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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.
Fibrous tissue of subchondral cystic lesions in horses produce local mediators and neutral metalloproteinases and cause bone resorption in vitro. To define the release of nitric oxide (NO), prostaglandin E2 (PGE2), and the neutral metalloproteinases (NMPs) in horses with subchondral cystic lesions (SCL) and to study bone resorption triggered by conditioned media of fibrous tissue of SCL in vitro. Methods: Equine explant cultures of fibrous tissue of SCL, and synovial membrane and articular cartilage of normal horses and horses affected with moderate and severe osteoarthritis were performed. NO, PGE2, and NMP concentrations of media samples were measured, and osteoclast formation and activation was studied in vitro. Methods: Experiment 1...
Changes in bone morphogenic enzymes and lipid composition of equine osteochondrotic subchondral bone. Osteochondrosis (OC) is a disturbance in the process of endochondral ossification, a process in which cartilage is mineralised and transformed into bone. In this process different biochemical events occur, of which the cartilage component has been studied so far almost exclusively. In this study we concentrated on the biochemical characterisation of normal and osteochondrotic subchondral bone, by analysis of enzyme activities, DNA content and phospholipids (PL). In subchondral bone, lysyl oxidase and both total and bone alkaline phosphatase activity were significantly increased in all degrees ...
Catalase activity in equine semen. To characterize the activity of catalase in equine semen. Methods: 15 stallions of known and unknown reproductive history. Methods: Seminal plasma was collected from raw equine semen by centrifugation, and samples of seminal plasma were frozen prior to assay for catalase activity. Tissue samples (n = 3 stallions) from the bulbourethral gland, prostate gland, vesicular gland, and testis were homogenized, and cauda epididymal fluid was collected for determination of catalase activity. Catalase activity was determined as an enzyme kinetic assay by the disappearance of H2O2 as measured by ultravio...
Concentrations of elastinolytic metalloproteinases in respiratory tract secretions of healthy horses and horses with chronic obstructive pulmonary disease. To determine whether samples of tracheal epithelial lining fluid (TELF) obtained from horses have elastinolytic activity characteristic of metalloproteinases, to compare elastinolytic activity in TELF obtained from healthy horses and horses with chronic obstructive pulmonary disease (COPD), and to determine whether chemically modified tetracycline-3 (CMT-3) inhibits elastinolytic activity in TELF ANIMALS: 10 horses with COPD and 10 healthy control horses. Methods: Zymography and fluorometry were used to measure elastinolytic activity, and EDTA was used to inhibit elastinolytic activity and ver...
Purification, characterization, and cDNA sequencing of cytosolic phospholipase A(2) from equine neutrophils. It has been demonstrated that equine neutrophils, but not eosinophils, require exogenous arachidonic acid for calcium ionophore A23187-induced leukotriene synthesis. Because cytosolic phospholipase A(2) (cPLA(2)) plays an essential role in leukotriene formation in leukocytes, we investigated the presence of a functional cPLA(2) in equine neutrophils. To determine whether cPLA(2) from neutrophils was catalytically active, we purified the enzyme >6,500 fold with 3% recovery from equine neutrophils. The full-length cDNA sequence encoded a 749-amino acid protein. The deduced amino acid sequence...
Detection of biological threat agents by immunomagnetic microsphere-based solid phase fluorogenic- and electro-chemiluminescence. This article reviews the recent development of two solid-phase chemiluminescence-based techniques, fluorogenic-chemiluminescence (FCL) and electro-chemiluminescence (ECL) for detection of biological threat agents. Both techniques entail a labeled sandwich immunoassay. The objectives of this work are to develop advanced techniques for sensitive and effective detection of a target analyte, particularly in cases where the analysis includes complex samples containing multiple contaminating factors. Other important considerations in developing such detection techniques include the ease of use, the ...
Interaction of alcohol dehydrogenase with tert-butylhydroperoxide: stimulation of the horse liver and inhibition of the yeast enzymes. Preincubation of horse liver alcohol dehydrogenase (HLADH) with the oxidative agent, tert-butyl hydroperoxide (tBOOH) results in a twofold stimulation of the ethanol dehydrogenase activity of this enzyme. This stimulation was dependent on tBOOH concentration up to 100 mM; above this concentration tBOOH did not further stimulate ethanol oxidation by HLADH. Active-site-directed reagents and classical ADH binary complexes were used to probe the possible mechanism of this activating effect. The rate and extent of stimulation by tBOOH is strongly reduced by binary complexes with NAD(+) or NADH, who...
