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Topic:Enzymes
Enzymes are biological catalysts that facilitate biochemical reactions in horses by lowering the activation energy required for these processes. They are involved in various physiological functions, including digestion, metabolism, and cellular repair. Common enzymes in equine biology include amylase, lipase, and lactate dehydrogenase, each playing a specific role in the breakdown of nutrients and energy production. The activity and concentration of these enzymes can vary in response to different physiological and pathological conditions, serving as potential indicators in veterinary diagnostics. This page compiles peer-reviewed research studies and scholarly articles that explore the function, regulation, and clinical implications of enzymes in equine health.
The application of in vitro technologies to study the metabolism of the androgenic/anabolic steroid stanozolol in the equine. In this study, the use of equine liver/lung microsomes and S9 tissue fractions were used to study the metabolism of the androgenic/anabolic steroid stanozolol as an example of the potential of in vitro technologies in sports drug surveillance. In vitro incubates were analysed qualitatively alongside urine samples originating from in vivo stanozolol administrations using LC-MS on a high-resolution accurate mass Thermo Orbitrap Discovery instrument, by LC-MS/MS on an Applied Biosystems Sciex 5500 Q Trap and by GC-MS/MS on an Agilent 7000A. Using high-resolution accurate mass full scan analysis o...
MMP-mediated collagen breakdown induced by activated protein C in equine cartilage is reduced by corticosteroids. The plasma serine protease activated protein C (APC) is synthesized by human chondrocytes at sites of pathological cartilage fibrillation. APC levels are increased in osteoarthritis (OA) synovial fluid, and in vitro APC has been shown to synergize with interleukin-1beta (IL-1) to promote degradation from ovine cartilage. A model of equine cartilage degradation was established and used to explore corticosteroid activities. Intraarticular corticosteroids are a commonly prescribed treatment for joint disease, however their role in disease modification remains unclear. APC synergized with IL-1 or ...
Purification and partial characterization of proteinase inhibitors of equine seminal plasma. The aims of the study were: 1/ to isolate and identify equine seminal plasma proteinase inhibitors, 2/ to evaluate their inhibitory potential, and 3/ to test a correlation between protein concentration in seminal plasma supernatant (obtained after precipitation with 36% ammonium sulfate) and stallion sexual maturity. Seminal plasma proteins obtained from six stallions were chromatographed in a Superose 12 (FPLC system) column followed by C(18) HPLC reverse-phase. Inhibition of trypsin amidase activity was evaluated in the collected fractions. Active proteins with a molecular mass of 6.3-7.0 KD...
Muscle fiber population and biochemical properties of whole body muscles in Thoroughbred horses. We examine the muscle fiber population and metabolic properties of skeletal muscles from the whole body in Thoroughbred horses. Postmortem samples were taken from 46 sites in six Thoroughbred horses aged between 3 and 6 years. Fiber type population was determined on muscle fibers stained with monoclonal antibody to each myosin heavy chain isoform and metabolic enzyme activities were determined spectrophotometrically. Histochemical analysis demonstrated that most of the muscles had a high percentage of Type IIa fibers. In terms of the muscle characteristic in several parts of the horse body, th...
Estrogen metabolism by the equine embryo proper during the fourth week of pregnancy. Estrogen production by the trophoblast is considered important in early equine pregnancy and leads to high concentrations in yolk-sac (Y-S) fluid. The embryo proper is a potential site for their action. We examined estrogen metabolism in the embryo proper because some actions of estrogens are derived from locally formed metabolites. The embryo proper, as well as separated extraembryonic tissues, of conceptuses collected about day 25 of pregnancy, were incubated with (3)[H]-estrone (E(1)) and (3)[H]-estradiol (E(2)). Steroids were recovered from media by solid-phase extraction and eluted separa...
