Microphytobenthos as a biogeomorphological force in intertidal sediment stabilization

The properties and behavior of intertidal marine sediments cannot be understood without taking their biology into account. Biological factors are important for the stability and erosion threshold of intertidal sediments as well as for sediment transport. In this paper I focus on intertidal sediments that are colonized and dominated by phototrophic microorganisms and their impact on the morphodynamics and sediment stabilization. The emphasis is on epipelic diatoms. These organisms exude copious amounts of extracellular polymeric substances (EPS) that may contribute to the stability of the sediment by gluing and binding. I review the factors that lead to the development of such microphytobenthic communities and the processes that lead to the exudation of EPS and its fate in intertidal mudflats. Epipelic diatoms exude EPS partly as the result of unbalanced growth. Extraction of EPS from cultures of epipelic diatoms yields two operational fractions. While one fraction contains largely neutral EPS, which may serve as a carbon- and energy reserve for the organism, the other is acidic and more recalcitrant to degradation. The latter EPS fraction is therefore predominant in the muddy sediment and may be responsible for increasing the erosion threshold. However, since extracted EPS alone is incapable of increasing the erosion threshold, diatoms are apparently actively involved in the structuring of the biofilm matrix. Therefore, sediment stabilization cannot be attributed simply to EPS alone.     

The ability of the mitochondrial Ca2+-binding glycoprotein to restore Ca2+ transport in glycoprotein-depleted rat liver mitochondria

Rat liver mitochondria may be subfractionated in sediment and supernatant fractions by swelling in the presence of EDTA and oxaloacetate. The sediment is largely depleted of the Ca²⁺-binding glycoprotein and its Ca²⁺-transporting activity may be as low as 10–20% of the starting value. Both the rate of Ca²⁺ uptake and the capacity to maintain a high Ca²⁺ concentration gradient across the membrane are depressed. Addition of an osmotic supernatant to the assay mixture may partially restore the original Ca²⁺-transporting ability. The active component in the supernatant is the Ca²⁺-binding glycoprotein. This is shown by the following facts: (a) the effect is enhanced by the addition of the purified glycoprotein to the supernatant; (b) precipitation of the glycoprotein from the supernatant by affinity chromatography-purified antibodies abolishes the stimulatory effect, and (c) in the presence of 130 μM Mg²⁺, the glycoprotein alone may restore fully the Ca²⁺-transporting ability of the particles. The maximal velocity is already reached at 0.1 μg glycoprotein/mg mitochondrial protein.     

Differentiation between Ca2+ transport and ATP-induced Ca2+ binding by sarcoplasmic reticulum

The Ca²⁺ actively accumulated by sarcoplasmic reticulum isolated from skeletal muscle is composed of two fractions; one represented by intravesicular free Ca²⁺ and another represented by Ca²⁺ selectively bound to the membranes. Both of these Ca²⁺ fractions depend on ATP, although it is not clear whether ATP hydrolysis is essential for accumulation of the second Ca²⁺ fraction. The existence of the membrane-bound Ca²⁺ induced by ATP is clearly shown in experiments in which the Ca²⁺ retention by sarcoplasmic reticulum is measured in the presence and in the absence of X-537A, a Ca²⁺ ionophore, which makes the membrane permeable to Ca²⁺. Thus, in the presence of X-537A all Ca²⁺ accumulated due to ATP is bound to the membranes. This membrane-bound Ca²⁺ represents about 30 nmol/mg protein in the range of external $\text{p}\text{Ca}$ values of 7 to 3.5. The magnitude of this Ca²⁺ fraction is slightly higher whether or not the experiments are performed in the presence of oxalate, which greatly increased the intravesicular Ca²⁺ accumulation. Furthermore, taking advantage of the impermeability of sarcoplasmic reticulum to EGTA, it is possible to show the existence of the membrane-bound Ca²⁺ as a distinct fraction from that which exists intravesicularly.     

