Quantification and intracellular distribution of ribulose-1,5-bisphosphate carboxylase in Thiobacillus neapolitanus,as related to possible functions of carboxysomes

Ribulose-1,5-bisphosphate carboxylase (RuBPCase) has been quantified by immunological methods in Thiobacillus neapolitanus cultivated under various growth conditions in the chemostat at a fixed dilution rate of 0.07 h^-1. RuBPCase was a major protein in T. neapolitanus accounting for a maximum of 17% of the total protein during CO₂ limitation and for a minimum of 4% during either ammonium- or thiosulfate limitation in the presence of 5% CO₂ (v/v) in the gasphase. The soluble RuBPCase (i.e. in the cytosol) and the particulate RuBPCase (i.e. in the carboxysomes) were shown to be immunologically identical. The intracellular distribution of RuBPCase protein between carboxysomes and cytosol was quantified by rocket immunoelectrophoresis. The particulate RuBPCase content, which correlated with the volume density of carboxysomes, was minimal during ammonium limitation (1.3% of the total protein) and maximal during CO₂ limitation (6.8% of the total protein). A protein storage function of carboxysomes is doubtful since nitrogen starvation did not result in degradation of particulate RuBPCase within 24 h. Proteolysis of RuBPCase was not detected. Carboxysomes, on the other hand, were degraded rapidly (50% within 1 h) after change-over from CO₂ limitation to thiosulfate limitation with excess CO₂. Particulate RuBPCase protein became soluble during this degradation of carboxysomes, but this did not result in an increase in soluble RuBPCase activity. Modification of RuBPCase resulting in a lower true specific activity was suggested to explain this phenomenon. The true specific activity was very similar for soluble and particulate RuBPCase during various steady state growth conditions (about 700 nmol/min·mg RuBPCase protein), with the exception of CO₂-limited growth when the true specific activity of the soluble RuBPCase was extremely low (260 nmol/min ·mg protein). When chemostat cultures of T. neapolitanus were exposed to different oxygen tensions, neither the intracellular distribution of RuBPCase nor the content of RuBPCase were affected. Short-term labelling experiments showed that during CO₂ limitation, when carboxysomes were most abundant, CO₂ is fixed via the Calvin cycle. The data are assessed in terms of possible functions of carboxysomes.Abbreviations RuBPCase ribulose-1,5-bisphosphate carboxylase - PEP phosphoenolpyruvate - RIE rocket immunoelectrophoresis - CIE crossed immunoelectrophoresis     

Breakdown of Ribulose Bisphosphate Carboxylase and Change in Proteolytic Activity during Dark-induced Senescence of Wheat Seedlings

When 8-day-old wheat seedlings (Triticum aestivum L. var. Chris) are placed in the dark the fully expanded primary leaves undergo the normal changes associated with senescence, for example, loss of chlorophyll, soluble protein, and photosynthetic capacity (Wittenbach 1977 Plant Physiol. 59: 1039-1042). Senescence in this leaf is completely reversible when plants are transferred to the light during the first 2 days, but thereafter it becomes an irreversible process. During the reversible stage of senescence the loss of ribulose bisphosphate carboxylase (RuBPCase) quantitated immunochemically, accounted for 80% of the total loss of soluble protein. There was no significant change in RuBPCase activity per milligram of antibody-recognized carboxylase during this stage despite an apparent decline in specific activity on a milligram of soluble protein basis. With the onset of the irreversible stage of senescence there was a rapid decline in activity per milligram of carboxylase, suggesting a loss of active sites. There was no increase in total proteolytic activity during the reversible stage of senescence despite the loss of carboxylase, indicating that this initial loss was not due to an increase in total activity. An 80% increase in proteolytic activity was correlated with the onset of the irreversible stage and the rapid decline in RuBPCase activity per milligram of carboxylase. Delaying senescence with zeatin reduced the rate of loss of carboxylase and delayed both the onset of the irreversible stage and the increase in proteolytic activity to the same degree, suggesting that these events are closely related. The main proteinases present in wheat and responsible for the increase in activity are the thiol proteinases. These proteinases have a high affinity for RuBPCase, exhibiting an apparent K_m at 38 C of 1.8 × 10⁻⁷ m. The K_m for casein was 1.1 × 10⁻⁶ m. If casein is representative of noncarboxylase protein, then the higher affinity for carboxylase may provide an explanation for its apparent preferential loss during the reversible stage of senescence.     

