Different Concentrations Of Substrate Research Articles

Rheological and Self-Healing Behavior of Hydrogels Synthesized from l-Lysine-Functionalized Alginate Dialdehyde.

Alginate dialdehyde and l-lysine-functionalized alginate dialdehyde were prepared to provide active aldehyde and l-lysine sites along the alginate backbone, respectively. Different concentrations of substrates and the reduction agent were added, and their influence on the degree of l-lysine substitution was evaluated. An amination reduction reaction (with l-lysine) was conducted on alginate dialdehyde with a 31% degree of oxidation. The NMR confirmed the presence of l-lysine functionality with the degree of substitution of 20%. The structural change of the polymer was observed via FTIR spectroscopy, confirming the formation of Schiff base covalent linkage after the crosslinking. The additional l-lysine sites on functionalized alginate dialdehyde provide more crosslinking sites on the hydrogel, which leads to a higher modulus storage rate than in the original alginate dialdehyde. This results in dynamic covalent bonds, which are attributed to the alginate derivative-gelatin hydrogels with shear-thinning and self-healing properties. The results suggested that the concentration and stoichiometric ratio of alginate dialdehyde, l-lysine-functionalized alginate dialdehyde, and gelatin play a fundamental role in the hydrogel's mechanical properties.

Production of Polyhydroxyalkanoates (PHAs) Using the Bacterium Ralstonia Eutropha to Obtain Bio-Plastic

The purpose of this research was to publicize the use of agro-industrial substrates in obtaining PHAs-type granules, which are synthesized intracellularly by some bacterial genera as reserve material when they encounter nutrient limitations, which would replace synthetic polymers, but the cost of elaboration of this process is expensive, for that reason in the present study different concentrations of substrate were evaluated using a DBCA experimental design. The study to obtain PHA was carried out in three concentrations of ED from cassava starch during two fermentation times. The best time was 72 hours with adding 10g/L hydrolyzed cassava starch syrup. Initiating the isolation of the bacterium from soils where the PHA-producing bacteria live, for its identification, the Gram and molecular staining technique (PCR) was used, and it was distinguished as a Gram-negative bacterium whose carbon source was the yucca flour syrup. Subsequently, the protocol of conditions that intervene in the batch-type fermentation was established. During this time, the accumulation of PHAs, the consumption of glucose, and the accumulation of cells were evaluated through a microbial growth curve. Next, the extraction of the biopolymer was carried out using solutions with acid. Finally, the lamination of the biopolymer was carried out, which does not completely cover the proposed product. Rather, it was possible to cover 10% of the entire product.

Chitinase Activity by Chitin Degrading Strain (Bacillus Salmalaya) in Shrimp Waste

Introduction: Chitin degradation by chitinase enzyme can be used on a large scale for bioremediation of seafood waste and is environmentally friendly. Objective: The main aim of this study was to screen the potential of the strain as a chitin degrading agent. Methods: In this present study, the cell-free supernatant of Bacillus salmalaya was determined for its protein concentration. Bacillus salmalaya139SI was isolated from agricultural soil and it was identified by staining technique and colony morphology. The production of chitinase by Bacillus salmalaya139SI was optimized under different concentrations of substrate, pH and temperature. Results: Strain 139SI exhibited strong hemolytic activity and the crude protein concentration of Bacillus salmalaya was 84.09mg/ mL with OD value 0.462. Strain 139SI were also screened on colloidal chitin agar medium supplemented with mineral salt. Chikinase production was determined by clear zones of hydrolysis produced after 7 days of incubation at 37°C. The maximum chitinase production was observed in Brain Heart Infusion broth supplemented with 1.0% colloidal chitin at pH 7 and temperature 35°C after four days of incubation. Chitinase activity was observed when high concentrations of crude extract of 139SI able to degrade shrimp shell by showing the degradation zone at day 4. Conclusion: From the results, we concluded that the Bacillus salmalayahas potential to be a biofunctional chitinase that could degrade complex polysaccharides present in the organic wastes and applicable in cleaning the environment.