Colony formation of highly dispersed <Emphasis Type="Italic">Microcystis aeruginosa</Emphasis> by controlling extracellular polysaccharides and calcium ion concentrations in aquatic solution

This study isolated extracellular polysaccharides (EPS) as a powder material from cyanobacterial blooms and the powdered EPS was used to trigger colony formation of dispersed unicellular M. aeruginosa by controlling EPS concentration in culture medium. The effect of Ca²⁺ ions on the colony formation of M. aeruginosa was also investigated, then the interaction between EPS and Ca²⁺ ions on colony formation was discussed. The results showed that the addition of the powdered EPS into the medium did not cause morphological changes of M. aeruginosa, suggesting that EPS alone would not induce the colony formation of M. aeruginosa. On the other hand, a high concentration of calcium ions (1000 mg/l) caused colony formation. When EPS and Ca²⁺ ions in the culture medium were adjusted to 200 and 1000 mg/l, respectively, the colony density, the average cell number per colony and the particle size of M. aeruginosa showed ca. 1.7–2.0 times greater values than those in the Ca²⁺ added medium. Calcium ion contributed to the aggregation of M. aeruginosa via crosslinked reaction with negatively charged M. aeruginosa cells, and the addition of EPS possessing negatively charged functional groups such as carboxy groups could enhance the reaction, promoting the crosslinked reaction between EPS and Ca²⁺ ions.     

Influence of sediment on burrowing by the soldier crab Mictyris longicarpus Latreille

This study analyses the spatial distribution of burrowing by the soldier crab Mictyris longicarpus (Mictyridae) in relation to the topography of intertidal sandflats. Four different locations were sampled in Port Jackson and Botany Bay (Sydney, New South Wales, Australia). In each location, abundances of crabs and the proportion of juvenile crabs varied among sites 50 m² and tens of metres apart, but not between high- and lowshore levels, nor among shores. Topographic formations, referred to as humps and depressions, were common on the flats and were of the same order of size and spacing as the sites, which showed variation in numbers/sizes of crabs. Manipulative experiments were done in one of the locations to distinguish between the hypotheses that the spatial distribution of the crabs varied according to (1) topography, or (2) the composition of sediment in the different topographic features. In the first experiment, small plots (0.09 m² and 20 cm deep) of sediment were transplanted between humps and depressions, including appropriate controls for disturbance and translocation. The numbers and sizes of crabs colonizing the experimental plots were then measured. Differences in abundance among plots were found higher on the shore. Therefore, the experiment was repeated at highshore levels with increased replication. There were more crabs in depressions than in humps. The proportion of juveniles varied according to the type of sediment, but only in depressions. Heterogeneity and sediment were, therefore, important for explaining some of the spatial variability of the abundances of the crabs.     

The quantitative variability and monosaccharide composition of sediment carbohydrates associated with intertidal diatom assemblages

The extracellular secretions of epipelic diatoms (Bacillariophyceae) axe an important source of carbohydrates on intertidal sediments. For analytical purposes, sediment carbohydrates have been operationally separated into colloidal and bulk fractions that are often assumed to be similar in their chemical properties. However, there has been little investigation into the nature of the two fractions. In this study, carbohydrate fractions were sampledin situ, isolated, purified and biochemically characterised using gas chromatography-mass spectrometry (GC-MS). Both carbohydrate fractions were found to contain similar sugars although in different proportions. Glucose represented more than 80% of the monosaccharides identified in the colloidal carbohydrate fraction while only 37% of monosaccharides present in the bulk carbohydrate fraction. Colloidal carbohydrate concentrations showed short-term variability and were correlated with diatom biomass (as chlorophylla) suggesting the colloidal fraction is labile and may be of recent origin, perhaps representing diatom activity. Concentrations of the bulk carbohydrate fraction did not show significant short-term variation and was therefore more refractory. This combination of biochemical and field data suggested that the bulk and colloidal carbohydrate fractions were chemically and physically different. These findings have relevance to studies of estuarine carbon cycling.Abbreviations LTSEM Low-temperature scanning electron microscopy - GC-MS Gas chromatography-mass spectroscopy - EPS Extracellular polymeric substances     

Effects of sediment properties on benthic primary production in the Columbia River estuary