Partial purification and characterization of endoproteinases from senescing barley leaves

Two major endoproteinases were purified from senescing primary barley leaves. The major enzyme (EP₁) appeared to be a thiol proteinase and accounted for about 85% of the total proteolytic activity measured in vitro. This proteinase was purified 5,800-fold and had a molecular weight of 28,300. It was highly unstable in the absence of dithiothreitol or at a pH greater than 7.5. Leupeptin, at a concentration of 10 micromolar, inhibited this enzyme 100%. A second proteinase (EP₂) was purified approximately 50-fold and had a molecular weight of 67,000. It was inhibited 20% by 1 millimolar dithiothreitol and 50% by 1 millimolar phenylmethyl sulfonylfluoride. EP₂ contributed about 15% of the total proteolytic activity measured in vitro. Both proteinases hydrolyzed a variety of artificial and protein substrates, and both had pH optima of 5.5 to 5.7 when either azocasein or [¹⁴C]ribulose-1,5-bisphosphate carboxylase ([¹⁴C]RuBPCase) was the substrate. The thiol endoproteinase hydrolyzed azocasein linearly but hydrolyzed [¹⁴C]RuBPCase biphasically. A third endoproteinase (EP₃), not detected by standard proteolytic assays, was observed when [¹⁴C]RuBPCase was the substrate.     

Relations between d-ribulose-1,5-bisphosphate carboxylase,carboxysomes and CO2 fixing capacity in the obligate chemolithotroph Thiobacillus neapolitanus grown under different limitations in the chemostat

An adaptation of the d-ribulose-1,5-bisphosphate carboxylase (RuBPCase) activity to changing CO₂ concentrations in the growth medium in the chemostat was observed in the obligate chemolithotroph Thiobacillus neapolitanus. RuBPCase activity has been separated in a soluble and particulate fraction. The activity of the particulate fraction appeared to be associated with the carboxysomes.The total activity of RuBPCase of CO₂ limited cultures was about 5-fold higher than the activity of thiosulphate limited cultures grown in the presence of 5% CO₂ whilst the particulate activity and the soluble activity were about 8- and 1.5-fold higher, respectively. The fluctuation of the total and particulate RuBPCase activity correlated with the changes in volume density of carboxysomes in the cell.An inverse correlation between maximal CO₂ fixing capacity by whole cells and the volume density of carboxysomes was observed. The change in ratio of soluble RuBPCase activity to particulate RuBPCase activity paralleled the change in maximal CO₂ fixation by whole cells during the different growth conditions.     

Physiological comparisons of two soybean cultivars differing in canopy photosynthesis. II. Variation in specific leaf weight,nitrogen, and protein components

Cultivar differences in canopy apparent photosynthesis (CAP) have been observed in soybean (Glycine max (L.) Merr.) but little is known about the physiological mechanisms which are responsible for such differences. This study was initiated to determine if variation in ribulose 1,5-bisphosphate carboxylase (RuBPCase) and soluble protein exists among cultivars which differ in CAP during reproductive growth. In addition, the relationship between specific leaf weight (SLW) and leaf protein was examined. Two Maturity Group VI cultivars, Tracy (high CAP) and Davis (low CAP), were grown in the field during 1979, 1980, and 1981 and in a greenhouse experiment. Leaves located at two canopy positions (topmost, fully expanded leaf and eighth node from the top) in 1979 and three canopy positions (those mentioned, plus the fourth node from the top) in 1980 and 1981 were sampled. Leaves at the two upper canopy positions exhibited greater SLW, RuBPCase m^–2, and soluble protein m^–2 than found at the eighth node down. Photosynthetic capacity of leaves at inner canopy regions was therefore affected by both light penetration into the canopy and leaf protein status. Over the three year period, the SLW was 23 percent and the soluble protein m^–2 leaf 21 percent greater in Tracy than in Davis. Although the trend in RuBPCase m^–2 leaf was not significant, it was consistently greater in Tracy in the field and greenhouse. No cultivar differences were observed when the proteins were expressed on a unit of leaf dry weight. The quantity of RuBPCase per unit leaf area was positively correlated with SLW with significant partial correlation coefficients of 0.62, 0.67, 0.35, and 0.82 for 1979, 1980, 1981, and the greenhouse study, respectively. Since these cultivars have similar leaf area indices during September, the greater SLW of Tracy is translated into more photosynthetic proteins per unit ground area and higher CAP rate.Abbreviations AP Leaf Apparent Photosynthesis - CAP Canopy Apparent Photosynthesis - DAP Days After Planting - DTT Dithothreitol - HEPES N-2-hydroethylpiperazine N-2 ethanesulfonic acid - LAI Leaf Area Index - LSD Least Significant Difference - PPFD Photosynthetic Photon Flux Density - PVP-40 Polyvinylpolypyrroledone (molecular weight, 4000) - RuBPCase Ribulose 1,5-bisphosphate Carboxylase - SLW Specific Leaf Weight     

