Wide Variety Of Biochemical Processes Research Articles

The roles of brain lipids and polar metabolites in the hypoxia tolerance of deep-diving pinnipeds.

Lipids make up more than half of the human brain's dry weight, yet the composition and function of the brain lipidome is not well characterized. Lipids not only provide the structural basis of cell membranes, but also take part in a wide variety of biochemical processes. In neurodegenerative diseases, lipids can facilitate neuroprotection and serve as diagnostic biomarkers. The study of organisms adapted to extreme environments may prove particularly valuable in understanding mechanisms that protect against stressful conditions and prevent neurodegeneration. The brain of the hooded seal (Cystophora cristata) exhibits a remarkable tolerance to low tissue oxygen levels (hypoxia). While neurons of most terrestrial mammals suffer irreversible damage after only short periods of hypoxia, in vitro experiments show that neurons of the hooded seal display prolonged functional integrity even in severe hypoxia. How the brain lipidome contributes to the hypoxia tolerance of marine mammals has been poorly studied. We performed an untargeted lipidomics analysis, which revealed that lipid species are significantly modulated in marine mammals compared with non-diving mammals. Increased levels of sphingomyelin species may have important implications for efficient signal transduction in the seal brain. Substrate assays also revealed elevated normoxic tissue levels of glucose and lactate, which suggests an enhanced glycolytic capacity. Additionally, concentrations of the neurotransmitters glutamate and glutamine were decreased, which may indicate reduced excitatory synaptic signaling in marine mammals. Analysis of hypoxia-exposed brain tissue suggests that these represent constitutive mechanisms rather than an induced response towards hypoxic conditions.

Organic and inorganic zinc in the diet of a commercial strain of diploid and triploid rainbow trout (Oncorhynchus mykiss): Effects on performance and mineral retention

Zinc is an important micro-mineral for finfish required in trace amounts and is involved in a wide variety of biochemical processes. In aquaculture, triploid fish are utilized for their sterility to decrease the risk of genetic pollution to wild populations and for their potential for faster growth. However, to our knowledge there is no information regarding zinc requirements for triploid rainbow trout (Oncorhynchus mykiss), despite reports from previous studies that mineral requirements may differ. Hence, this study was designed to determine the effects of organic and inorganic zinc on a commercial strain of genetically similar diploid and triploid rainbow Trout. Eleven dietary treatments, including one basal diet (33 mg/kg, Zn33) and 10 experimental diets, supplemented with incremental levels (63, 93, 123, 153 and 183 mg/kg) of inorganic (ZnSO4) and organic (Bioplex® Zn, Alltech) zinc supplemented to the basal diet were fed for 9 weeks (64 days). Twenty-two experimental groups (a unique combination of ploidy, zinc type, and dose level) were run in triplicate, with 11 fish per tank (66 L), with an average starting weight of 53.07 ± 4.35 g. Results showed neither ploidy (diploid vs. triploid) nor type of mineral (organic vs. inorganic) significantly influenced growth parameters. There was a significant interaction between zinc type and dose in diploids for lipid retention, energy retention, and vertebral zinc content. In triploids, there was a significant interaction between zinc type and dose for lipid, energy and protein retention, and vertebral zinc content. In conclusion, numerical data suggest that organic Zn requirement is lower than inorganic Zn in triploid trout, recommended requirement of inorganic Zn is 123 mg/kg whereas organic Zn is 63 mg/kg diet. The results showed that interactions between type of zinc and dose had a significant effect on nutrient retention, representing a potential for increased capacity to utilize feed ingredients.

