Differential inhibition by alpha and epsilonPKC pseudosubstrate sequences: a putative mechanism for preferential PKC activation in neonatal cardiac myocytes

The aims of the current study were: 1) to determine if the epsilonPKC pseudosubstrate peptide (epsilonphi) (NH(2)-RKRQGAVRRRVHQVNG-COOH) could be used as an epsilonPKC-selective inhibitor in neonatal cardiac myocytes (NCMs) and 2) to determine if differences in the alpha and epsilonPKC autoinhibitory pseudosubstrate mechanisms could play roles in alpha and epsilonPKC-selective functions. Introduction of the epsilonphi into NCMs by transient permeabilization modestly attenuated 3 nM 4-beta PMA-induced slowing of contraction rate, an epsilonPKC mediated response (Circ Res. 76:654-663; J. Biol. Chem. 271:24962-24966). In contrast, the alphaPKC pseudosubstrate peptide (alphaphi) (NH(2)-RFARKGALRQKNVHEVK-COOH) was 6- to 10-fold more potent at antagonizing the 3 nM 4-beta PMA-induced slowing of contraction rate. Addition of purified PKC to the particulate cell fraction of NCMs promoted (32)P incorporation into 3 proteins of approximately 18, approximately 46 and approximately 97 kDa. The alphaphi antagonized these phosphorylations with IC(50) values of 1 - 5 microM. These IC(50) values were 1.8 - 4.7-fold lower than those observed for the epsilonphi. In in vitro phosphorylation assays with recombinant alpha or epsilon PKC isozymes the phi failed to inhibit the PKC isozyme as potently as the alphaphi peptide but both the alphaphi and the epsilonphi were equally effective inhibitors of the recombinant alphaPKC isozyme. In addition, in vitro cleavage of the epsilonphi by the protease Arg-C in lysates from NCMs treated with 3 nM 4-beta PMA was greatly enhanced when compared to that of the alphaPKC isozyme. Our studies suggest that the epsilonphi cannot be used as a selective inhibitor of the epsilonPKC isozyme in NCMs and that there are differences in the epsilonPKC and alphaPKC autoinhibitory pseudosubstrate mechanisms.     

Gating of delayed rectification in acutely isolated canine cardiac Purkinje myocytes. Evidence for a single voltage-gated conductance.

Studies of time-dependent, plateau outward current (delayed rectification) in the heart are complicated by the accumulation and depletion of K+ ions in intercellular clefts. To minimize this problem, we studied delayed rectification in acutely isolated (enzymic solution, gentle agitation) canine cardiac Purkinje myocytes using the single microelectrode voltage-clamp technique. We found a sigmoidal voltage-dependence for activation of outward plateau current, with maximal activation occurring at potentials near -10 mV. The activation and deactivation of plateau outward current was adequately described as the sum of a fast and slow exponential component. A comparison of the time course of activation of plateau outward current and the "envelope" of tail currents suggests that a single voltage-gated conductance with one open and two closed states can account for delayed rectification in Purkinje myocytes. These results differ from those previously obtained with intact sheep Purkinje fibers in which two time-dependent conductances were postulated to account for delayed rectification (Noble, D., and R. W. Tsien, 1969, J. Physiol. (Lond.), 200:205-231).     

Oligomer size of the serotonin 5-hydroxytryptamine 2C (5-HT2C) receptor revealed by fluorescence correlation spectroscopy with photon counting histogram analysis: evidence for homodimers without monomers or tetramers

Fluorescence correlation spectroscopy (FCS) and photon counting histogram (PCH) are techniques with single molecule sensitivity that are well suited for examining the biophysical properties of protein complexes in living cells. In the present study, FCS and PCH were applied to determine the diffusion coefficient and oligomeric size of G-protein-coupled receptors. FCS was used to record fluctuations in fluorescence intensity arising from fluorescence-tagged 5-hydroxytryptamine 2C (5-HT(2C)) receptors diffusing within the plasma membrane of HEK293 cells and rat hippocampal neurons. Autocorrelation analysis yielded diffusion coefficients ranging from 0.8 to 1.2 μm(2)/s for fluorescence-tagged receptors. Because the molecular brightness of a fluorescent protein is directly proportional to the number of fluorescent proteins traveling together within a protein complex, it can be used to determine the oligomeric size of the protein complex. FCS and PCH analysis of fluorescence-tagged 5-HT(2C) receptors provided molecular brightness values that were twice that of GFP and YFP monomeric controls, similar to a dimeric GFP control, and unaltered by 5-HT. Bimolecular fluorescence complementation of the N- and C-terminal halves of YFP attached to 5-HT(2C) receptors was observed in endoplasmic reticulum/Golgi and plasma membranes with a brightness equal to monomeric YFP. When GFP-tagged 5-HT(2C) receptors were co-expressed with a large excess of untagged, non-fluorescent 5-HT(2C) receptors, the molecular brightness was reduced by half. PCH analysis of the FCS data were best described by a one-component dimer model without monomers or tetramers. Therefore, it is concluded that 5-HT(2C) receptors freely diffusing within the plasma membrane are dimeric.     

