Recommendations were developed to help aquatic scientists select which curve fitting method should be used to fit data that are expected to follow rectangular hyperbolic relationships.Rectangular hyperbolae of the form V = (V-S)/(Km + S), where V is a biological rate and S is the concentration of some substrate, are widely used by researchers to model the kinetics of processes such as enzyme activity versus substrate concentration, nutrient uptake versus nutrient concentration, and grazing and growth rate versus prey concentration.A variety of procedures exist to estimate the parameters V, , , , , (the rate of the process at saturating substrate concentration) and K, (the concentration of S at which half the saturated rate is achieved).There has been extensive discussion in the biochemical and ecological literature as to which fitting method is most appropriate, based largely on theoretical and statistical considerations.However, the assumptions inherent in these fitting procedures are typically violated by the data obtained in many field and laboratory studies, e.g. the measurement of S has an associated error, or error levels in the measurement of Vmay not be constant across S. Thus, there is a problem predicting a priori which fitting method should be used.In this study, this problem was approached using Monte Carlo simulations.Data sets with known V,,,, and Km were constructed for 5 different data cases, ranging from data sets where saturation was not achieved to data sets where very few sub-saturated measurements were available.Random, normally distributed errors were assigned to each point based on a 10 %, 20 % or 50 % constant or variable error in the estimate of V, or 20 % error in both S and V Six fitting procedures were applied including linear methods (Lineweaver-Burk; Eadie-Hofstee; Hanes-Woolf), the median method (Eisenthal and Cornish-Bowden), and non-linear least-squares methods (Cleland-Wilkinson; Tseng-Hsu).Non-linear methods were generally superior, but for data sets with low error (10%) all methods gave almost equally accurate results.Data with constant error were more difficult to fit than those where error varied with V. Criteria for selecting a fitting method based on data characteristics are discussed and applied to actual data sets.
The enzyme nitrate reductase (NR) has been proposed as an index of nitrate incorporation rates in marine phytoplankton, but it has proven difficult to interpret NR measurements in field settings because many previous NR assays have been poorly optimized and NR activity in phytoplankton has been poorly characterized under steady state conditions. An NR assay was developed for the diatom Thalassiosira pseudonana using an extraction in phosphate buffer with Triton X‐100, EDTA, dithiothreitol, polyvinyl pyrrolidone, and bovine serum albumin. NR activity in homogenates was stable for up to 1 h, but filtered samples could be stored for 96 h in liquid nitrogen without significant loss of activity. Addition of FAD to crude extracts of T. pseudonana had no effect, whereas the effect on desalted extracts or crude extracts from other species, varied from decreases in NR activity to over 250% increases. Half‐saturation constants ( K m ) varied between species; high levels of NADH or nitrate were found to be inhibitory in some cases. These results indicate a wide diversity of forms of NR in marine phytoplankton. Under continuous, light‐limited growth, NR activity was quantitatively related to calculated rates of nitrate incorporation ( µ N ) in T. pseudonana, Skeletonema costatum, and three other diatom species examined. The relationship differed for 10 other species; NR activity was equal to µ N in some cases, but higher or lower in others. In dinoflagellates, in particular, NR activity was highly correlated with µ N , but accounted for <20% of µ N in Amphidinium carterae.