11 Metal Amino Acid Chelates/complexes with Emphasis on Molecule Absorption and Efficacy Differences in Swine

Abstract The objective of this presentation will be to discuss differences in the molecular structures between various organic trace minerals (OTM) and inorganic trace minerals (ITM) and discuss how these properties impact stability, absorption, bioavailability and retention. The Association of American Feed Control Officials (AAFCO) lists several different categories of OTM including chelates, complexes and proteinates. Typical ligands include organic acids, amino acids, peptides and polysaccharides. It is widely known that bioavailability of ITM is low, primarily due to the presence of antagonisms such as phytate and/or fiber or excesses of other minerals. For this reason, inclusions of trace minerals (e.g. Zn, Cu, Mn, Fe) are often added in commercial livestock diets at 2–3-fold higher concentrations than recommended by NRC. Feeding highly bioavailable trace minerals is important. These minerals are required components of thousands of the proteins, enzymes and transcription factors that support a wide variety of biochemical processes in the cells and tissues of animals. These functions include gene regulation, cell growth and division, immune development and function, tissue development and integrity, reproduction and oxidative stress management. Low bioavailability of these trace minerals can reduce animal performance, immune function, reproductive performance and increase lameness. Numerous in vitro and in vivo methodologies have been used to compare bioavailability and demonstrate higher stability, tissue retention and digestibility of OTM vs ITM. Sow longevity is a key factor in commercial swine herd profitability. Reproductive problems and lameness are the most common reasons for premature sow culling from breeding herds. Compared to ITM, OTM reduced gilt and sow mortality 9–17% (P < 0.10), culling rate 20–35% (P < 0.01) and increased sow retention (through parity 3) 5–10%; (P < 0.01). Greater bioavailability translates to biological benefits to the producer, however, our findings demonstrate that not all OTM perform better than ITM.

What can urinary exosomes tell us?

Exosomes are involved in a wide variety of biochemical processes in human body homeostasis. Exosomes also provide important information regarding communications among several organ systems. Additionally, they can serve as molecular vehicles to deliver drugs. Therefore, exosomes have received wide attention in current biomedical research for unraveling pathogenic mechanisms of diseases, searching for novel biomarkers, and discovering new drugs. This paper reviews and discusses the significance of urinary exosomes for a better understanding of human disease pathophysiology and their potential use as therapeutic targets. Isolation methods of exosomes and the latest technological advances are also discussed. Furthermore, novel urinary exosomal biomarkers are highlighted with special emphasis on their clinical applicability (particularly sensitivity, specificity, reliability, and other aspects). Finally, future trends for this field are analyzed and our perspectives are provided.

Single molecule secondary structure determination of proteins through infrared absorption nanospectroscopy

The chemical and structural properties of biomolecules determine their interactions, and thus their functions, in a wide variety of biochemical processes. Innovative imaging methods have been developed to characterise biomolecular structures down to the angstrom level. However, acquiring vibrational absorption spectra at the single molecule level, a benchmark for bulk sample characterization, has remained elusive. Here, we introduce off-resonance, low power and short pulse infrared nanospectroscopy (ORS-nanoIR) to allow the acquisition of infrared absorption spectra and chemical maps at the single molecule level, at high throughput on a second timescale and with a high signal-to-noise ratio (~10–20). This high sensitivity enables the accurate determination of the secondary structure of single protein molecules with over a million-fold lower mass than conventional bulk vibrational spectroscopy. These results pave the way to probe directly the chemical and structural properties of individual biomolecules, as well as their interactions, in a broad range of chemical and biological systems.

Two-Color Valence-to-Core X-ray Emission Spectroscopy Tracks Cofactor Protonation State in a Class I Ribonucleotide Reductase.

Proton transfer reactions are of central importance to a wide variety of biochemical processes, though determining proton location and monitoring proton transfers in biological systems is often extremely challenging. Herein, we use two-color valence-to-core X-ray emission spectroscopy (VtC XES) to identify protonation events across three oxidation states of the O2 -activating, radical-initiating manganese-iron heterodinuclear cofactor in a class I-c ribonucleotide reductase. This is the first application of VtC XES to an enzyme intermediate and the first simultaneous measurement of two-color VtC spectra. In contrast to more conventional methods of assessing protonation state, VtC XES is a more direct probe applicable to a wide range of metalloenzyme systems. These data, coupled to insight provided by DFT calculations, allow the inorganic cores of the MnIV FeIV and MnIV FeIII states of the enzyme to be assigned as MnIV (μ-O)2 FeIV and MnIV (μ-O)(μ-OH)FeIII , respectively.

Two‐Color Valence‐to‐Core X‐ray Emission Spectroscopy Tracks Cofactor Protonation State in a Class I Ribonucleotide Reductase

AbstractProton transfer reactions are of central importance to a wide variety of biochemical processes, though determining proton location and monitoring proton transfers in biological systems is often extremely challenging. Herein, we use two‐color valence‐to‐core X‐ray emission spectroscopy (VtC XES) to identify protonation events across three oxidation states of the O2‐activating, radical‐initiating manganese–iron heterodinuclear cofactor in a class I‐c ribonucleotide reductase. This is the first application of VtC XES to an enzyme intermediate and the first simultaneous measurement of two‐color VtC spectra. In contrast to more conventional methods of assessing protonation state, VtC XES is a more direct probe applicable to a wide range of metalloenzyme systems. These data, coupled to insight provided by DFT calculations, allow the inorganic cores of the MnIVFeIV and MnIVFeIII states of the enzyme to be assigned as MnIV(μ‐O)2FeIV and MnIV(μ‐O)(μ‐OH)FeIII, respectively.