Effects of sediment properties on benthic primary production were investigated along transects in the intertidal regions of the Columbia River estuary. On the tidal flats, benthic diatoms were the most abundant plants, while microalgae and submergent vascular macrophytes were relatively rare. At five intensive study sites, mean rates of benthic primary production varied between 5 mg C m⁻² h⁻¹ at Clatsop Spit and 84 mg C m⁻² h⁻¹ at Youngs Bay. A regional analysis of data from these sites and from 31 less intensively studied survey sites indicated that Youngs Bay and Baker Bay were more productive than Grays Bay, Cathlamet Bay and the tidal flats in the upper estuary above these bays. This pattern corresponded to the stability and particle size distribution of the sediment in these areas. Benthic gross primary production for the total estuarine area of 410 km² was estimated to be 4895 t carbon per year and represents a mean rate of 72 g C m⁻² year⁻¹. Of the total net primary production in the estuary, benthic plants accounted for about 7%, while phytoplankton and emergent vascular plants in the marshlands contributed approximately 56% and 37%, respectively. Data from this study also suggested that physical processes related to estuarine circulation can ultimately determine the productivity of benthic plant assemblages, and minimize the regulatory and limiting effects of temperature, nutrient supply and consumption by benthic animals.     

STIM1-Regulated Ca2+ Influx across the Apical and the Basolateral Membrane in Colonic Epithelium

In nonexcitable cells, store-operated Ca²⁺ entry is the most important pathway for influx of extracellular Ca²⁺ serving as a second messenger in the cytoplasm. The present study investigated the expression, localization and polar distribution of two key components of store-operated Ca²⁺ entry identified, e.g., in lymphocytes or epithelial cell lines—STIM1 (stromal interacting molecule 1), working as a Ca²⁺ sensor in the endoplasmic reticulum, and Orai1, working as the (or part of the) store-operated Ca²⁺ channel in the plasma membrane—in a native intestinal epithelium, i.e., rat colon. Immunohistochemical investigations revealed expression of STIM1 and Orai1 in the rat colonic epithelium. Ca²⁺ store depletion led to a translocation of STIM1 both to the basolateral as well as to the apical cell pole as observed by confocal microscopy. A Ca²⁺ depletion/repletion protocol was used in Ussing chamber experiments to investigate the contribution of basolateral and apical store-operated Ca²⁺ entry to the induction of anion secretion. These experiments revealed that Ca²⁺-dependent anion secretion was induced not only by basolateral Ca²⁺ repletion but also, to a lesser extent, by apical Ca²⁺ repletion. Both responses were suppressed by La³⁺. The effect of basolateral Ca²⁺ repletion was significantly inhibited by brefeldin A, a blocker of vesicular transport from the endoplasmic reticulum to the Golgi apparatus. In a final series of experiments, fura-2-loaded HT29/B6 cells were used. A carbachol-induced increase in the cytosolic Ca²⁺ concentration was significantly reduced when cells were pretreated with siRNA against STIM1. In conclusion, these results demonstrate that STIM1 as a key component of intracellular Ca²⁺ signaling is expressed by rat colonic epithelium and is involved in the regulation not only of basolateral but also of apical Ca²⁺ influx.     

Distribution of Exopolymeric Substances in the Littoral Sediments of an Oligotrophic Lake

Bacteria and algae release exopolymeric substances (EPS) that perform a wide range of important functions in aquatic and terrestrial systems. In this study we measured EPS in sediments at nine littoral sites around a shallow oligotrophic basin, and tested whether the concentration and composition of EPS was related to sediment characteristics. The concentrations of both loosely bound (colloidal) and tightly bound (capsular) EPS carbohydrates ranged up to ~800 µg glucose equiv. cm^–2 and were well within the range of concentrations reported from marine intertidal flats, where EPS play an important role in stabilizing sediments, affecting nutrient exchanges between sediments and the water column, feeding benthic invertebrates, and sequestering and increasing the transfer of contaminants to food webs. Proteins were an important component of the EPS in these littoral sediments, with protein:carbohydrate ratios of ~0.4. In summer, the concentrations of most EPS fractions were positively related (P < 0.05) to the porewater and organic matter content of the sediments. Capsular EPS concentrations were lower in the fall, with a simultaneous increase in colloidal proteins but not in colloidal carbohydrates. This suggests that the carbohydrates in this colloidal EPS may be more labile than the proteins. Our results suggest that exopolymeric substances could be an important, but neglected, component of littoral sediments in lakes.     