Strong cross-bridges potentiate the Ca(2+) affinity changes produced by hypertrophic cardiomyopathy cardiac troponin C mutants in myofilaments: a fast kinetic approach

This spectroscopic study examined the steady-state and kinetic parameters governing the cross-bridge effect on the increased Ca(2+) affinity of hypertrophic cardiomyopathy-cardiac troponin C (HCM-cTnC) mutants. Previously, we found that incorporation of the A8V and D145E HCM-cTnC mutants, but not E134D into thin filaments (TFs), increased the apparent Ca(2+) affinity relative to TFs containing the WT protein. Here, we show that the addition of myosin subfragment 1 (S1) to TFs reconstituted with these mutants in the absence of MgATP(2-), the condition conducive to rigor cross-bridge formation, further increased the apparent Ca(2+) affinity. Stopped-flow fluorescence techniques were used to determine the kinetics of Ca(2+) dissociation (k(off)) from the cTnC mutants in the presence of TFs and S1. At a high level of complexity (i.e. TF + S1), an increase in the Ca(2+) affinity and decrease in k(off) was achieved for the A8V and D145E mutants when compared with WT. Therefore, it appears that the cTnC Ca(2+) off-rate is most likely to be affected rather than the Ca(2+) on rate. At all levels of TF complexity, the results obtained with the E134D mutant reproduced those seen with the WT protein. We conclude that strong cross-bridges potentiate the Ca(2+)-sensitizing effect of HCM-cTnC mutants on the myofilament. Finally, the slower k(off) from the A8V and D145E mutants can be directly correlated with the diastolic dysfunction seen in these patients.     

Development of models of active ion transport for whole-cell modelling: cardiac sodium-potassium pump as a case study

This study presents a method for the reduction of biophysically-based kinetic models for the active transport of ions. A lumping scheme is presented which exploits the differences in timescales associated with fast and slow transitions between model states, while maintaining the thermodynamic properties of the model. The goal of this approach is to contribute to modelling of the effects of disturbances to metabolism, associated with ischaemic heart disease, on cardiac cell function.

The approach is illustrated for the sodium-potassium pump in the myocyte. The lumping scheme is applied to produce a 4-state representation from the detailed 15-state model of Läuger and Apell, Eur. Biophys. J. 13 (1986) 309, for which the principles of free energy transduction are used to link the free energy released from ATP hydrolysis (ΔG_ATP) to the transition rates between states of the model. An iterative minimisation algorithm is implemented to determine the transition rate parameters based on the model fit to experimental data. Finally, the relationship between ΔG_ATP and pump cycling direction is investigated and compared with recent experimental findings.     

Identification of residues within tropomodulin-1 responsible for its localization at the pointed ends of the actin filaments in cardiac myocytes

Tropomodulin is a tropomyosin-dependent actin filament capping protein involved in the structural formation of thin filaments and in the regulation of their lengths through its localization at the pointed ends of actin filaments. The disordered N-terminal domain of tropomodulin contains three functional sites: two tropomyosin-binding and one tropomyosin-dependent actin-capping sites. The C-terminal half of tropomodulin consists of one compact domain containing a tropomyosin-independent actin-capping site. Here we determined the structural properties of tropomodulin-1 that affect its roles in cardiomyocytes. To explore the significance of individual tropomyosin-binding sites, GFP-tropomodulin-1 with single mutations that destroy each tropomyosin-binding site was expressed in cardiomyocytes. We demonstrated that both sites are necessary for the optimal localization of tropomodulin-1 at thin filament pointed ends, with site 2 acting as the major determinant. To investigate the functional properties of the tropomodulin C-terminal domain, truncated versions of GFP-tropomodulin-1 were expressed in cardiomyocytes. We discovered that the leucine-rich repeat (LRR) fold and the C-terminal helix are required for its proper targeting to the pointed ends. To investigate the structural significance of the LRR fold, we generated three mutations within the C-terminal domain (V232D, F263D, and L313D). Our results show that these mutations affect both tropomyosin-independent actin-capping activity and pointed end localization, most likely by changing local conformations of either loops or side chains of the surfaces involved in the interactions of the LRR domain. Studying the influence of these mutations individually, we concluded that, in addition to the tropomyosin-independent actin-capping site, there appears to be another regulatory site within the tropomodulin C-terminal domain.     