Measurement of Mesoscale Conformational Dynamics of Freely Diffusing Molecules with Tracking FCS

Few techniques are suited to probe the structure and dynamics of molecular complexes at the mesoscale level (∼100–1000 nm). We have developed a single-molecule technique that uses tracking fluorescence correlation spectroscopy (tFCS) to probe the conformation and dynamics of mesoscale molecular assemblies. tFCS measures the distance fluctuations between two fluorescently labeled sites within an untethered, freely diffusing biomolecule. To achieve subdiffraction spatial resolution, we developed a feedback scheme that allows us to maintain the molecule at an optimal position within the laser intensity gradient for fluorescence correlation spectroscopy. We characterized tFCS spatial sensitivity by measuring the Brownian end-to-end dynamics of DNA molecules as short as 1000 bp. We demonstrate that tFCS detects changes in the compaction of reconstituted nucleosome arrays and can assay transient protein-mediated interactions between distant sites in an individual DNA molecule. Our measurements highlight the applicability of tFCS to a wide variety of biochemical processes involving mesoscale conformational dynamics.

Zytotoxizitäts-Assays mittels Imaging und Lumineszenz

Cytotoxicity is still a major cause for the failure of potential drugs in clinical trials and there is an urgent need for reliable and time-saving tests. Since compounds can interfere with a wide variety of biochemical processes, it is very important to analyze several parameters in orthogonal approaches.

Structural Basis for the Enhanced Stability of Protein Model Compounds and Peptide Backbone Unit in Ammonium Ionic Liquids

Protein folding/unfolding is a fascinating study in the presence of cosolvents, which protect/disrupt the native structure of protein, respectively. The structure and stability of proteins and their functional groups may be modulated by the addition of cosolvents. Ionic liquids (ILs) are finding a vast array of applications as novel cosolvents for a wide variety of biochemical processes that include protein folding. Here, the systematic and quantitative apparent transfer free energies (ΔG'(tr)) of protein model compounds from water to ILs through solubility measurements as a function of IL concentration at 25 °C have been exploited to quantify and interpret biomolecular interactions between model compounds of glycine peptides (GPs) with ammonium based ILs. The investigated aqueous systems consist of zwitterionic glycine peptides: glycine (Gly), diglycine (Gly(2)), triglycine (Gly(3)), tetraglycine (Gly(4)), and cyclic glycylglycine (c(GG)) in the presence of six ILs such as diethylammonium acetate (DEAA), diethylammonium hydrogen sulfate (DEAS), triethylammonium acetate (TEAA), triethylammonium hydrogen sulfate (TEAS), triethylammonium dihydrogen phosphate (TEAP), and trimethylammonium acetate (TMAA). We have observed positive values of ΔG'(tr) for GPs from water to ILs, indicating that interactions between ILs and GPs are unfavorable, which leads to stabilization of the structure of model protein compounds. Moreover, our experimental data ΔG'(tr) is used to obtain transfer free energies (Δg'(tr)) of the peptide backbone unit (or glycyl unit) (-CH(2)C═ONH-), which is the most numerous group in globular proteins, from water to IL solutions. To obtain the mechanism events of the ILs' role in enhancing the stability of the model compounds, we have further obtained m-values for GPs from solubility limits. These results explicitly elucidate that all alkyl ammonium ILs act as stabilizers for model compounds through the exclusion of ILs from model compounds of proteins and also reflect the effect of alkyl chain on the stability of protein model compounds.