Further Characterization of the Red Beet Plasma Membrane Ca-ATPase Using GTP as an Alternative Substrate

The GTP-driven component of Ca²⁺ uptake in red beet (Beta vulgaris L.) plasma membrane vesicles was further characterized to confirm its association with the plasma membrane Ca²⁺-translocating ATPase and assess its utility as a probe for this transport system. Uptake of ⁴⁵Ca²⁺ in the presence of GTP demonstrated similar properties to those previously observed for red beet plasma membrane vesicles utilizing ATP with respect to pH optimum, sensitivity to orthovanadate, dependence on Mg:substrate concentration and dependence on Ca²⁺ concentration. Calcium uptake in the presence of GTP was also strongly inhibited by erythrosin B, a potent inhibitor of the plant plasma membrane Ca²⁺-ATPase. Furthermore, after treatment with EGTA to remove endogenous calmodulin, the stimulation of ⁴⁵Ca²⁺-uptake by exogenous calmodulin was nearly equivalent in the presence of either ATP or GTP. Taken together these results support the proposal that GTP-driven ⁴⁵Ca²⁺ uptake represents the capacity of the plasma membrane Ca²⁺-translocating ATPase to utilize this nucleoside triphosphate as an alternative substrate. When plasma membrane vesicles were phosphorylated with [γ-³²P]-GTP, a rapidly turning over, 100 kilodalton phosphorylated peptide was observed which contained an acyl-phosphate linkage. While it is proposed that this peptide could represent the catalytic subunit of the plasma membrane Ca²⁺-ATPase, it is noted that this molecular weight is considerably lower than the 140 kilodalton size generally observed for plasma membrane Ca²⁺-ATPases present in animal cells.     

Microphytobenthos,their Extracellular Polymeric Substances,and the Morphogenesis of Intertidal Sediments

Intertidal sediments are important areas that separate the land from the sea and form natural coastal defenses. They are known as highly productive ecosystems, fueling the coastal food web. It is also conceived that microphytobenthos contribute to the stability of intertidal sediments by increasing the erosion threshold and that they are major players in coastal morphodynamics. Depending on the sedimentary composition of intertidal flats, different types of microphytobenthos colonize the sediment surface. Fine sand sediment is often colonized by cyanobacteria, prokaryotic algae, which form dense and rigid microbial mats. Mudflats on the other hand are characterized by the development of thin biofilms of epipelic diatoms. Both groups of phototrophic microorganisms excrete extracellular polymeric substances (EPS), but they do so in different ways and for different reasons. Two operationally defined fractions, water- and EDTA-extractable EPS, have been obtained from intertidal diatom biofilms and from cultures. They differ in composition and their production seems to be under different metabolic control. Water-extractable EPS are considered to be closely associated with the diatoms and are rich in neutral sugars, notably glucose. These EPS show a dynamic relationship with the microphytobenthic biomass. EDTA-extractable EPS are tightly bound to the sediment, probably through bridging by divalent ions. This material is rich in uronic acids and other acid sugars and is weakly related to chlorophyll. These EPS have been conceived to be a major factor in the structuring and diagenesis of coastal sediments and essential for increasing the sediment erosion threshold. However, this relationship is now questioned.     

Cooperative interactions between calcium-binding sites on glycerinated muscle fibers the influence of cross-bridge attachment

A double isotope technique and EGTA buffers were used to measure the binding of Ca²⁺ to rabbit psoas muscle fibers extracted with detergent and glycerol. These experiments were designed to test the effect of rigor complex formation, determined by the degree of filament overlap, on the properties of the Ca²⁺-binding sites in the intact filament lattice. In the presence of 5 mM MgCl₂ (no ATP), reduction of filament overlap was associated with a reduced binding of Ca²⁺ over the entire range of free Ca²⁺ concentrations (5 · 10^?8 – 2 · 10^?5 M). With maximum filament overlap (sarcomere length 2.1–2.2 μm) the maximum bound Ca²⁺ was equivalent to 4 mol Ca²⁺/mol troponin and there was significant positive interaction between binding sites, as shown by Scatchard and Hill plots. With no filament overlap (sarcomere length 3.8–4.4 μm) the maximum bound Ca²⁺ was equivalent to 3 μmol Ca²⁺/mol troponin and graphical analysis indicated a single class of non-interacting sites. The data provide evidence that when cross-bridge attachments between actin and myosin filaments are formed not only does an additional Ca²⁺ binding site appear, but cooperative properties are imposed upon the binding sites.     