Paraspecific neutralization of the venom of African species of cobra by an equine antiserum against Naja melanoleuca: a comparative study. Venoms of snakes belonging to the same Genera tend to share biochemical, toxinological and antigenic characteristics. Accordingly, paraspecific neutralization of venom lethality by experimental antisera and commercial antivenoms has been reported. We studied the spectrum of neutralization of lethality of an experimental monovalent equine antiserum against the strongly neurotoxic African forest cobra (Naja melanoleuca) when tested against venoms of most species of African Naja, both neuro and cytotoxic as described by some authors. We report a comparison of the median lethal doses (LD50) of the...
Two novel IgG endopeptidases of Streptococcus equi. Streptococcus equi ssp. equi causes strangles, a highly contagious and serious disease in the upper respiratory tract of horses. Streptococcus equi ssp. zooepidemicus, another subspecies of this genus, is regarded as an opportunistic commensal in horses. The present study describes the characterization of two novel immunoglobulin G (IgG) endopeptidases of these subspecies, IdeE2 and IdeZ2. Both enzymes display sequence similarities with two previously characterized IgG endopeptidases, IdeE of S. equi ssp. equi and IdeZ of S. equi ssp. zooepidemicus. IdeE2 and IdeZ2 display high substrate-speci...
Molecular characterization and expression pattern of the equine lactate dehydrogenase A and B genes. The species-specific properties of LDH isozymes are essentially determined by M (muscle) and H (heart) subunit proteins encoded by the LDHA and LDHB genes, respectively. In the present study, we molecularly characterized the full-length equine lactate dehydrogenase A (eLDHA) and B (eLDHB) cDNAs. The eLDHA cDNA consisted of a 999-bp open reading frame (ORF), while the eLDHB and newly acquired bat LDHB consisted of a 1002-bp ORF, which is 3 bp shorter than the LDHB ORF of other registered mammals. The alignment of amino acid sequences showed that eLDHA acquired positively charged His 88 and 226,...
Equine alpha S1-casein: characterization of alternative splicing isoforms and determination of phosphorylation levels. alpha(S1)-Casein was isolated from Haflinger mare's milk by hydrophobic interaction chromatography and displayed great micro-heterogeneity by 2-dimensional electrophoresis, probably because of a variable degree of phosphorylation and alternative splicing events. The aim of the present work was to investigate the complexity of the mare's alpha(S1)-casein. The different isoforms present in milk were submitted to a double treatment of dephosphorylation, first by using alkaline phosphatase and then acid phosphatase to achieve complete dephosphorylation. The apoforms were then analyzed by electrosp...
Analysis of a candidate gene for behavioural trait variability in horses: catechol-O-methyltransferase (COMT). The research focuses on the analysis of the catechol-O-methyltransferase (COMT) gene in horses. The study was specifically interested in the role this gene might have in behavioral trait variability due [...]
Identification and isolation of cDNA clones encoding the abundant secreted proteins in the saliva proteome of Culicoides nubeculosus. Culicoides spp. are vectors of several infectious diseases of veterinary importance and a major cause of allergy in horses and other livestock. Their saliva contains a number of proteins which enable blood feeding, enhance disease transmission and act as allergens. We report the construction of a novel cDNA library from Culicoides nubeculosus linked to the analysis of abundant salivary gland proteins by mass spectrometry. Fifty-four novel proteins sequences are described including those of the enzymes maltase, hyaluronidase and two serine proteases demonstrated to be present in Culicoides sali...
In vitro evaluation of differences in phase 1 metabolism of ketamine and other analgesics among humans, horses, and dogs. To investigate cytochrome P450 (CYP) enzymes involved in metabolism of racemic and S-ketamine in various species and to evaluate metabolic interactions of other analgesics with ketamine. Methods: Human, equine, and canine liver microsomes. Methods: An analgesic was concurrently incubated with luminogenic substrates specific for CYP 3A4 or CYP 2C9 and liver microsomes. The luminescence signal was detected and compared with the signal for negative control samples. Ketamine and norketamine enantiomers were determined by use of capillary electrophoresis. Results: A concentration-dependent decrease...