Fetal cardiac troponin isoforms rescue the increased Ca2+ sensitivity produced by a novel double deletion in cardiac troponin T linked to restrictive cardiomyopathy: a clinical, genetic, and functional approach

A novel double deletion in cardiac troponin T (cTnT) of two highly conserved amino acids (Asn-100 and Glu-101) was found in a restrictive cardiomyopathic (RCM) pediatric patient. Clinical evaluation revealed the presence of left atrial enlargement and marked left ventricle diastolic dysfunction. The explanted heart examined by electron microscopy revealed myofibrillar disarray and mild fibrosis. Pedigree analysis established that this mutation arose de novo. The patient tested negative for six other sarcomeric genes. The single and double recombinant cTnT mutants were generated, and their functional consequences were analyzed in porcine skinned cardiac muscle. In the adult Tn environment (cTnT3 + cardiac troponin I), the single cTnT3-ΔN100 and cTnT3-ΔE101 mutations had opposing effects on the Ca(2+) sensitivity of force development compared with WT, whereas the double deletion cTnT3-ΔN100/ΔE101 increased the Ca(2+) sensitivity + 0.19 pCa units. In addition, cTnT3-ΔN100/ΔE101 decreased the cooperativity of force development, suggesting alterations in intrafilament protein-protein interactions. In the fetal Tn environment, (cTnT1 + slow skeletal troponin I), the single (cTnT1-ΔN110) and double (cTnT1-ΔN110/ΔE111) deletions did not change the Ca(2+) sensitivity compared with control. To recreate the patient's heterozygous genotype, we performed a reconstituted ATPase activity assay. Thin filaments containing 50:50 cTnT3-ΔN100/ΔE101:cTnT3-WT also increased the myofilament Ca(2+) sensitivity compared with WT. Co-sedimentation of thin filament proteins indicated that no significant changes occurred in the binding of Tn containing the RCM cTnT mutation to actin-Tm. This report reveals the protective role of Tn fetal isoforms as they rescue the increased Ca(2+) sensitivity produced by a cTnT-RCM mutation and may account for the lack of lethality during gestation.     

Generation and characterization of antibodies specific for caspase-cleaved neo-epitopes: a novel approach

Apoptosis research has been significantly aided by the generation of antibodies against caspase-cleaved peptide neo-epitopes. However, most of these antibodies recognize the N-terminal fragment and are specific for the protein in question. The aim of this project was to create antibodies, which could identify caspase-cleaved proteins without a priori knowledge of the cleavage sites or even the proteins themselves. We hypothesized that many caspase-cleavage products might have a common antigenic shape, given that they must all fit into the same active site of caspases. Rabbits were immunized with the eight most prevalent exposed C-terminal tetrapeptide sequences following caspase cleavage. After purification of the antibodies we demonstrated (1) their specificity for exposed C-terminal (but not internal) peptides, (2) their ability to detect known caspase-cleaved proteins from apoptotic cell lysates or supernatants from apoptotic cell culture and (3) their ability to detect a caspase-cleaved protein whose tetrapeptide sequence differs from the eight tetrapeptides used to generate the antibodies. These antibodies have the potential to identify novel neo-epitopes produced by caspase cleavage and so can be used to identify pathway-specific caspase cleavage events in a specific cell type. Additionally this methodology may be applied to generate antibodies against products of other proteases, which have a well-defined and non-promiscuous cleavage activity.     

DNA damage by anti-cancer agents resolved at the nucleotide level of a single copy gene: evidence for a novel binding site for cisplatin in cells.