Influence of Inoculum and Substrate Concentration on Anaerobic Digestion of Ocimum Sanctum (Holy Basil)

“Anaerobic digestion is the constrained degradation of usual organic waste in the oxygen deficient environment and the existence of anaerobic microorganisms. Anaerobic digestion is accomplished by microbial diversity. “Substrate loading rate is a measure of biological conversion capacity of anaerobic system and has significant influence on the process performance. Henceforth in the current work, the outcome of different concentrations of substrate to inoculum (0.3, 0.6 and 0.9) on anaerobic digestion of Holy Basil (Ocimum sanctum) used as substrate was explored in batch reactors for 45 days along with heterogenous cowdung as Inoculum. Temperature and biogas were noted daily. “The Volatile Solids (VS), Chemical Oxygen Demand (COD), Total Kjeldhal Nitrogen (TKN) and Phosphorous were analyzed at an interval of 5 days. Though the degradation rates of various parameters showed a higher efficiency at 0.3 inoculum to “substrate concentration, the biogas generation was impending to be maximum at 0.6 among the various digesters.

Effect of different concentrations of substrate in microbial fuel cells toward bioenergy recovery and simultaneous wastewater treatment

ABSTRACT Microbial fuel cells (MFCS) is a promising and expanding technology able to eliminate various pollutants of wastewater while converting its chemical energy into power energy using biocatalysts. The potential application of double-chamber microbial fuel cell (DC-MFC) for chemical oxygen demand (COD) removal and generated power from wastewater in the different conditions is investigated. DC-MFC is operated with anaerobic sludge as an active biocatalyst in an anode section, an aerobic cathode section and a Nafion117 membrane as a separator. The performance of the bioreactor is determined with different concentrations of chemical oxygen demand (COD) loadings in the MFC process, in terms of COD removal, power generation and columbic efficiencies. The results illustrated that COD removal efficiency increased at the high concentrations of organic matter. So that at COD concentration of 2000.0 mg/L the highest COD removal efficiency (84%) was obtained. But with increasing substrate initial concentration to 10000.0 mg/L the efficiency decreased to 79%. The important outputs of the system like the highest voltage, maximum generated power, current density, and energy efficiency with the 100,000 mg/L COD are 447 mV, 50.7 mW/m2, 570.0 mA/m2, and 18.6%, respectively. The optical density levels increased due to bacterial growth while pH severely decreased in the anode chamber when using high-concentration substrates in the MFC.

Oenological tannins to prevent Botrytis cinerea damage in grapes and musts: Kinetics and electrophoresis characterization of laccase

Enzymatic parameters (KM and Vmax), residual activity, effect of bentonite and electrophoresis characterization of laccase in the presence of different oenological tannins (OT) were investigated in relation to B. cinerea negative effects in grapes and musts. Five OT were tested (gallotannin, ellagitannin, quebracho, grape-skin and grape-seed) in comparison with ascorbic acid (AA), sulfur dioxide (SO2) and bentonite. We added OT, AA, SO2 and bentonite to botrytized must obtained by inoculation of grapes with B. cinerea strain 213. Laccase activity was measured by the syringaldazine method at different concentrations of substrate. Enzymatic parameters were determined using Michaelis-Menten and Lineweaver-Burk plots. The B. cinerea strain was also grown in a liquid medium for laccase production. Molecular weight of laccases and effect of OT upon these laccases were studied by SDS-PAGE. Results confirm that bentonite, contrary to OT, did not permit to reduce laccase activity. Regardless the tannin considered, Vmax, KM and laccase activity were reduced and gallotannin, grape-skin and grape-seed tannin presented the greatest ability. Efficiency of grape-seed tannin addition in order to reduce the laccase activity, was comparable to that of AA or SO2 at the typical doses employed in oenology for each one. Oenological tannins appear to be excellent processing aids to prevent laccase effects and contribute to reduce the use of SO2 in grapes and musts.