Osmotic stress stimulates generation of superoxide anion by spermatozoa in horses. The objective of this study was to examine the interplay between osmotic and oxidative stress as well as to determine mechanisms by which osmotic stress increases superoxide generation in spermatozoa of horses. Superoxide production, as measured by dihydroethidium (DHE), increased when spermatozoa of horses were incubated under either hyperosmotic or hyposmotic conditions. This increase in superoxide production was inhibited by the MAP kinase p38 inhibitor, SB203580, and by the superoxide scavenger, tiron. Incubation of spermatozoa under hyperosmotic conditions increased overall protein tyrosi...
Effect of live yeast culture supplementation on hindgut microbial communities and their polysaccharidase and glycoside hydrolase activities in horses fed a high-fiber or high-starch diet. Four cecum and right ventral colon-fistulated horses were assigned in a 4 x 4 Latin square design and fed a high-fiber (HF) or a high-starch (HS) diet with or without 10 g of Saccharomyces cerevisiae (SC; CBS 493.94) containing 4.5 x 10(9) cfu/g. The HF and HS diets consisted of pelleted feeds and long wheat straw (18.0 and 3.5 g of DM.kg(-1) of BW.d(-1), respectively) given in 2 equal meals to provide an NDF:starch ratio of 3.5 and 1.0, respectively. After a 21-d adaptation period intestinal contents were collected 4 h after the morning meal on d 23 and 25 to determine bacterial and SC concen...
Method for co-purification of equine neutrophil elastase and myeloperoxidase from a limited blood volume. Neutrophil myeloperoxidase (MPO) and elastase can be released in severe inflammatory diseases and cause tissue injuries. Equine enzymes have already been individually purified from large blood quantities. We describe the isolation of both enzymes from a same limited blood volume. Both MPO and elastase were extracted by crushing PMN isolated by centrifugation on a percoll-gradient from a 460 ml blood collection. MPO and elastase were separated by an ionic exchange chromatography phase and further purified by gel filtration chromatography on Superdex 200 and 75, respectively. Enzymes were identi...
Lipid peroxidation, assessed with BODIPY-C11, increases after cryopreservation of stallion spermatozoa, is stallion-dependent and is related to apoptotic-like changes. Lipid peroxidation (LPO) of stallion spermatozoa was assessed in fresh semen and in samples of the same ejaculates after freezing and thawing. Particular attention was paid to individual differences in the susceptibility to LPO and its possible relationship with freezability. Innate levels of LPO were very low in fresh spermatozoa but increased after thawing, a change that was largely stallion-dependent. The level of LPO in fresh spermatozoa was not correlated with that of the thawed spermatozoa. Negative correlations existed between LPO and intact membranes post-thaw (r=-0.789, P<0.001), a...
Association between myeloperoxidase concentration in equine frozen semen and post-thawing parameters. Despite improvement of techniques, semen of 20% of stallions remains unfreezable. Recent studies focused on the impact of reactive oxygen species and oxidant enzymes on semen characteristics. Myeloperoxidase (MPO) is a pro-oxidant enzyme contained in and released by neutrophils during degranulation or after cell lysis. It is responsible for the formation of hypochlorous acid, a strong oxidant agent, which could damage spermatozoa. The aim of this study was to determine the relation between MPO concentration and characteristics of frozen semen from stallions. Thirty-five straws from different s...
Prostaglandin H synthase Type 2 is differentially expressed in endometrium based on pregnancy status in pony mares and responds to oxytocin and conceptus secretions in explant culture. The equine embryo must signal its presence to the uterus for pregnancy to continue to term. Mobility of the conceptus throughout the uterus is crucial for its survival, and this action presumably permits the conceptus to transmit its antiluteolytic signal to the endometrium. Studies were completed to establish whether this unidentified antiluteolytic signal targets prostaglandin G/H synthase 2 (PGHS2), a rate limiting enzyme in converting arachidonic acid to prostaglandins (PGs). In the first study, quantitative RT-PCR was used to determine the relative abundance of PGHS2 mRNA in endometrium d...