A new PCR based technique has been developed to investigate the sequence selectivity of adduct formation by DNA damaging agents in a single copy gene in isolated genomic DNA or in drug treated cells. Single-strand ligation PCR (sslig-PCR) demonstrated that cisplatin and nitrogen mustards reacted with guanine in an N-ras fragment with varying sequence specificity similar to that observed previously in plasmid DNA. In cisplatin-treated cells sslig-PCR demonstrated all the adducts found in isolated DNA and with the same sequence selectivity showing a preference for GG and AG sites. However, in cells an additional site of DNA binding of cisplatin was observed at the two occurrences of the sequence 5'-TACT-3' on the transcribed and non-transcribed strands. This sequence is not a recognised target for cisplatin and represents a novel adduct formed in cells.     

A novel method for detection and counting of single bacteria in a wide field using an ultra-high-sensitivity TV camera without a microscope

We describe a novel method for enumeration of bacteria, based on the principle that small, light emitting particles on a flat surface can be easily and rapidly detected and counted using an ultra-high-sensitivity TV camera. To test this method, we obtained TV images of individual cells of a luminous bacterium on a membrane filter without the use of a microscope. The positions of the luminous points in the TV images were almost the same as the positions of the bacterial colonies after growth. Our results show that the single cells can be efficiently detected and counted by our method if they emit light or can be stimulated to emit light.     

Assessment of the proportion of transgene-bearing sperm by fluorescence in situ hybridization: a novel approach for the detection of germline mosaicism in transgenic male founders

Genetic mosaicism is frequent among transgenic animals produced by pronuclear microinjection. A successful method for the screening of founder animals for germline mosaicism prior to mating would greatly reduce the costs associated with the propagation of the transgenic lines, and improve the efficiency of transgenic livestock production. With this aim, we have devised a simple method to detect integrated transgenes in individual spermatozoa using fluorescence in situ hybridization (FISH). The experiments reported here were undertaken to investigate the efficiency of this FISH-based approach to accurately evaluate the proportion of transgene-bearing sperm and to be applied for the detection of potential germline mosaics. Sperm samples from mice homozygous and hemizygous for a beta-lactoglobulin transgene were analyzed in a first set of experiments. A high hybridization efficiency was achieved, and the proportions of transgene-positive sperm cells in both homozygous (94.8-98.2%) and hemizygous (49.8-51.9%) animals were close to the expected frequencies (100 and 50%, respectively). To evaluate the sensitivity of the assay more directly, simulated mosaic samples with 5, 10, 15, 20 and 40% of transgene-bearing spermatozoa were then prepared and analyzed by FISH. Significant differences in the frequency of transgene-positive sperm were observed between all mosaic samples, indicating that even small deviations (5%) from the expected 50% transgene transmission rate in a founder animal could be reliably detected with our assay. Therefore, the method proposed represents a novel approach for the identification of germline mosaic founder males in livestock transgenic projects and a much more economic and faster alternative to breeding.     

Building genetic networks using relatedness information: a novel approach for the estimation of dispersal and characterization of group structure in social animals

Natal dispersal is an important life history trait driving variation in individual fitness, and therefore, a proper understanding of the factors underlying dispersal behaviour is critical to many fields including population dynamics, behavioural ecology and conservation biology. However, individual dispersal patterns remain difficult to quantify despite many years of research using direct and indirect methods. Here, we quantify dispersal in a single intensively studied population of the cooperatively breeding chestnut-crowned babbler (Pomatostomus ruficeps) using genetic networks created from the combination of pairwise relatedness data and social networking methods and compare this to dispersal estimates from re-sighting data. This novel approach not only identifies movements between social groups within our study sites but also provides an estimation of immigration rates of individuals originating outside the study site. Both genetic and re-sighting data indicated that dispersal was strongly female biased, but the magnitude of dispersal estimates was much greater using genetic data. This suggests that many previous studies relying on mark-recapture data may have significantly underestimated dispersal. An analysis of spatial genetic structure within the sampled population also supports the idea that females are more dispersive, with females having no structure beyond the bounds of their own social group, while male genetic structure expands for 750 m from their social group. Although the genetic network approach we have used is an excellent tool for visualizing the social and genetic microstructure of social animals and identifying dispersers, our results also indicate the importance of applying them in parallel with behavioural and life history data.     