Water Deficit and Associated Changes in Some Photosynthetic Parameters in Leaves of ;Valencia' Orange (Citrus sinensis [L.] Osbeck)

Photosynthetic CO₂ assimilation, transpiration, ribulose-1,5-bisphosphate carboxylase (RuBPCase), and soluble protein were reduced in leaves of water-deficit (stress) `Valencia' orange (Citrus sinensis [L.] Osbeck). Maximum photosynthetic CO₂ assimilation and transpiration, which occurred before midday for both control and stressed plants, was 58 and 40%, respectively, for the stress (−2.0 megapascals leaf water potential) as compared to the control (−0.6 megapascals leaf water potential). As water deficit became more severe in the afternoon, with water potential of −3.1 megapascals for the stressed leaves vs. −1.1 megapascals for control leaves, stressed-leaf transpiration declined and photosynthetic CO₂ assimilation rapidly dropped to zero. Water deficit decreased both activation and total activity of RuBPCase. Activation of the enzyme was about 62% (of fully activated enzyme in vitro) for the stress, compared to 80% for the control. Water deficit reduced RuBPCase initial activity by 40% and HCO₃⁻/Mg²⁺-saturated activity by 22%. However, RuBPCase for both stressed and control leaves were similar in K_cat (25 moles CO₂ per mole enzyme per second) and K_m for CO₂ (18.9 micromolar). Concentrations of RuBPCase and soluble protein of stressed leaves averaged 80 and 85%, respectively, of control leaves. Thus, reductions in activation and concentration of RuBPCase in Valencia orange leaves contributed to reductions in enzyme activities during water-deficit periods. Declines in leaf photosynthesis, soluble protein, and RuBPCase activation and concentration due to water deficit were, however, recoverable at 5 days after rewatering.     

Photosynthesis, leaf resistances, and ribulose-1,5-bisphosphate carboxylase degradation in senescing barley leaves

The relationship between loss of ribulose-1,5-bisphosphate carboxylase (RuBPCase) and the decline in photosynthesis during the senescence of barley primary leaves was assessed. Loss of RuBPCase accounted for about 85% of the decrease in soluble protein. RuBPCase was highly correlated with in vitro RuBPCase activity (r = 0.95) and gross photosynthesis (r = 0.96). However, the rate of photosynthesis per milligram RuBPCase increased during the early stages of leaf senescence. The concentration of nonreducing sugars was negatively correlated (1% level) with photosynthesis. Free α-amino N, in contrast to nonreducing sugars, declined markedly during senescence. A decrease in chlorophyll and an increase in in vitro protease activity was observed, but these changes did not appear to be closely related to the decline in photosynthesis and RuBPCase. Mesophyll resistance increased at the same rate that photosynthesis and RuBPCase declined. Stomatal resistance increased more rapidly than mesophyll resistance and accounted for about 24% of the total increase in resistance to CO₂ diffusion. The concentration of CO₂ in the intercellular air spaces decreased during the last stage of senescence. Although loss of RuBPCase probably is the primary event responsible for the decline in photosynthesis during leaf senescence, other factors such as in vivo regulation and stomatal aperture must also be considered.     