Monoclonal antibodies to the Ca2+ + Mg2+-dependent ATPase of sarcoplasmic reticulum identify polymorphic forms of the enzyme and indicate the presence in the enzyme of a classical high-affinity Ca2+ binding site

In order to determine whether polymorphic forms of the Ca²⁺ + Mg²⁺-dependent ATPase exist, we have examined the cross-reactivity of five monoclonal antibodies prepared against the rabbit skeletal muscle sarcoplasmic reticulum enzyme with proteins from microsomal fractions isolated from a variety of muscle and nonmuscle tissues. All of the monoclonal antibodies cross-reacted in immunoblots against rat skeletal muscle Ca²⁺ + Mg²⁺-dependent ATPase but they cross-reacted differentially with the enzyme from chicken skeletal muscle. No cross-reactivity was observed with the Ca²⁺ + Mg²⁺-dependent ATPase of lobster skeletal muscle. The pattern of antibody cross-reactivity with a 100,000 dalton protein from sarcoplasmic reticulum and microsomes isolated from various muscle and nonmuscle tissues of rabbit demonstrated the presence of common epitopes in multiple polymorphic forms of the Ca²⁺ + Mg²⁺-dependent ATPase. One of the monoclonal antibodies prepared against the purified Ca²⁺ + Mg²⁺-dependent ATPase of rabbit skeletal muscle sarcoplasmic reticulum was found to cross-react with calsequestrin and with a series of other Ca²⁺-binding proteins and their proteolytic fragments. Its cross-reactivity was enhanced in the presence of EGTA and diminished in the presence of Ca²⁺. Its lack of cross-reactivity with proteins that do not bind Ca²⁺ suggests that it has specificity for antigenic determinants that make up the Ca²⁺-binding sites in several Ca²⁺-binding proteins including the Ca²⁺ + Mg²⁺-dependent ATPase.This paper is dedicated to the memory of Dr. David E. Green.     

Do Ca2+-chelating polysaccharides reduce calcium ion release from gypsum-based biomaterials?

Background

Calcium sulphate, a widely used bone filler, may negatively affect human osteoblasts due to release of high quantities of calcium ions. To reduce this effect, an attempt was made to enrich calcium sulphate with Ca²⁺-chelating plant and rhizobial exopolysaccharides (EPS).

Methodology

Incubation of polysaccharide-enriched calcium sulphate composites was performed in DMEM/F12 medium. Ca²⁺ (and Mg²⁺ and Pi) levels were estimated using standardised, spectrophotometry-based kits. Composite surface morphology was tested using SEM technique.

Results

Rhizobial EPS was found slightly less effective at Ca²⁺ chelation than sodium alginate. Both polysaccharides may be used as gypsum supplements in the form of setting liquids (0.3% total mass), but only sodium alginate may be used as a powder (up to 5% total mass of the composite). Polysaccharide-triggered reduction of Ca²⁺ release reached the level of 50% during the first 2.5 h of incubation, then decreased significantly.

Conclusions

Both tested polysaccharides possess calcium-chelating properties. However, although alginate caused a reduction in Ca²⁺ levels in the media incubated with the gypsum samples, the reduction was too short lived to provide a long-term effect. Further modification of the composite content using calcium-deficient hydroxyapatite and low-molecular weight rhizobial EPS with higher solubility could bring more satisfactory results.     