Towards the development of biofuel production from paper mill effluent

Biohydrogen and biomethane production via two stage sequencing system from paper mill effluent (PME) was evaluated to enhance its COD reduction. Heat-shock pretreatment of anaerobic sludge at 90 °C for 15 min was chosen to obtain a mixed microbial inoculum for dark fermentative hydrogen production. The effect of initial pH (5, 6 and 7) on the efficiency of hydrogen fermentation at different concentrations of substrate (3, 5 and 7 g-COD/L) was investigated, which the maximum hydrogen yield was obtained at initial pH 5. After that, the effects of substrate concentration (3, 5, 7, 10 and 15 g-COD/L) and fermentation temperature (37 and 55 °C) were investigated on the hydrogen production yield. The efficiency of dark hydrogen fermentation for all substrate concentrations was higher at 55 °C. The hydrogen production was enhanced as substrate concentration increased up to 5 g-COD/L, but then it was reduced, and the optimum hydrogen yield of 38.8 mL H2/g-CODinitial was obtained at substrate concentration of 5 g-COD/L at 55 °C. The highest hydrogen (35.1 mL H2/g-CODinitial) was obtained at 5 g-COD/L and 55 °C and the highest methane (553.8 mL CH4/g-CODinitial) was obtained at 3 g-COD/L and 37 °C. Maximum COD reduction of 88.1% was obtained in two-stage sequencing dark fermentation/anaerobic digestion system at 3 g-COD/L and 37 °C.

Electrochemical assay for 20S proteasome activity and inhibition with anti-cancer drugs

The majority of eukaryotic regulated protein turnover is performed by the proteasome, a multi-catalytic enzyme. Due to the fact that proteasome enzyme abnormal functioning was observed in different malignant cells, the proteasome is becoming a target for medical treatment. In order to evaluate the mechanisms of action of pharmaceutical compounds on proteasome enzyme inhibition, detecting and characterizing its activity is essential. An electrochemical assay that allows the monitoring of the chymotrypsin-like activity and inhibition of the 20S proteasome enzyme, based on the electrochemical detection of an electroactive compound released upon proteolysis of an adequate chymotrypsin-substrate is described. By employing differential pulse voltammetric measurement, the activity of the 20S proteasome enzyme was investigated for different incubation times of 20S with oligopeptide substrate as well as for different concentrations of substrate. Enzyme kinetic parameters were determined by voltammetry and the electrochemical assay compared with fluorescence spectroscopy. Electrochemical quartz crystal microbalance and atomic force microscopy were also used to investigate substrate interaction with the 20S proteasome and their adsorption at the electrode surface. Finally, the new electrochemical assay allowed to investigate the mechanisms of two different proteasome inhibitor drugs, bortezomib and oprozomib, underlying the applicability of the assay for understanding proteasome inhibitor action.

IN VITRO SCREENING OF CELL WALL DEGRADING ENZYME PRODUCTIVITY FROM FUNGAL CULTURE FILTRATES ON DEPROTEINISED PLANT FLUID BY CUP PLATE ASSAY