Phase locking,period doubling bifurcations and chaos in a mathematical model of a periodically driven oscillator: A theory for the entrainment of biological oscillators and the generation of cardiac dysrhythmias

A mathematical model for the perturbation of a biological oscillator by single and periodic impulses is analyzed. In response to a single stimulus the phase of the oscillator is changed. If the new phase following a stimulus is plotted against the old phase the resulting curve is called the phase transition curve or PTC (Pavlidis, 1973). There are two qualitatively different types of phase resetting. Using the terminology of Winfree (1977, 1980), large perturbations give a type 0 PTC (average slope of the PTC equals zero), whereas small perturbations give a type 1 PTC. The effects of periodic inputs can be analyzed by using the PTC to construct the Poincaré or phase advance map. Over a limited range of stimulation frequency and amplitude, the Poincaré map can be reduced to an interval map possessing a single maximum. Over this range there are period doubling bifurcations as well as chaotic dynamics. Numerical and analytical studies of the Poincaré map show that both phase locked and non-phase locked dynamics occur. We propose that cardiac dysrhythmias may arise from desynchronization of two or more spontaneously oscillating regions of the heart. This hypothesis serves to account for the various forms of atrioventricular (AV) block clinically observed. In particular 22 and 42 AV block can arise by period doubling bifurcations, and intermittent or variable AV block may be due to the complex irregular behavior associated with chaotic dynamics.     

Fixed wavelength fluorescence (FF) of bile as a monitoring tool for polyaromatic hydrocarbon exposure in fish: an evaluation of compound specificity, inner filter effect and signal interpretation

Fixed wavelength fluorescence (FF) of bile has been evaluated as a monitoring tool for the screening of polyaromatic hydrocarbon (PAH) contamination in fish. The methodology was studied through laboratory and field experiments with Atlantic cod (Gadus morhua L.) and flounder (Platichthys flesus L.) exposed to various forms of PAH contamination. The present study demonstrates the ability of FF screening to discriminate between 2-, 4- and 5-ring PAH metabolites by using the wavelength pairs 290/335 nm, 341/383 nm and 380/430 nm, respectively. In general, the degree of fluorescence interference between these metabolite groups appears to be low. Dose- and time-response patterns of the FF signals were shown to give a good reflection of the PAH exposure. Further, the necessity of an appropriate dilution of bile samples prior to fluorescence measurements is demonstrated by a study of inner filter effect. Normally a dilution of 1000-2000-fold is necessary. Individual differences in the bile density, e.g. measured as the concentration of the bile pigment biliverdin, have to be allowed for when applying the FF method. However, it is shown that normalizing the FF signals to biliverdin concentrations on an individual basis added extra error to the data set. The simple, rapid and cost-effective FF method is found to be well suited for screening fish for PAH contamination.     

Fixed wavelength fluorescence (FF) of bile as a monitoring tool for polyaromatic hydrocarbon exposure in fish: an evaluation of compound specificity,inner filter effect and signal interpretation

Fixed wavelength fluorescence (FF) of bile has been evaluated as a monitoring tool for the screening of polyaromatic hydrocarbon (PAH) contamination in fish. The methodology was studied through laboratory and field experiments with Atlantic cod (Gadus morhua L.) and flounder (Platichthys flesus L.) exposed to various forms of PAH contamination. The present study demonstrates the ability of FF screening to discriminate between 2-, 4- and 5-ring PAH metabolites by using the wavelength pairs 290/335 nm, 341/383 nm and 380/430 nm, respectively. In general, the degree of fluorescence interference between these metabolite groups appears to be low. Dose- and time-response patterns of the FF signals were shown to give a good reflection of the PAH exposure. Further, the necessity of an appropriate dilution of bile samples prior to fluorescence measurements is demonstrated by a study of inner filter effect. Normally a dilution of 1000-2000-fold is necessary. Individual differences in the bile density, e.g. measured as the concentration of the bile pigment biliverdin, have to be allowed for when applying the FF method. However, it is shown that normalizing the FF signals to biliverdin concentrations on an individual basis added extra error to the data set. The simple, rapid and cost-effective FF method is found to be well suited for screening fish for PAH contamination.     