Transient currents and Ca2+ gradient relaxation in characean algae cells: Theory and experiment

The transient Ca²⁺ and Ca²⁺-dependent Cl^? currents in the plasma membrane of voltage-clamped cells of the freshwater alga Chara corallina were studied. We used our own earlier proposed method, which utilized a rapid (～10 ms) injection of Ca²⁺ ions into the cell during the deactivation period of calcium channels after their activation with a positive voltage pulse (injection with a “tail” Ca²⁺ current). This procedure makes it possible to determine the amplitude of the Ca²⁺ component in the transient current as well as the amplitude and kinetics of the Cl^? component, dependent on the Ca²⁺ submembrane concentration. The calculated results, which used a cell model that takes the diffusion of Ca²⁺, the Ca²⁺-buffering properties of the cytoplasm, and the nonlinear dependence of i _Cl on [Ca²⁺]_cyt, as well as the presence of chloroplasts into account, were in good agreement with the actual behavior of transient current in the experiments. It was demonstrated that the duration of the slow stage of [Ca²⁺]_cyt relaxation to the resting level (～10^?7 M) (which is related to the function of Ca²⁺-ATPases), was ～10² s. This suggests that the slow stage determines the duration of the refractory period after generation of the action potential.     

Biochemical characteristics of the Ca2+ pumping ATPase in the peribacteroid membrane from broad bean root nodules

Ca²⁺-ATPase in the peribacteroid membrane (PBM) of symbiosomes isolated from Vicia faba root nodules was characterized in terms of its hydrolytic and transport activities. Both activities were found to be pH-dependent and exhibit pH optimum at pH 7.0. Translocation of Ca²⁺ through the PBM by the Ca²⁺-ATPase was shown to be fueled by ATP and other nucleotide triphosphates in the following order: ATP?>?ITP???GTP???UTP???CTP, the K _m of the enzyme for MgATP being about 100 μM. Ca-dependent ITP-hydrolytic activity of symbiosomes was investigated in the presence of the Ca-EGTA buffer system and showed the affinity of PBM Ca²⁺-ATPase for Ca²⁺ of about 0.1 μM. The transport activity of Ca²⁺-ATPase was inhibited by erythrosin B as well as orthovanadate, but markedly stimulated by calmodulin from bovine brain. These results allowed us to conclude that this enzyme belongs to IIB-type Ca²⁺-ATPases which are present in other plant membranes.     

Adenosine triphosphatase activity in the neural lobe of the bovine pituitary gland

1. Homogenates of neural lobes of bovine pituitary glands were fractionated by differential and density-gradient ultracentrifugation and the distribution of adenosine triphosphatase (ATPase) activity was studied. It was shown that all the activity was membrane-bound. 2. On the basis of ionic requirements the ATPase activity was grouped into three categories: (a) Mg²⁺-dependent, (b) Ca²⁺-dependent and (c) Mg²⁺+Na⁺+K⁺-dependent (ouabain-sensitive) ATPases. The activity in the absence of bivalent cations was negligible. The ratio between the activities of the three ATPases varied between the different subcellular fractions. 3. Preincubation of the subcellular fractions with deoxycholate increased the activity of the Mg²⁺+Na⁺+K⁺-dependent enzyme, whereas the Mg²⁺- and Ca²⁺-activated ATPases were either unaffected or slightly inhibited. Triton X-100 solubilized the Mg²⁺- and Ca²⁺-ATPases; however, the activity of the Mg²⁺+Na⁺+K⁺-ATPase was abolished by the concentration of Triton X-100 used. 4. All the subfractions displayed unspecific nucleotide triphosphatase activity towards GTP, ITP and UTP. These substrates inhibited the hydrolysis of ATP by all three ATPases. ADP also inhibited the ATPases. 5. Polyacrylamide-gel electrophoresis of extracts containing the Mg²⁺- and Ca²⁺-dependent ATPase activity solubilized by Triton X-100 revealed the presence of two enzymes; one activated by either Mg²⁺ or Ca²⁺ and the other activated only by Ca²⁺. 6. In sucrose density gradients the distribution of vasopressin was different from that of all three types of ATPases. It is therefore suggested that the neurosecretory granules do not possess ATPase activity.     