The present study purpose is to evaluate the potentiality of the Deproteinised juice made from selected plants to induce the enzyme productivity by growing the fungi on it. It is because, in earlier findings, the DPJ was found potential in enhancing fungi and the plant growth when used as the medium. The internal factors are responsible in DPJ which induces plants and fungi growth. During the process of Green Crop Fractionation (GCF), the deproteinised (DPJ) obtained from tissues of cabbage, beet, lucerne (Alfalfa), carrot and Anathum (Dill) forages left after leaf protein extraction employed as a medium for the cultivation of mycelial biomass of Penicillium and Aspergillus fungi. The mycelial growth in vitro was compared with the glucose nitrate medium. The culture filtrates were used to screen different secreted hydrolytic or cell wall degrading enzymes.. The agar ‘cup‐plate’ diffusion technique has been applied to the quantitative determination of enzyme activity, principally to amylase, cellulase and protease. With all enzymes so far examined, the relationship between diameter of zone over a wide range secreted quantitatively was examined. All fungi grew well on DPJ in comparison to their growth on glucose nitrate (GN) medium. Comparatively with GN medium, lucerne DPJ was found having more mycelial cellular proliferation. When the fungi grown on different concentrations of substrates, enriched with carboxymethyl cellulose, casein and starch in deproteinised leaf extracts and GN medium, it was found that there was the enhancement in the mycelial dry weight grown on DPJ as compared with glucose nitrate medium. Penicillium showed more yield of enzyme activities especially of cellulases and amylases as compared to protease by cup plate method. There was enhancement of mycelial biomass when the concentrations of substrates increased from 1% to 2%, while there was no change in the activity of all enzymes by increasing the concentrations of substrates. Activities of the enzymes in vitro can be indicative of the pattern of organogenesis in callus cultures in further studies by fungal culture filtrates cultured on deproteinised fluid medium.

Comparison of ethanol production from Gracilaria fisheri and Gracilaria tenuistipitata cultivated in aquaculture system in Thailand

Several species of seaweeds contain higher amounts of polysaccharides and low lignin. These species can be sources for bioethanol production and can be utilized for other biochemical production. In Thailand, large amounts of the red seaweeds Gracilaria fisheri and G. tenuistipitata can easily grow in effluents from aquaculture ponds and are used for wastewater treatment. This study was done to assess the potential substrates from these two Gracilaria species to produce ethanol. Different concentrations of substrate from each species (20–200 mg) were pretreated with 1 M H2SO4 at 95 °C for 150 min. The optimal pretreatment was 60 mg of substrate treated with 1 M H2SO4 at 95 °C for 150 min. This condition gave the highest amount of fermentable sugars in both Gracilaria species. Under these conditions, G. fisheri showed a higher catalytic efficiency (E = 5.2) than G. tenuistipitata (E = 4.1). Ethanol fermentation was performed by using the yeast strain Saccharomyces cerevisiae TISTR 5339. The ethanol produced from the G. tenuistipitata fermentation had the highest yield, producing 0.19 g ethanol g−1 glucose, whereas that of G. fisheri yielded 0.16 g ethanol g−1 glucose. This study suggests that the biomass of G. fisheri and G. tenuistipitata from a marine aquaculture system is a potential source for ethanol production.

Enzymatic Inhibition Constant of Acetylcholinesterase for the Electrochemical Detection and Sensing of Chlorpyrifos

Infiltration into soils of pesticides used during agricultural production has led to the contamination of aquatic ecosystems due to their long persistence in the environment. Some pesticides (e.g. Chlorpyrifos) are inhibitors of cholinesterase enzyme activity and their presence in water samples can be indirectly detected by a decrease in enzymatic activity. Biosensors based on cholinesterase enzymes are an alternative for the sensitive detection of important contaminants in the environmental sector. Acetylcholinesterase enzyme (AChE) catalyzes the hydrolysis of acetylthiocholine (ATCh) to produce thiocholine (TCh). This feature can be employed to measure the decrease in AChE activity. The inhibitory characteristics of the AChE-Chlorpyrifos system have been studied through cyclic voltammetry, by evaluation of the oxidation of the thiol group, which corresponds to TCh production on platinum electrodes in the presence of an inhibitor. In the present study, enzymatic curves were obtained at different concentrations of substrate and inhibitor, which were then used to determine the enzymatic kinetics corresponding to a mixed inhibition type, with an inhibition constant (Ki) of (18.26 ± 0.01) μM. TCh electrochemical detection appears to be a promising option for the development of biosensors to identify and quantify pesticides present in the ecosystem.