Sequential synthesis of some proteins in cultured carrot explant (Daucus carota) cells during callus induction

Freshly isolated explants of the secondary phloem of carrot roots were exposed to ¹⁴C-leucine for various periods from t₀—to 18 h and the ¹⁴C labelling of protein was studied by 2-dimensional PAGE followed by fluorograph. The labelling pattern of proteins indicated a sequential activation of synthesis of about 130 proteins during the 18 h experimental period prior to the onset of cell division activity.Abbreviations IAA indole acetic acid - 2iP 2-isopentenyladenine - PVP polyvinylpyrrolidone - CBB Coomassie brilliant blue - RuBPCase ribulosebisphosphate carboxylase - LSC liquid scintillation counter - spec.act. specific radioactivity - u.l. uniformly labelled     

Biosynthesis of ribulose-1.5-bisphosphate carboxylase in greening cells of Euglena gracilis

Light induction of chloroplast development in Euglena leads to quantitative changes in the protein composition of the soluble cell part. One major part of these is the observed accumulation of ribulose-1.5-bisphosphate carboxylase/oxygenase (RuBPCase) enzyme (EC 4.1.1.39). As measured by immunoelectrophoresis, a small amount of RuBPCase (about 10^-6 pmol) is present in a dark-grown cell, whereas a greening cell (72h) contains 10–20 pmol enzyme. Both the cytoplasmic and chloroplastic translation inhibitors, cycloheximide and spectinomycin, have a strong inhibitory effect on the synthesis of the enzyme throughout the greening process of Euglena cells. Electrophoretic and immunological analyses of the soluble phase prepared from etiolated or greening cells do not show the presence of free subunits of the enzyme. For each antibiotic-treated greening cell, the syntheses of both subunits are blocked. Our data indicate that tight reciprocal control between the syntheses of the two classes of subunits occurs in Euglena. In particular, the RuBPCase small subunit synthesis in greening Euglena seems more dependent on the protein synthesis activity of the chloroplast than the syntheses of other stromal proteins from cytoplasmic origin.Abbreviations LSU large subunit of ribulose-1.5-bisphosphate carboxylase - RuBP ribulose-1.5-bisphosphate - RuBP-Case ribulose-1.5-bisphosphate carboxylase - SSU small subunit of ribulose-1.5-bisphosphate carboxylase     

Ribulose Bisphosphate Carboxylase and Proteolytic Activity in Wheat Leaves from Anthesis through Senescence

Changes in ribulose bisphosphate carboxylase (RuBPCase) and proteolytic activity were followed in the flag leaf and second leaf of field-grown winter wheat (cv. Arthur). These changes were followed in relation to changes in leaf chlorophyll, protein, and photosynthesis, and seed development. Levels of RuBPCase were determined by rocket immunoelectrophoresis as described previously (Wittenbach 1978 Plant Physiol 62: 604-608). RuBPCase constituted 40 to 45% of the total soluble protein in the flag leaf and an even higher percentage of the soluble protein in the second leaf. This ratio remained unchanged until senescence when RuBPCase protein was apparently lost at a faster rate than total soluble protein. No change in the specific activity of RuBPCase on either a milligram protein or RuBPCase basis was observed until senescence. A close correlation existed among the various indices of senescence in the field, namely, the decline in chlorophyll, protein, photosynthesis, and RuBPCase activity. In addition, proteinase activity increased with the onset of senescence. These enzymes readily degraded RuBPCase, exhibiting a pH optimum of 4.8 to 5.0 and a temperature optimum of 50 C. Proteinase activity was modified by sulfydryl reagents suggesting the presence of sulfydryl groups at or near the active sites.     

Changes in the Amounts of Ribulose Bisphosphate Carboxylase Synthesized and Degraded during the Life Span of Rice Leaf (Oryza sativa L.)

