the influx of vanadate, the electron paramagnetic resonance (EPR) signal of intracellular vanadyl increases, indicating that transported V(V) is reduced upon entering the cell. The EPR signal of the blood cells at room temperature is characteristic of unbound V(IV), in agreement with reports that reduced vanadate is not bound to a protein or other macromolecule in these cells.

The blood of Ascidia nigra: blood cell frequency distribution, morphology, and the distribution and valence of vanadium in living blood cells.

1. The blood plasma of Ascidia nigra has been characterized with regard to pH, salinity, and ultraviolet-visible absorption. Whole blood of A. nigra has been characterized with regard to cell count, cellular volume, hemoglobin (iron) content, vanadium content. Blood cell types have been examined, categorized, and differentially counted.2. Epr and nmr studies prove that trivalent vanadium is present. Staining of whole blood slides with OsO4 reagent identify the vanadium-carrying cells as being mainly the green globular blood cells. The observed uv-vis absorption spectrum does not correlate well with known vanadium(III) complex spectra.3. Density separation of blood cells has been achieved; this result coupled with various cell morphologies suggests that different cells may represent different maturational stages in a developmental process.4. Vanadium analyses of layerings of cells of different densities suggest that vanadium may be present in more than one type of blood cell, where, when not present as van...

Vanadium in tunicates: Oxygen-binding studies

1. 1. No reversible oxygen-binding by the blood of the tunicate A. nigra is detectable. 2. 2. Blood cells contain iron(II) and vanadium(III), and a yellow-green chromogen which can be separated from the metals by fractionation on Sephadex G-100. 3. 3. The chromogen will reduce oxygen in alkaline medium, it is heat-stable and has absorption peaks at 280 and 330 nm (in 0.1 N HCl). The peak at 330 nm is lost on oxidation.

Vanadium-containing tunicate blood cells are not highly acidic

The intracellular pH of intact blood cells of the tunicate Ascidia nigra was measured by transmembrane equilibration of [14C] methylamine. The pH of unfractionated blood cells is 7.39 +/- 1.10. The pH of vanadocytes, determined in a fractionation study, is 7.2. Previously used methods, in which pH values less than 3.0 are inferred from cell lysis or vital staining experiments, are shown to be unsuitable for intracellular pH determination due to the chemical composition of these vanadium-containing cells.

Tunichromes and metal ion accumulation in tunicate blood cells

Abstract 1. 1. Metal analyses have been performed on the tunicates Ascidia nigra (accumulates V(III)), Ciona intestinalis (accumulates V(IV)) and Molgula manhattensis (accumulates Fe(II)). 2. 2. Negligible amounts of Cr, Ni, Mn, Ti and Cu were detected in blood cells, but Cu occurs at significant levels in the bodies of all species. Al was found in the blood of M. manhattensis . 3. 3. A green chromogen (tunichrome) was found in blood cells of all species. A. nigra possesses vanadium and tunichrome in approx equimolar concentrations. 4. 4. Tunichromes from C. intestinalis and M. manhattensis have absorption peaks at 270 nm; M. manhattensis tunichrome also has a peak at 340 nm. Both, like A. nigra tunichrome, can reduce Fe(III) and V(V).

THE PHOTOSYNTHETIC RHYTHM OF ACETABULARIA CRENULATA. II. MEASUREMENTS OF PHOTOASSIMILATION OF CARBON DIOXIDE AND THE ACTIVITIES OF ENZYMES OF THE REDUCTIVE PENTOSE CYCLE

1. The photosynthetic rhythm of Acetabularia crenulata affects both light (quantum yield) and dark reactions in a parallel manner.2. No significant difference was found between the activity of RuDP carboxylase in the extracts of samples taken at the middle of the light and dark periods nor was any difference detected in the affinity of this enzyme to CO2. The activity of RuDP carboxylase in the cell extracts was sufficient to account for the observed rates of photoassimilation of CO2 at saturating light intensities.3. The activities of eight other enzymes of the reductive pentose phosphate cycle were also shown not to differ to a significant extent in extracts of cells taken at the middle of the light and dark periods. Five of these enzymes (phosphoglycerate kinase, glyceraldehyde-3-P dehydrogenase, triose isornerase, R-5-P isomerase and Ru-5-P kinase) had activities considerably above those required for the observed light-saturated rate of CO2 assimilation while the activities of aldolase transketolase a...

IRON ACCUMULATION IN TUNICATE BLOOD CELLS. I. DISTRIBUTION AND OXIDATION STATE OF IRON IN THE BLOOD OF BOLTENIA OVIFERA, STYELA CLAVA, AND MOLGULA MANHATTENSIS

The iron concentration, oxidation state, and distribution in blood plasma and blood cells of three iron containing tunicateS were determined. Preliminary studies are reported on the possible role of plasma proteins in iron uptake. Iron(II) concentration in the millimolar range was found in the blood cell cy toplasm ofall three species; no iron(III) in solution was detected in blood cells. Over 70% of the total iron in the cells is associated with the membranes. Although the iron concentration in S. clava blood cells is substantially greater than that in B. ovifera cells, the iron to protein ratio by weight is similar in both species. SDS-electrophoresis of B. ovifera blood showed two protein subunits com mon to both plasma and blood cells. These two subunits are most likely the major components ofthe high molecular weight protein found in the plasma. This protein was shown to bind iron(III) when iron(III) citrate was added to the plasma.

WATER TRANSPORT RATES OF THE TUNICATE CIONA INTESTINALIS

Sea water transport rates of the tunicate Ciona intestinalis were determined by measuring the volume of sea water transported through the specimen, and measuring the number of particles cleared by the specimen in a given time interval. The rate was also determined directly by matching the flow produced by the tunicate to that produced by a calibrated pump. Ciona transports sea water at variable rates; at times, it does not transport at all. The rate limits covering all techniques are: lower limit, 2.5 ml/hr/g wet weight and upper limit, 185 ml/hr/g wet weight; the average value based on clearance and direct measurements is 50 ml/ hr/g wet weight. Even at the lowest rate found, transport is rapid enough to ensure complete mixing between sea water and reaction or absorption sites in the pharyngeal chamber, alimentary tract or atrial chamber. We conclude that the rate controlling process for absorption of oxygen, vanadate ions, micro-organisms or organic detritus is not the rate of passage of the feeding current, but rather the rate of the intrinsic absorption process such as complex formation, ion exchange or adsorption.