STUDY OF ALKALINE PHOSPHATASE KINETICS IN POLYCYSTIC OVARIES PATIENTS.

Purification of the alkaline phosphatase from serum for people living with polycystic ovaries were used gel filtration technique using the gel Sephadex column G100 with dimensions (20 × 2) cm, for the purpose of purification of alkaline phosphatase the use of a buffer of the (Tris -HCl) with a pH (7.2) to separate، enzyme kinetics, where studied the effect of different concentrations of substrate (sodium bi-article basis Phenyl phosphatases), results showed increasing the activity of alkaline phosphatases when increased substrate concentration. The resulting shape is hyperbolic. Km (1.14) mM, also studied the effect of pH and found that the optimal pH of the enzyme is (10). The optimum temperature was 37 ° C and the effect of reaction time was 20 minutes. The effect of Ascorbic acid show it works as an Inhibitor to the purified enzyme as that type of inhibition is non-competitive inhibitor type.

Channel of viral DNA packaging motor for real time kinetic analysis of peptide oxidation states.

Nanopore technology has become a powerful tool in single molecule sensing, and protein nanopores appear to be more advantageous than synthetic counterparts with regards to channel amenability, structure homogeneity, and production reproducibility. However, the diameter of most of the well-studied protein nanopores is too small to allow the passage of protein or peptides that are typically in multiple nanometers scale. The portal channel from bacteriophage SPP1 has a large channel size that allows the translocation of peptides with higher ordered structures. Utilizing single channel conductance assay and optical single molecule imaging, we observed translocation of peptides and quantitatively analyzed the dynamics of peptide oligomeric states in real-time at single molecule level. The oxidative and the reduced states of peptides were clearly differentiated based on their characteristic electronic signatures. A similar Gibbs free energy (ΔG0) was obtained when different concentrations of substrates were applied, suggesting that the use of SPP1 nanopore for real-time quantification of peptide oligomeric states is feasible. With the intrinsic nature of size and conjugation amenability, the SPP1 nanopore has the potential for development into a tool for the quantification of peptide and protein structures in real time.

The Effect of Ring-Size on the Anodic Oxidation of ‘Cyclic Amides’ in Methanol

The anodic oxidation of three ‘cyclic amides’ of type N-acylazacycloalkanes [5- (I), 6- (II) and 7-membered (III) rings] has been studied in methanol under constant current electrolysis, at C anodes and in the presence of various supporting electrolytes, and different concentrations of substrates. Four major products were formed in good yields by all three substrates, namely N-acyl, α-azacycloalkenes, N-acyl, α-methoxyazacycloalkanes, N-acyl, α-methoxy, α'-azacycloalkenes and N-acyl, α,α′-dimethoxyazacycloalkanes. The relative ratios among products and selectivity were found to be highly dependent on the nature of the electrolyte used, and to a lesser extent on substrate concentration. In terms of ring-size effect it was found that the rate of oxidation and current efficiency (yield) was in the order: I>II>III. Also the latter two behaved similarly (but different from I) when various supporting electrolytes were used.

Potencial dos resíduos do processamento de camarão para remediação de águas contaminadas com drenagem ácida mineral

Resumo A drenagem ácida mineral (DAM) é um dos mais graves impactos ambientais da mineração. Cursos de água impactados pela DAM são avaliados como impróprios acarretando graves impactos ambientais e problemas socioeconômicos severos. Este estudo visa identificar o potencial de um resíduo, casca de camarão, como fonte de quitina e alcalinidade para remediação de águas contaminadas por DAM visando seu posterior reuso para fins secundários não potáveis. Foram efetuados ensaios com a casca de camarão, diferenciando entre corpo (CR) e cabeça (CB), e quitina comercial (QT, 70% de pureza), para o tratamento de amostras de água superficiais e subterrâneas impactadas da Região Carbonífera Catarinense/SC (Brasil). Realizaram-se ensaios em duplicata e em regime de batelada variando o teor de substrato, tempo de contato e velocidade de agitação. Conseguiu-se a remoção quase total de Fe (>92%) e Al (>99%) e uma remoção de Mn entre 40-60%. CR apresentou os melhores resultados após 48h de tratamento com 200 rpm e 10 gL–1de substrato. O aumento no pH foi mais significativo nas amostras contendo CR, passando o pH de ~3 para ~7. Os resultados indicaram o potencial da utilização direta da casca de camarão para o tratamento de efluentes para reuso secundário não potável.