In situ synthesis and degradation of ribulose bisphosphate carboxylase(RuBPCase) were studied quantitatively in the 12th leaf bladeof the rice plant during the life span of the leaf. Levels ofRuBPCase protein were determined by rocket immunoelectrophoresis.The amounts of RuBPCase synthesized and degraded were estimatedusing ¹⁵N tracer. RuBPCase was scarcely recognized in the leaf when the tip ofthe leaf had just emerged from the 1 lth leaf sheath. Then itincreased rapidly and reached its maximum content a week afterthe completion of leaf expansion. At this time RuBPCase accountedfor 56% of the soluble leaf protein N (26% of the total leafN). The total amount of RuBPCase synthesized up to this timewas about 90% of the amount synthesized throughout the leaf'slife. Degradation of RuBPCase started about the time when it reachedthe maximum content and proceeded at a faster rate during senescencethan that of the remaining soluble protein. When the leaf hadsenesced completely, it contained little measurable RuBPCasealthough the total leaf N was about 30% of the maximum level.These results clearly suggest that RuBPCase is a major N componentwhich is used as remobilized N for the growth of young tissues. Influx and efflux of N and the synthesis and degradation ofRuBPCase are discussed in relation to leaf age. (Received February 18, 1983; Accepted June 16, 1983)     

Comparison of Patterns of Accumulation of Ribulose Bisphosphate Carboxylase Antigen and Catalytic Activity and Measurement of Antigen Half-Life during the Cell Cycle of Chlorella sorokiniana

By use of specific immunochemical procedures, ribulose-1,5-bisphosphate carboxylase (RuBPCase), antigen and catalytic activity were shown to have coincident step-patterns of accumulation during the cell cycle of Chlorella sorokiniana. Pulse-chase studies, employing radioactive sulfate, were performed during the period of rapid accumulation of enzyme activity and during the period of constant enzyme activity in the cell cycle. No degradation of RuBPCase antigen could be detected during either of these cell cycle periods. Thus, the step-pattern of accumulation of RuBPCase activity resulted from periodic synthesis of an enzyme that was stable under steady-state cell cycle conditions. Although inhibition of protein synthesis by cycloheximide, at different times in the cell cycle in the light, resulted in rapid decay of RuBPCase activity, this loss in activity occurred without detectable loss in enzyme antigen. When synchronous cells were placed into the dark, to slow the rate of protein synthesis in the absence of cycloheximide, the levels of enzyme antigen and activity decreased by 30 and 50%, respectively, during the 10-hour dark period. Thus, in C. sorokiniana changes in RuBPCase activity do not necessarily reflect parallel changes in enzyme antigen, particularly when cell growth is perturbed by changes from steady-state cultural conditions.     

The effect of sewage-enriched river Ganga water on the biomass production of theAzolla-Anabaena complex

The biomass formation ofAzolla was greatly enhanced by water of the River Ganga and by prevailing environmental conditions. It increased gradually from January to April (first maximum 2.409 g.m^–2.day^–1), declined during June (1.185 g.m^–2.day^–1), and reached a second its maximum during September (2.629 g.m^–2.day^–1). The biomass formation was related to the nutrient availability in the medium in a particular season (measured were: nitrate-N, available phosphorus, total suspended solids, and conductivity). The average annual production of 6.73 ton.ha^–1.yr^–1 is equivalent to the average production of 0.025 ton.ha^–1.yr^–1 phosphorus, 0.252 ton.ha^–1.yr^–1 nitrogen, and 1.57 ton.ha^–1.yr^–1 crude protein.     

The relationship between carbon-dioxide-limited photosynthetic rate and ribulose-1,5-bisphosphate-carboxylase content in two nuclear-cytoplasm substitution lines of wheat,and the coordination of ribulose-bisphosphate-carboxylation and electron-transport capacities

Photosynthesis in two cultivars of Triticum aestivum was compared with photosynthesis in two lines having the same nuclear genomes but with cytoplasms derived from T. boeoticum. The in-vitro specific activity of ribulose-1,5-bisphosphate carboxylase (RuBPCase; EC 4.1.1.39) isolated from lines with T. boeoticum cytoplasm was only 71% of that of normal T. aestivum. By contrast, the RuBPCase activities calculated from the CO₂-assimilation rate at low partial pressures of CO₂, p(CO₂), were the same for all lines for a given RuBPCase content. This indicates that both types of RuBPCase have the same turnover numbers in-vivo of 27.5 mol CO₂·(mol enzyme)^–1·s^–1 (23°). The rate of CO₂ assimilation measured at normal p(CO₂), p _a =340 bar, and high irradiance could be quantitatively predicted from the amount of RuBPCase protein. The maximum rate of RuBP regeneration could also predict the rate of CO₂ assimilation at normal ambient conditions. Therefore, the maximum capacities for RuBP carboxylation and RuBP regeneration appear to be well-balanced for normal ambient conditions. As photosynthetic capacity declined with increasing leaf age, the capacities for RuBP carboxylation and RuBP regeneration declined in parallel.Abbreviations PAR photosynthetically active radiation - RuBP(Case) ribulose-1,5-bisphosphate (carboxylase)     