Blood glutathione status and activity of glutathione-metabolizing antioxidant enzymes in erythrocytes of young trotters in basic training. The aim of this study was to evaluate response of blood glutathione status and activity of glutathione-metabolizing antioxidant enzymes in erythrocytes of young trotters in basic training. Nine untrained trotters (aged 16-20 months) were exposed to a 4-month training program based on exercises at low-to-moderate intensity. The conditioning consisted of breaking the horses and running them on distances varying from 4 to 40 km a week. The workloads were increased on a 3-week basis. Exercise intensity was monitored by measuring heart rate and blood lactate. Blood samples were collected at rest, b...
Equine PSGL-1 modifications required for P-selectin binding. Equine PSGL-1 (ePSGL-1) is widely expressed on equine PBMC as a homodimer with sialylation (sLeX) modifications that contribute to P-selectin binding affinity. To investigate the role of other potential post-translational modifications required for high-affinity P-selectin binding, ePSGL-1 was transfected into CHO cells expressing equine FucT-VII and/or C2GnT. P-selectin-IgG chimera binding by ePSGL-1 transfected into CHO cells only occurred when both FucT-VII and C2GnT were expressed, establishing that fucosylation and core-2 branching are required as post-translational modifications for high...
Regulation of platelet activating factor-induced equine platelet activation by intracellular kinases. Lipopolysaccharide (LPS) can activate equine platelets directly or indirectly, via leukocyte-derived platelet activating factor (PAF). Thromboxane (Tx) production by LPS-stimulated equine platelets requires p38 MAPK and this kinase has been suggested as a therapeutic target in endotoxaemia. The present study has utilised selective inhibitors to investigate the role of p38 MAPK and two other kinases, phosphatidylinositol-3 kinase (PI3K) and protein kinase C (PKC), in regulating PAF-induced Tx production, aggregation and 5-HT release in equine platelets, and the modification of these responses b...
Equine cytochrome P450 2C92: cDNA cloning, expression and initial characterization. Substantial gaps exist in our knowledge of the metabolic clearance of therapeutic agents in horses. Accordingly, a cytochrome P450 monooxygenase in the 2C family was cloned from an equine liver, sequenced and expressed in a baculovirus expression system. Catalytic activities of the recombinant protein were measured with a number of substrates. The protein, assigned CYP2C92, displayed optimal catalytic activity with diclofenac using molar ratios of CYP2C92 to NADPH CYP450 reductase of 1:18. Addition of cytochrome b(5) to diclofenac incubations had no significant effect on metabolic turnover. CY...
Identification of protein tyrosine phosphatases and dual-specificity phosphatases in mammalian spermatozoa and their role in sperm motility and protein tyrosine phosphorylation. Protein tyrosine kinases have important roles in spermatozoa; however, little is known about the presence and regulation in these cells of their counterparts in signaling, namely, protein tyrosine phosphatases (PTPs) and dual-specificity phosphatases (DSPs). The objectives of the present study were to identify PTPs and DSPs in boar, stallion, and dog spermatozoa; to characterize their subcellular distribution; and to investigate the roles of tyrosine phosphatases in maintenance of protein tyrosine phosphorylation level and in sperm motility. Using Western blotting with specific antibodies in b...
Use of a bacteriophage lysin, PlyC, as an enzyme disinfectant against Streptococcus equi. Streptococcus equi is the causative agent of the purulent infection equine strangles. This disease is transmitted through shedding of live bacteria from nasal secretions and abscess drainage or by contact with surfaces contaminated by the bacteria. Disinfectants are effective against S. equi, but inactivation by environmental factors, damage to equipment, and toxicity are of great concern. Bacteriophage-encoded lysins (cell wall hydrolases) have been investigated as therapeutic agents due to their ability to lyse susceptible gram-positive organisms. Here, we investigate the use of one lysin, P...