Are monoterpenoids and phenylpropanoids efficient inhibitors of acetylcholinesterase from stored product insect strains?

ABSTRACTIn previous papers the toxicity of the monoterpenoids linalool and carvone and the phenylpropanoids trans‐anethole and estragole, as well as the capacity to induce resistance to Sitophilus oryzae and Cryptolestes pusillus trough acetylcholinesterase (AChE) inhibition from either susceptible or tolerant (up to the tenth generation) insect populations, was tested at different concentrations of substrate. In this study, the activity of AChE inhibited by monoterpenoids and phenylpropanoids at lower concentrations (from 0.02 to 10 mM) was measured by Ellman's method at the same concentration of substrate (1 mM). Mainly, all of the monoterpenoids and phenylpropanoids applied turned out to be weak inhibitors at low concentrations, except for linalool and estragole, which were relatively the most powerful inhibitors for S. oryzae susceptible and tolerant strains, and for carvone, but just for the tolerant strain. However, in all these cases, high concentrations of inhibitors were necessary. The compounds studied on C. pusillus turned out to be extremely weak inhibitors, with inhibition not exceeding 40%. Higher values at half‐maximal inhibitory concentration (IC50) were found for tolerant than susceptible strains in S. oryzae and C. pusillus by using linalool, indicating that esterases, or specifically AChE, could be involved in the mode of action. In addition, in relation to the types of inhibition, linalool and estragole were competitive inhibitors, whereas carvone presented a non‐competitive inhibition. This combined information should be taken into account when essential oils, monoterpenoids or phenylpropanoids are used in organic farming, because these compounds can be used as effective insecticides, although they were deficient AChE inhibitors, should this situation be acceptable for ecological agriculture. Copyright © 2014 John Wiley & Sons, Ltd.

Naringenin inhibits α-glucosidase activity: A promising strategy for the regulation of postprandial hyperglycemia in high fat diet fed streptozotocin induced diabetic rats

Obesity and the onset of diabetes are two closely linked medical complications prevalent globally. Postprandial hyperglycemia is one of the earliest abnormalities of glucose homeostasis associated with type 2 diabetes (T2D). Postprandial glucose levels can be regulated through α-glucosidase inhibition. The present study aims to demonstrate the potent inhibitory role of naringenin against α-glucosidase activity. The mode of inhibition of naringenin was examined by measuring enzyme activity in vitro with different concentrations of substrate using Lineweaver–Burk plot analysis. It shows competitive inhibition towards mammalian α-glucosidase thereby competing with α-limit dextrins and oligosaccharide residues for binding in the active site. Similar results have been obtained from the molecular docking analyses, where naringenin shows preferential binding for the active sites in each of the evaluated human intestinal α-glucosidase enzymes. Post-docking intramolecular hydrogen bonding analysis shows water molecule mediated hydrogen bonding for N-terminal maltase glucoamylase and N-terminal sucrase isomaltase. Naringenin’s docked pose in the C-terminal maltase glucoamylase active site does not show any particular water mediated interaction similar to the co-crystallized acarbose. Further, our results suggest that naringenin (25mg/kg) exerts significant inhibition of intestinal α-glucosidase activity in vivo thereby delaying the absorption of carbohydrates in T2D rats, thus resulting in significant lowering of postprandial blood glucose levels. Both in vitro and in vivo results were compared to the commercially available α-glucosidase inhibitor acarbose. Our findings clearly indicate that naringenin dampens postprandial glycemic response and offers a potential complementary approach in the management of T2D.