Fusion expression of Escherichia coli prlC gene and preparation of PrlC proteinase affinity column

Escherichia coli PrlC is a trypsin-like proteinase regulating the cell cycle. The Escherichia coli prlC gene has been cloned into the pET28a prokaryotic expression vector. The recombinant fusion protein was produced mostly in the soluble, active form and the expression level amounted to approximately 70% of total protein. The recombinant proteinase was efficiently adsorbed to a resin containing immobilized Ni²⁺ via its amino terminal fusion hexahistidine tail to give a PrlC proteinase affinity column. The adsorbed fusion proteinase hydrolyzed 4-methylcoumaryl-7-amide of tert-butoxycarbonyl-l-valyl-l-prolyl-l-arginine (Boc-Val-Pro-Arg-NH-Mec), the specific substrate for the trypsin-like proteinase activity of E. coli PrlC.     

Hydrolysis of Ribulose-1,5-bisphosphate Carboxylase by Endoproteinases from Senescing Barley Leaves

The hydrolysis of ¹⁴C-labeled ribulose-1,5-bisphosphate carboxylase (RuBPCase) by two partially purified endoproteinases from senescing barley (Hordeum vulgare v. Numar) leaves is described. The major thiol proteinase, EP₁, exhibits biphasic kinetics which appear to be caused by a region of the large subunit of RuBPCase that is highly sensitive to attack by EP₁. This proteinase further hydrolyzes both the large and small subunit to smaller peptides. A second proteinase, EP₂, appears to convert the small subunit of RuBPCase rapidly to a 13.7-kilodalton fragment during initial stages of hydrolysis and then to degrade both this fragment and the large subunit. The presence of a third endoproteinase, EP₃, was discovered when [¹⁴C]RuBPCase, which appeared to be homogeneous by sodium dodecyl sulfate polyacrylamide electrophoresis, seemed to undergo very low but significant rates of “autolysis.” The large molecular weight fragments produced by EP₃ were different from those of EP₁ and EP₂.     

Entamoeba histolytica: Soluble and membrane-associated neutral sphingomyelinase-C and other unidentified esterase activity

Sphingomyelinase (SMase) activity was measured in Entamoeba histolytica particulate and soluble subcellular fractions. The effects on SMase of incubation time, total protein concentration, pH, and several divalent cations were determined. SMase-C and other unidentified esterase activity were detected in soluble and particulate fractions. SMase-C was 94.5-96.0% higher than the unidentified esterase activity. Soluble and insoluble SMase-C specific activities increased with protein dose and incubation time. Soluble and insoluble SMase-C activities were maximum at pH 7.5 and were dependent on Mg²⁺, Mn²⁺, or Co²⁺, and inhibited by Zn²⁺, Hg²⁺, Ca²⁺, and EDTA. SMase-C was active in the pH range of 3-10 and its maximum activity was at pH 7.5. The soluble and insoluble SMases have remarkably similar physicochemical properties, strongly suggesting that E. histolytica has just one isoform of neutral SMase-C that had not been described before and might be essential for E. histolytica metabolism or virulence.     

Effect of calcium on immobilization of rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.) peroxidase for bioassays in sodium alginate and Agarose gel