Dynamics Investigation of the Cytochrome P450cam Active Site Mutant Thr252ALA

Cytochrome P450s are vital heme-containing enzymes that facilitate catalysis in a wide variety of biochemical processes including carcinogenesis, drug metabolism, steroid biosynthesis and toxin degradation. Because of its ability to catalyze the hydroxylation mechanism of camphor in its active site, Cytochrome P450cam served as the model system for studying P450s. The crystal structure of a P450cam mutant, in which the active site Threonine252 (Thr252) has been replaced with an alanine (ala) residue (Thr252Ala), was solved at 2.2Å resolution. According to sequence alignment analysis, Thr252 is highly conserved among P450s, and it was suggested to be an essential active site residue, forming part of the dioxygen-binding site. Mutation of the active site Thr to Ala changes local conformation of the active site residues and affects hydroxylation of the substrate, producing hydrogen peroxide and “excess” water instead of the substrate polarizing product, 5-exo-hydroxycamphor. In the mutant active site, a solvent not present in the native enzyme is positioned in the dioxygen-binding region and the network of hydrogen bonds is restructured accordingly. These factors are believed to be critical to enzymatic reaction changes of P450cam mutant from the wild-type, most notably the proton delivery. Molecular dynamics (MD) simulation has been performed on the mutant to predict the relevant structural dynamics, especially those of the active site residues. Neutron scattering (NS) spectra calculations have been extrapolated from the simulation and compared with the results with the indicated crystallographic findings and those of the wild-type P450cam simulations already empirically verified via experimental neutron scattering. Preliminary results predict that local interactions around the heme and cam site have been altered midway through the trajectory. The preliminary data has also provided insight to understanding the local water diffusion dynamics of the CYP450cam mutant active site.

Trace Mineral Nutrition in Poultry and Swine

Trace minerals such as zinc, copper, and manganese are essential cofactors for hundreds of cellular enzymes and transcription factors in all animal species, and thus participate in a wide variety of biochemical processes. Immune development and transcription factors in all animal species, and thus participate in a wide variety of biochemical processes. Immune development and response, tissue and bone development and integrity, protection against oxidative stress, and cellular growth and division are just a few examples. Deficiencies in trace minerals can lead to deficits in any of these processes, as well as reductions in growth performance. As such, most animal diets are supplemented with inorganic and/or organic forms of trace minerals. Inorganic trace minerals (ITM) such as sulfates and oxides form the bulk of trace mineral supplementation, but these forms of minerals are well known to be prone to dietary antagonisms. Feeding high-quality chelated trace minerals or other classes of organic trace minerals (OTM) can provide the animal with more bioavailable forms of the minerals. Interestingly, many, if not most, published experiments show little or no difference in the bioavailability of OTMs versus ITMs. In some cases, it appears that there truly is no difference. However, real differences in bioavailability can be masked if source comparisons are not made on the linear portion of the dose-response curve. When highly bioavailable chelated minerals are fed, they will better supply the biochemical systems of the cells of the animal, leading to a wide variety of benefits in both poultry and swine. Indeed, the use of certain chelated trace minerals has been shown to enhance mineral uptake, and improve the immune response, oxidative stress management, and tissue and bone development and strength. Furthermore, the higher bioavailability of these trace minerals allows the producer to achieve similar or improved performance, at reduced levels of trace mineral inclusion.

Structures of two Arabidopsis thaliana major latex proteins represent novel helix‐grip folds

Here we report the first structures of two major latex proteins (MLPs) which display unique structural differences from the canonical Bet v 1 fold described earlier. MLP28 (SwissProt/TrEMBL ID Q9SSK9), the product of gene At1g70830.1, and the At1g24000.1 gene product (Swiss- Prot/TrEMBL ID P0C0B0), proteins which share 32% sequence identity, were independently selected as foldspace targets by the Center for Eukaryotic Structural Genomics. The structure of a single domain (residues 17-173) of MLP28 was solved by NMR spectroscopy, while the full-length At1g24000.1 structure was determined by X-ray crystallography. MLP28 displays greater than 30% sequence identity to at least eight MLPs from other species. For example, the MLP28 sequence shares 64% identity to peach Pp-MLP119 and 55% identity to cucumber Csf2.20 In contrast, the At1g24000.1 sequence is highly divergent (see Fig. 1), containing a gap of 33 amino acids when compared with all other known MLPs. Even when the gap is excluded, the sequence identity with MLPs from other species is less than 30%. Unlike some of the MLPs from other species, none of the A. thaliana MLPs have been characterized biochemically. We show by NMR chemical shift mapping that At1g24000.1 binds progesterone, demonstrating that despite its sequence dissimilarity, the hydrophobicmore » binding pocket is conserved and, therefore, may play a role in its biological function and that of the MLP family in general.« less

Design of a highly specific and noninvasive biosensor suitable for real‐time in vivo imaging of mercury (II) uptake