ABSTRACT Light‐limited cultures of the marine diatom Thalassiosira pseudonana (Hustedt) Hasle and Heimdal (3H clone) were grown over a range of growth rates between 0.06 and 1.64 d −1 . Variations in cell volume, cell quotas of carbon, nitrogen, and protein, and maximal activity of the enzyme nucleoside diphosphate kinase (NDPK) were measured and examined as a function of growth rate. NDPK from T. pseudonana showed K m values of 0.24 and 0.68 mM for thymidine 5′‐diphosphate and adenosine 5′‐triphosphate (ATP), respectively, which are similar to those found for NDPK from a variety of organisms, from bacteria to mammals. An apparent activation enthalpy of 3.52 kCal·mol −1 was determined from Arrhenius plots. No thermodynamic transition points were noted over a temperature range from 10° to 25°C. NDPK activity was significantly correlated with growth rate but not with cell volume, carbon, nitrogen, or protein; for interspecific comparisons, normalization of enzyme activity to cell number may be most meaningful. NDPK activity per cell versus growth rate followed a U‐shaped relationship, being relatively constant between 0.5 and 1.0 d −1 and rising at higher and lower growth rates. Over this range, enzyme activity may be regulated by substrate concentration (ATP or other nucleoside triphosphates) or by adenylate energy charge. At higher growth rates where energy charge and substrate concentrations are probably high, changes in enzyme concentration appear to be required. The reasons for a rise in enzyme activity at low growth rate is unclear. Simultaneous measurement of nucleoside di‐ and triphosphate levels alongside NDPK measurements may help clarify the relationship, but these preliminary experiments indicate that NDPK is of limited usefulness as an index of in situ growth rate.
The past decade has seen substantial breakthroughs in understanding the biochemistry, molecular biology and regulation of nitrate reductases (NR) in higher plants and green algae. In contrast, although there has been considerable interest in using various measurements of NR to provide ecophysiological information, comparable knowledge of NR is largely lacking in algal groups other than chlorophytes. Applying information about NR from chlorophytes and higher plants to other algae may be difficult. There is evidence that nonchlorophyte forms of NR are diverse and distinct in terms of biochemical characteristics and regulatory features. Key areas to be pursued in non-chlorophyte algae include the identification and adoption of model organisms for NR research in different algal groups; the creation of selected and engineered mutants; the purification, biochemical characterization and production of antibodies to different algal forms of NR; the identification of NR genes; and the undertaking of coordinated research on nitrate uptake proteins and other enzymes in pathways of nitrate assimilation.
Proteases are essential components of cells and participate in processes ranging from photoacclimation and nutrient acquisition to development and stress responses. Virtually nothing is known about this diverse group of enzymes in macroalgae. Methods to measure proteases developed for phytoplankton (caseinolysis, leucine aminopeptidase (LAP) activity and casein zymograms) have been modified to allow use with spectrophotometers as well as spectrofluorometers, so making them accessible to a wider number of users. Applying the methods to a wide range of macroalgae from intertidal zones in south-western Spain and Northern Ireland detected proteases in all species examined. Protease activities differed among species but there was little systematic variation with taxonomic group. Moreover, similar species in Spain and Northern Ireland often showed differences. With the exception of LAP activities in brown algae, protease activities were one to two orders of magnitude greater in macroalgae than those previously measured in phytoplankton, when expressed per unit protein. However, scaling of activities to protein was complicated by significant interferences. Copper-based protein assays gave erroneously high results when applied to the brown algae, and partial purification of proteins by trichloroacetic acid precipitation did not overcome the problem. The higher levels of protease activity suggest that proteolysis plays a more significant role in multicellular than in unicellular algae. Proteases were characterized in terms of activities at different pH and the effect of protease inhibitors. In common with microalgae but in contrast to findings for animals and higher plants, proteases tended to show higher activities in the neutral and alkaline ranges. Many commonly used protease inhibitors (e.g. leupeptin and phenylmethylsulphonyl fluoride) had relatively little effect, which may explain why biochemical work with macroalgal species has traditionally been difficult. Our results suggest that macroalgal proteases are easily measurable but highly variable. A major source of variability that has not been assessed is differing environmental conditions. If this is correct, measurements of proteolytic enzymes may provide a valuable tool for examining biologically relevant changes in environments. Controlled laboratory experiments and seasonal monitoring are the next logical steps towards this goal.
Data set contains vertical profile of dissolved silicate taken between 1988 and 2019 at Fox Point station off Milwaukee, Wisconsin, plus samples from three Milwaukee-area beaches of dissolved silica, suspended biogenic silica and biogenic silica contained in assemblages of Cladophora and associated epiphytes, collected between 2005 and 2019.