The activity and expression of chitinase in the equine lung and its activity in normal horses and animals with recurrent airway obstruction. Recurrent airway obstruction (RAO) is a chronic inflammatory condition in equine lung, which may share a common immunological basis with human asthma, in which dysregulated Th2 responses occur. Mammals express chitinases and chitinase-like proteins, two of which are active enzymes, chitotriosidase and acidic mammalian chitinase (AMCase). Both enzymes are upregulated in a range of inflammatory conditions, including asthma. We investigated the activity of chitinase in bronchoalveolar lavage fluid from horses with and without RAO in response to organic dust challenges. No significant differences ...
Antigenic, microbicidal and antiparasitic properties of an l-amino acid oxidase isolated from Bothrops jararaca snake venom. Venoms from the bee Apis mellifera, the caterpillar Lonomia achelous, the spiders Lycosa sp. and Phoneutria nigriventer, the scorpions Tityus bahiensis and Tityus serrulatus, and the snakes Bothrops alternatus, Bothrops jararaca, Bothrops jararacussu, Bothrops moojeni, Bothrops neuwiedi, Crotalus durissus terrificus, and Lachesis muta were assayed (800mug/mL) for activity against Staphylococcus aureus. Venoms from B. jararaca and B. jararacussu showed the highest S. aureus growth inhibition and also against other Gram-positive and Gram-negative bacteria. To characterize the microbicidal compon...
Adaptive response of equine intestinal Na+/glucose co-transporter (SGLT1) to an increase in dietary soluble carbohydrate. Experimental and epidemiological evidence suggests that consumption of hydrolyzable carbohydrate, hCHO (grain), by horses is an important risk factor for colic, a common cause of equine mortality. It is unknown whether the small intestinal capacity to digest hCHO and/or to absorb monosaccharides is limiting, or even if horses can adapt to increased carbohydrate load. We investigated changes in the brush-border membrane carbohydrate digestive enzymes and glucose absorptive capacity of horse small intestine in response to increased hCHO. Expression of the Na(+)/glucose co-transporter, SGLT1, was...
Chronic oral therapy with enalapril in normal ponies. Enalapril is an angiotensin converting enzyme (ACE) inhibitor that is frequently used in human, feline and canine patients with cardiac disease. Its use has been associated with impotence in human patients. The purpose of this study was to evaluate if enalapril (0.5mg/kg PO, q24h) is likely to alter behavior in stallions and to assess its effect on ACE activity at the standard dose used in dogs and cats. Twelve pony stallions were evaluated by physical examination and echocardiography followed by treatment with enalapril (n=6) or placebo (n=6) for 2 months. After one month, blood was drawn and...
The involvement of beta-1,4-Galactosyltransferase and N-Acetylglucosamine residues in fertilization has been lost in the horse. In human and rodents, sperm-zona pellucida binding is mediated by a sperm surface Galactosyltransferase that recognizes N-Acetylglucosamine residues on a glycoprotein ZPC. In large domestic mammals, the role of these molecules remains unclear: in bovine, they are involved in sperm-zona pellucida binding, whereas in porcine, they are not necessary. Our aim was to clarify the role of Galactosyltransferase and N-Acetylglucosamine residues in sperm-zona pellucida binding in ungulates. For this purpose, we analyzed the mechanism of sperm-zona pellucida interaction in a third ungulate: the horse, si...
Stereoselective biotransformation of ketamine in equine liver and lung microsomes. Stereoselectivity has to be considered for pharmacodynamic and pharmacokinetic features of ketamine. Stereoselective biotransformation of ketamine was investigated in equine microsomes in vitro. Concentration curves were constructed over time, and enzyme activity was determined for different substrate concentrations using equine liver and lung microsomes. The concentrations of R/S-ketamine and R/S-norketamine were determined by enantioselective capillary electrophoresis. A two-phase model based on Hill kinetics was used to analyze the biotransformation of R/S-ketamine into R/S-norketamine and,...