Isolation of a library of target-sites for sequence specific DNA binding proteins from chick embryonic heart: a potential tool for identifying novel transcriptional regulators involved in embryonic development

Enormity of the metazoan genomes and divergence in their regulation impose a serious constraint on the comprehensive understanding of context specific gene regulation. DNA elements located in the promoter, enhancer, and other regulatory regions of the genome dictate the temporal and spatial patterns of gene activities. However, owing to the diminutive and variable nature of the regulatory DNA elements, their identification and location remains a major challenge. We have developed an efficient strategy for isolating a repertoire of target sites for sequence specific DNA binding proteins from embryonic chick heart. A comprehensive library of such sequences was constructed and authenticated using various parameters including in silico determination of functional binding sites. This approach, therefore, for the first time, established an experimental and conceptual framework for defining the entire repertoire of functional DNA elements in any cellular context.     

Typing of Listeria monocytogenes strains isolated in Italy by inlA gene characterization and evaluation of a new cost‐effective approach to antisera selection for serotyping

Aims: In this study, 105 Listeria monocytogenes strains isolated from humans, foods and environmental samples were characterized using several typing methods. Moreover, serotyping procedure was evaluated, and a cost‐effective methodological approach based on preliminary PCRs screening was proposed. Methods and Results: The isolates were analysed by conventional serotyping, multiplex‐PCRs for serogroup and lineage identification and PCR–RFLP of inlA gene to identify potentially noninvasive L. monocytogenes. Among the strains, only the serotypes 1/2a, 1/2c, 1/2b, 4b and 3a were identified. The isolates were classified into serogroups I (58·10%), II (22·85%), III (12·38%) and IV (6·67%). Among clinical strains, lineage I was more represented (68·75%) than lineage II; whereas, lineage II was more associated with food (90·24%) and environmental (85·72%) isolates. Most of food (89·02%) and environmental (85·71%) isolates were classified into truncated InlA profiles, whereas the 93·75% of clinical strains were associated with a complete form of the protein. Conclusion: Molecular techniques were sensitive and specific for classifying strains into serogroup and lineage and in agreement with the serotyping. Moreover, a preliminary PCRs‐based screening was proposed to select only the necessary antisera by a flow chart; this methodological approach allows cost saving up to 42%. Our results further suggest the role of InlA protein in human listeriosis, particularly in immunocompetent individuals, and a correlation between truncated protein and serotype. Significance and Impact of the Study: This study further validates molecular methods for L. monocytogenes analysis and proposed a new cost‐effective approach for serotyping. It could help to improve a national surveillance network for L. monocytogenes infections in Italy.     

The use of the iron chelating agent desferrioxamine in rice cell cryopreservation: a novel approach for improving recovery

The iron chelating drug, desferrioxamine is used to suppress oxidative stress in mammalian transplant organs subjected to cold storage. The efficacy of desferrioxamine in improving post-thaw survival in cryopreserved cells from two rice culture lines was evaluated. Unfrozen rice cells maintained proliferation capacity over a fifteen day time course when exposed to concentrations of desferrioxamine > 10 mg·l⁻¹. Albeit, growth was reduced compared to controls. Short-term applications of the drug at concentrations of 0.5 and 10 mg·l⁻¹ before cryopreservation and during the early post-thaw period had a positive affect on recovery as assessed by cell proliferation and triphenyl tetrazolium chloride reduction capabilities. The pharmaceutical properties of desferrioxamine are attributed to iron sequestration and the prevention of harmful Fenton and free radical chemistry. However, desferrioxamine did not significantly reduce lipid peroxidation in cryopreserved rice cells.     

A novel approach for enhancing the catalytic efficiency of a protease at low temperature: Reduction in substrate inhibition by chemical modification

The alkaline protease, savinase was chemically modified to enhance the productivity of the enzyme at low temperatures on a complex polymeric protein (azocasein) substrate. At 5 and 15°C, savinase modified with ficol or dextran hydrolyzed fivefold more azocasein than the unmodified savinase. Kinetic studies showed that the catalytic improvements are associated with changes in uncompetitive substrate inhibition with K_i values of modified savinases sixfold higher than the unmodified savinase. Modeling of small‐angle scattering data indicates that two substrate molecules bind on opposing sides of the enzyme. The combined kinetic and structural data indicate that the polysaccharide modifier sterically blocks the allosteric site and reduces substrate inhibition. In contrast to the properties of cold‐active enzymes that generally manifest as low activation enthalpy and high flexibility, this study shows that increased activity and productivity at low temperature can be achieved by reducing uncompetitive substrate inhibition, and that this can be achieved using chemical modification with an enzyme in a commercial enzyme‐formulation. Biotechnol. Bioeng. 2009;103: 676–686. © 2009 Wiley Periodicals, Inc.