Mercury is a ubiquitous pollutant that when absorbed is extremely toxic to a wide variety of biochemical processes. Mercury (II) is a strong, "invisible" poison that is rapidly absorbed by tissues of the intestinal tract, kidneys, and liver upon ingestion. In this study, a novel fluorescence-based biosensor is presented that allows for the direct monitoring of the uptake and distribution of the metal under noninvasive in vivo conditions. With the introduction of a cysteine residue at position 205, located in close proximity to the chromophore, the green fluorescent protein (GFP) from Aequorea victoria was converted into a highly specific biosensor for this metal ion. The mutant protein exhibits a dramatic absorbance and fluorescence change upon mercuration at neutral pH. Absorbance and fluorescence properties with respect to the metal concentration exhibit sigmoidal binding behavior with a detection limit in the low nanomolar range. Time-resolved binding studies indicate rapid subsecond binding of the metal to the protein. The crystal structures obtained of mutant eGFP205C indicate a possible access route of the metal into the core of the protein. To our knowledge, this engineered protein is a first example of a biosensor that allows for noninvasive and real-time imaging of mercury uptake in a living cell. A major advantage is that its expression can be genetically controlled in many organisms to enable unprecedented studies of tissue specific mercury uptake.

Cell++—simulating biochemical pathways

With the generation of a wealth of information, detailing cellular components, their functions and interactions, there is a growing need for the development of new computational tools capable of interpreting these data within spatial and dynamic contexts. Here, we introduce Cell++, a novel stochastic simulation environment with the capacity to study a wide variety of biochemical processes within a spatial context. Focusing on three case studies, we highlight the potential impact of spatial organization in the evolution and engineering of signaling and metabolic pathways. In addition to altering signaling and metabolic efficiency, simulations also demonstrated features consistent with the phenomenon of metabolic channeling. Cell++ is licensed under the GNU general public license (GPL) and has been successfully implemented under Linux and IRIX operating systems. Source code together with a simple tutorial is available at http://www.compsysbio.org/CellSim/.

Medicinal Chemistry Based on the Sugar Code: Fundamentals of Lectinology and Experimental Strategies with Lectins as Targets

Theoretical calculations reveal that oligosaccharides are second to no other class of biochemical oligomery in terms of coding capacity. As integral part of cellular glycoconjugates they can serve as recognitive units for receptors (lectins). Having first been detected in plants, lectins are present ubiquitously. Remarkably for this field, they serve as bacterial and viral adhesins. Following a description of these branches of lectinology to illustrate history, current status and potential for medicinal chemistry, we document that lectins are involved in a wide variety of biochemical processes including intra- and intercellular glycoconjugate trafficking, initiation of signal transduction affecting e. g. growth regulation and cell adhesion in animals. It is thus justified to compare crucial carbohydrate epitopes with the postal code ensuring correct mail routing and delivery. In view of the functional relevance of lectins the design of high-affinity reagents to occupy their carbohydrate recognition domains offers the perspective for an attractive source of new drugs. Their applications can be supposed to encompass the use as cell-type-selective determinant for targeted drug delivery and as blocking devices in anti-adhesion therapy during infections and inflammatory disease. To master the task of devising custom-made glycans/glycomimetics for this purpose, the individual enthalpic and entropic contributions in the molecular rendezvous between the sugar receptor under scrutiny and its ligand in the presence of solvent molecules undergoing positional rearrangements need to be understood and rationally exploited. As remunerative means to this end, cleverly orchestrated deployment of a panel of methods is essential. Concerning the carbohydrate ligand, its topological parameters and flexibility are assessed by the combination of computer-assisted molecular-mechanics and molecular-dynamics calculations and NMR-spectroscopic measurements. In the presence of the receptor, the latter technique will provide insights into conformational aspects of the bound ligand and into spatial vicinity of the ligand to distinct side chains of amino acids establishing the binding site in solution. Also in solution, the hydrogen-bonding pattern in the complex can be mapped with monodeoxy and monofluoro derivatives of the oligosaccharide. Together with X-ray crystallographic and microcalorimetric studies the limits of a feasible affinity enhancement can be systematically probed. With galactoside-binding lectins as instructive mo del, recent progress in this area of drug design will be documented, emphasizing the general applicability of the outlined interdisciplinary approach.