Sub-mitochondrial location of Ruthenium Red-sensitive calcium-ion transport and evidence for its enrichment in a specific population of rat liver mitochondria

1. Seven fractions sedimenting at between 3000 and 120000g-min were prepared from a rat liver homogenate by differential centrifugation in buffered iso-osmotic sucrose. The following measurements were carried out on each of these fractions: Ruthenium Red-sensitive Ca²⁺ transport in the absence and in the presence of P_i as well as in the presence of N-ethylmaleimide to prevent P_i cycling, succinate-supported respiration in the absence and in the presence of ADP, the ΔE and −59 ΔpH components of the protonmotive force, cytochrome oxidase, uncoupler-stimulated adenosine triphosphatase, α-glycerophosphate dehydrogenase, P_i content and the effect on the `resting' rate of respiration of repeated additions of a fixed Ca²⁺ concentration. 2. Ca²⁺ transport either in the presence or in the absence of added P_i and in the presence of N-ethylmaleimide exhibits significantly higher rates in the fraction sedimenting at 8000g-min. By contrast, respiration in the presence or in the absence of added ADP and the values for ΔE and −59 ΔpH were similar in those fractions sedimenting between 4000 and 20000g-min, indicating that the driving force for Ca²⁺ transport was similar in each of these fractions. 3. Experiments designed to determine the capacity of the individual fractions for Ca²⁺, as measured by the effect of repeated additions of Ca²⁺ on the resting rate of respiration, showed that fraction 2, i.e. that sedimenting at 8000g-min, also exhibited the greatest tolerance towards the uncoupling action of the ion. 4. Of the three enzyme activity profiles, only that of α-glycerophosphate dehydrogenase was similar to that of Ca²⁺ transport. Because previous workers have assigned this enzyme to loci in the inner peripheral membrane [Werner & Neupert (1972) Eur. J. Biochem. 25, 379–396], it is concluded that the Ruthenium Red-sensitive Ca²⁺- transport system also is located in this domain of the inner membrane. The relation of these findings to the mechanisms of mitochondrial Ca²⁺ transport and the biogenesis of mitochondria is discussed.     

Mg2+/Ca2+-ATPase Activity Is Not Enriched in Synaptic Vesicles Isolated from Rat Cerebral Cortex

Neuronal ATPases comprise a wide variety of enzymes which are not uniformly distributed in different membrane preparations. Since purified vesicle fractions have Mg²⁺/Ca²⁺-ATPase, the purpose of the present study was to know whether such enzyme activities have a preferential concentration in a synaptic vesicle fraction in order to be used as markers for these organelles. Resorting to a procedure developed in this Institute, we fractionated the rat cerebral cortex by differential centrifugation following osmotic shock of a crude mitochondrial fraction and separated a purified synaptic vesicle fraction over discontinuous sucrose gradients. Mg²⁺/Ca²⁺-ATPase activities and ultrastructural studies of isolated fractions were carried out. It was observed that similar specific activities for Mg²⁺/Ca²⁺-ATPases were found in all fractions studied which contain synaptic vesicles and/or membranes. Although the present results confirm the presence of Mg²⁺ and Ca²⁺-ATPase activities in synaptic vesicles preparations, they do not favor the contention that Mg²⁺/Ca²⁺-ATPase is a good marker for synaptic vesicles.     

Annual sediment respiration in estuarine sandy intertidal flats in the Seto Inland Sea, Japan

To quantify organic matter mineralization at estuarine intertidal flats, we measured in situ sediment respiration rates using an infrared gas analyzer in estuarine sandy intertidal flats located in the northwestern Seto Inland Sea, Japan. In situ sediment respiration rates showed spatial and seasonal variations, and the mean of the rates is 38.8 mg CO₂-C m⁻² h⁻¹ in summer. In situ sediment respiration rates changed significantly with sediment temperature at the study sites (r ² = 0.70, p < 0.05), although we did not detect any significant correlations between the rates and sediment characteristics. We prepared a model for estimating the annual sediment respiration based on the in situ sediment respiration rates and their temperature coefficient (Q ₁₀ = 1.8). The annual sediment respiration was estimated to be 92 g CO₂-C m⁻² year⁻¹. The total amount of organic carbon mineralization for the entire estuarine intertidal flats through sediment respiration (43 t C year⁻¹) is equivalent to approximately 25% of the annual organic carbon load supplied from the river basin of the estuary.