Assessment of Nitrate Reductase Activity in the Leaves of Erythrina Blakei R. Parker

Combinations of different concentrations of substrate (0.025M, 0.05M, 0.1M, 0.15M, 0.20M and 0.25M KNO3) with different pH of buffer (0.1M, KH2PO4 of the pH 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.5, 7.6, 7.7, 7.8) solutions were tried for the nitrate reductase activity of Erythrina blakei leaves. Maximum nitrate reductase activity was observed in the combination of buffer solution of 0.1M having pH 6.8 and substrate solution of the concentration 0.15M. NR activity and moisture content in single leaf blades of Erythrina blakei leaves of different branches from different levels of tree have different concentration of activity and Moisture percentage. In single leaf blades maximum NR activity was recorded in leaf number 10, Branch number 3. And maximum moisture percentage observed in leaf number 12, Branch number 3.

Single-shot characterization of enzymatic reaction constants Km and kcat by an acoustic-driven, bubble-based fast micromixer.

In this work we present an acoustofluidic approach for rapid, single-shot characterization of enzymatic reaction constants K(m) and k(cat). The acoustofluidic design involves a bubble anchored in a horseshoe structure which can be stimulated by a piezoelectric transducer to generate vortices in the fluid. The enzyme and substrate can thus be mixed rapidly, within 100 ms, by the vortices to yield the product. Enzymatic reaction constants K(m) and k(cat) can then be obtained from the reaction rate curves for different concentrations of substrate while holding the enzyme concentration constant. We studied the enzymatic reaction for β-galactosidase and its substrate (resorufin-β-D-galactopyranoside) and found K(m) and k(cat) to be 333 ± 130 μM and 64 ± 8 s(-1), respectively, which are in agreement with published data. Our approach is valuable for studying the kinetics of high-speed enzymatic reactions and other chemical reactions.

Insights into O-Linked N-Acetylglucosamine ([0-9]O-GlcNAc) Processing and Dynamics through Kinetic Analysis of O-GlcNAc Transferase and O-GlcNAcase Activity on Protein Substrates

Cellular O-linked N-acetylglucosamine (O-GlcNAc) levels are modulated by two enzymes: uridine diphosphate-N-acetyl-D-glucosamine:polypeptidyltransferase (OGT) and O-GlcNAcase (OGA). To quantitatively address the activity of these enzymes on protein substrates, we generated five structurally diverse proteins in both unmodified and O-GlcNAc-modified states. We found a remarkably invariant upper limit for k(cat)/K(m) values for human OGA (hOGA)-catalyzed processing of these modified proteins, which suggests that hOGA processing is driven by the GlcNAc moiety and is independent of the protein. Human OGT (hOGT) activity ranged more widely, by up to 15-fold, suggesting that hOGT is the senior partner in fine tuning protein O-GlcNAc levels. This was supported by the observation that K(m,app) values for UDP-GlcNAc varied considerably (from 1 μM to over 20 μM), depending on the protein substrate, suggesting that some OGT substrates will be nutrient-responsive, whereas others are constitutively modified. The ratios of k(cat)/K(m) values obtained from hOGT and hOGA kinetic studies enable a prediction of the dynamic equilibrium position of O-GlcNAc levels that can be recapitulated in vitro and suggest the relative O-GlcNAc stoichiometries of target proteins in the absence of other factors. We show that changes in the specific activities of hOGT and hOGA measured in vitro on calcium/calmodulin-dependent kinase IV (CaMKIV) and its pseudophosphorylated form can account for previously reported changes in CaMKIV O-GlcNAc levels observed in cells. These studies provide kinetic evidence for the interplay between O-GlcNAc and phosphorylation on proteins and indicate that these effects can be mediated by changes in hOGT and hOGA kinetic activity.