Effect of Iron Depletion on Protein Profiles of Bilophila wadsworthia

Pathogenicity of microorganisms is a multifactorial phenomenon. Host environment plays a role in the development of the infectious process. The presence of key nutrients, such as iron, may be important for the expression of virulence factors. Iron is an essential component of a wide variety of biochemical processes in microorganisms, and its availability is of major importance in bacterial pathogenesis. In response to a low level of available iron in vivo, the creation of high-affinity iron-uptake systems and of a number of iron-regulated membrane proteins is induced. Although relationships between iron and virulence factors of aerobes have been better studied, a few such studies have been carried out with regard to anaerobes (mainly Bacteroides fragilis) [ 1, 2]. As discussed by Duerden [3], there is as yet no clear understanding of many of the virulence factors of anaerobes. Little is known about the mechanisms of iron uptake used by anaerobic bacteria, and nothing about those of Bilophila wadsworthia. The purpose of this work was to study the properties of this recently recognized anaerobe in relation to iron metabolism. B. wadsworthia is an anaerobic, gram-negative, asaccharolytic, bileresistant, and strongly catalase-positive bacillus [4]. This bacterium was first isolated from patients with gangrenous and perforative appendicitis. This study sought to determine whether B. wadsworthia expressed iron-regulated outer-membrane proteins (OMPs) in an iron-depleted environment and whether these proteins were immunogenic. Iron-free reagents and materials were used to prepare the ironfree medium. In this work, all glassware was deferrated. Chemicals used to prepare the medium were of highest purity. The medium was one previously described, to which oxgall (2%) and pyruvate (10 mM) were added [5]. The medium components were deferrated with Chelex-100 resin (Bio-Rad Laboratories, Hercules, CA), except for MgSO4, amino acids, and vitamins. Medium without iron contained <0.04 tM Fe+++. Iron was added to a concentration of 100 iM in preparation of the ironcontaining medium. The final pH was adjusted to 7.0-7.1. Four strains of B. wadsworthia originally isolated from appendicitis case specimens were studied, including the reference strain ATCC 49260. They were all incubated for 14 days in 200-mL flask cultures (in media with and without iron) at 37?C in an anaerobic atmosphere. Detection of siderophores in culture supernatants was attempted by means of the method described by Schwyn and Neilands [6]. Preparation of the outer membrane was accomplished according to the method described by Sprott et al. [7]. Cells harvested by centrifugation were disrupted ultrasonically. Unbroken cells were discarded by centrifugation. The supernatant was treated with 2% Triton

Biochemical Effects of Iron Deprivation

The consequences of iron deprivation on iron-containing enzymes of different tissues in rat and/or human is reviewed. Iron participates in a wide variety of biochemical processes, including mitochondrial electron transport, catecholamine metabolism and DNA synthesis. Recently, a broad spectrum of biochemical abnormalities resulting from iron deficiency have been described. Effects on skeletal muscle, cardiac muscle, brain tissue, liver tissue gastrointestinal tractus, body temperature regulation, DNA synthesis are successively discussed. The key liabilities of tissue iron deficiency, even at a mild degree relate to decrease in intellectual performance, and in physical capacity during exercise, alteration of temperature regulation, immune function.

Enzyme kinetics and the rate of biological processes.

It is found empirically that a simple modification of the usual theoretical kinetic formulation (in which a transformation in the temperature scale is made) describes the temperature dependence of a wide variety of biochemical processes with a greater accuracy than hitherto achieved. Used in conjunction with the formulation of the theory of absolute reaction rates this empirical relation facilitates the determination of the thermodynamic functions. The results of applying these relations to biochemical processes support the contention that in the lower temperature range of enzyme activity a thermodynamic equilibrium exists between catalytically active and inactive forms of the enzyme. It is suggested that at low temperatures the formation of intramolecular hydrogen bridges converts reactive enzyme particles to a catalytically inactive condition, in which the active centers either lose their specific configuration or are no longer exposed to the substrate. Upon the basis of this interpretation, values of the entropy changes that are calculated theoretically are found to be in agreement with those calculated from the experimental data. The reactive configuration of the enzyme is apparently possessed in only a relatively narrow temperature band, being lost at both high and low temperatures. The kinetics of biological processes appear to differ only quantitatively from those of in vitro enzyme-catalyzed reactions. In both cases the non-linearity of the Arrhenius plots appears to be due to the fact that in the lower temperature range of enzyme activity a series of reactions are involved in the formation of the activated complex. These include reactions which lead to the formation of the catalytically reactive form of the enzyme followed by that which leads to the formation of the activated complex. The conversion of enzymes to the catalytically inactive form is essentially completed at temperatures of -10-0 degrees C. in living systems, whereas in in vitro experiments with purified enzyme preparations this condition is not attained until temperatures 30-60 degrees C. lower have been reached.