The direct immobilization of soluble peroxidase isolated and partially purified from shoots of rice seedlings in calcium alginate beads and in calcium agarose gel was carried out. Peroxidase was assayed for guaiacol oxidation products in presence of hydrogen peroxide. The maximum specific activity and immobilization yield of the calcium agarose immobilized peroxidase reached 2,200 U mg⁻¹ protein (540 mU cm⁻³ gel) and 82%, respectively. In calcium alginate the maximum activity of peroxidase upon immobilization was 210 mU g⁻¹ bead with 46% yield. The optimal pH for agarose immobilized peroxidase was 7.0 which differed from the pH 6.0 for soluble peroxidase. The optimum temperature for the agarose immobilized peroxidase however was 30°C, which was similar to that of soluble peroxidase. The thermal stability of calcium agarose immobilized peroxidase significantly enhanced over a temperature range of 30∼60°C upon immobilization. The operational stability of peroxidase was examined with repeated hydrogen peroxide oxidation at varying time intervals. Based on 50% conversion of hydrogen peroxide and four times reuse of immobilized gel, the specific degradation of guaiacol for the agarose immobilized peroxidase increased three folds compared to that of soluble peroxidase. Nearly 165% increase in the enzyme protein binding to agarose in presence of calcium was noted. The results suggest that the presence of calcium, ions help in the immobilization process of peroxidase from rice shoots and mediates the direct binding of the enzyme to the agarose gel and that agarose seems to be a better immobilization matrix for peroxidase compared to sodium alginate.     

Light and metabolite regulation of the synthesis of ribulose-1,5-bisphosphate carboxylase/oxygenase and the corresponding mRNAs in the unicellular alga Chlorogonium

In the unicellular green alga Chlorogonium elongatum, the synthesis of the plastid enzyme ribulose bisphosphate carboxylase/oxygenase (RuBPCase) and its mRNAs is under the control of light and acetate. Acetate is the sole metabolizable organic carbon source for this organism. Light greatly promotes the synthesis of RuBPCase and the increase in the concentration of the mRNAs of both subunits of the enzyme while acetate has a strong inhibitory effect on this process. There is a good agreement between RuBPCase synthesis and the amount of translateable RuBPCase mRNA present in cells which are cultured under different conditions (autotrophic, heterotrophic, mixotrophic). During the transition period after transfer of the cells from heterotrophic to autotrophic growth conditions the amounts of the large and small subunits of the enzyme increase well coordinated. In contrast to the protein subunits the two subunit-mRNAs accumulate with different kinetics.Abbreviations LSU large subunit of RuBPCase - poly(A)^- RNA - poly(A)⁺RNA non-, poly-adenylated RNA - RuBPCase ribulose-1,5-bisphosphate carboxylase/oxygenase EC 4.1.1.39 - SSU small subunit of RuBPCase     

Population Biology and Growth of Ozark Cavefish in Logan Cave National Wildlife Refuge, Arkansas

Ozark cavefish, Amblyopsis rosae, is a threatened species endemic to the Springfield Plateau of the Ozark Highlands in Arkansas, Missouri, and Oklahoma. One of the largest known Ozark cavefish populations, located in Logan Cave, Arkansas, was surveyed 25 times over a two-year period between 1993 and 1995. During the study, 147 Ozark cavefish > 30 mm (TL) were marked with visual implant tags and 140 Ozark cavefish were available for recapture; 68 were recaptured 189 times and the rest (72) were never recaptured. Individual Ozark cavefish persisted in Logan Cave for a relatively short time. Only 14% of 80 fish tagged during a previous study in 1992 were recaptured during this study, and half of all recaptured fish disappeared within three months. However, if a fish persisted for at least seven months in the cave, its probability of being recaptured over an additional year was high. Maximum persistence of a tagged fish was 28 months, suggesting these fish have a maximum life-span of 4–5 years. Growth averaged 0.6 mm per month, with maximum recorded growth of 6 mm per month and a maximum size of 65 mm TL. Smaller fish grew faster than larger fish but growth rates were sporadic, with several mid-sized fish (45–49 mm) showing little growth (0–3 mm year^-1) while some fish > 50 mm grew up to 12 mm year^-1. Most fish gained in length during April–October, the same period a maternity colony of gray bats occupied the cave. Gross Ozark cavefish movement over the study period ranged up to 1002 m, with a mean movement of 1.2 m day^-1; movement was positively correlated with Ozark cavefish total length. Death seemed the most likely explanation for loss of tagged Ozark cavefish, including fish that emigrated out of the cave. Little up-stream movement was recorded between reaches and did not account for loss of tagged fish. Reproduction within the cave and immigration from the aquifer accounted for persistence of Ozark cavefish in Logan Cave.