[The specific blocks of heterochromatin on metaphase chromosomes of horse and Prjewalski horse detected by in situ digestion with restriction endonucleases]. Restriction endonuclease in situ digestion of metaphase chromosomes gives an opportunity to reveal strips with different structure within GC-rich pericentric heterochromatin of the domestic horse and the wild Przewalski horse. Blocks of heterochromatin, which are insensitive to HaeIII and brightly stained with chromomycin A3 after restriction enzyme digestion, are localized on the border with euchromatin in the majority of chromosomes of Equus caballus and E. przewalskii. In contrast to chromosome 5 of E. caballus, acrocentric chromosomes of E. prezewalskii which are homologous to this chromos...
Direct MS-MS identification of isoxsuprine-glucuronide in post-administration equine urine. Isoxsuprine is routinely recovered from enzymatically-hydrolyzed, post-administration urine samples as parent isoxsuprine in equine forensic science. However, the specific identity of the material in horse urine from which isoxsuprine is recovered has never been established, although it has long been assumed to be a glucuronide conjugate (or conjugates) of isoxsuprine. Using ESI/MS/MS positive mode as an analytical tool, urine samples collected 4-8 h after isoxsuprine administration yielded a major peak at m/z 554 that was absent from control samples and resisted fragmentation to daughter ions...
Evaluation of the role of keratan sulphate as a molecular marker to monitor cartilage metabolism in horses. The role of keratan sulphate (KS) as a metabolic marker of cartilage was evaluated using an in vitro model of equine articular cartilage. Articular cartilage was harvested from clinically healthy 6-month-old foals (n = 3). Chondrocytes were centrifuged and cultured as pellets. Chondrocyte pellets were stimulated by insulin-like growth factor-I alpha (IGF-I alpha) or interleukin-1 alpha (IL-1 alpha) for 2 weeks. The concentrations of sulphated glycosaminoglycans (GAG) and KS in the culture media were measured by a 1,9-dimethyl-methylene blue (DMMB) colorimetric assay and an inhibition enzyme-li...
Intrafollicular concentrations of steroids and steroidogenic enzymes in relation to follicular development in the mare. The objective of the present study was to determine the changes in follicular fluid steroid concentrations and in granulosa cell steroidogenic enzyme expression during the follicular phase, in relation to follicular size and physiological status in the mare. Follicular fluid and follicular cells were recovered by ultrasound-guided follicular punctures either around the time of emergence of the dominant follicle, at the end of the dominant follicle growth, or at the preovulatory stage, after injection of gonadotropin to induce ovulation. Cellular relative amounts of steroidogenic acute regulato...
Consideration of the role of antigenic keratan sulphate reacting to a 1/14/16H9 antibody as a molecular marker to monitor cartilage metabolism in horses. The role of keratan sulphate (KS) as a marker of cartilage metabolism was evaluated by using an in vitro model of equine articular cartilage. Articular cartilage was harvested from clinically healthy 6-month-old foals (n=3). Chondrocytes were centrifuged and cultured as pellets. Chondrocyte pellets were stimulated by insulin-like growth factor (IGF)-Ialpha or interleukin (IL)-1alpha for 2 weeks. The sulfated glycosaminoglycans (GAG) and antigenic KS concentrations in the culture media were measured by a 1,9-dimethyl-methylene blue (DMMB) colorimetric assay and an inhibition ELISA using a 1/14/...
A long-lived tyrosyl radical from the reaction between horse metmyoglobin and hydrogen peroxide. The reaction between metmyoglobin (metMb) and hydrogen peroxide has been known since the 1950s to produce globin-centered free radicals. The direct electron spin resonance spectrum of a solution of horse metMb and hydrogen peroxide at room temperature consists of a multilined signal that decays in minutes at room temperature. Comparison of the direct ESR spectra obtained from the system under N(2)- and O(2)-saturated conditions demonstrates the presence of a peroxyl radical, identified by its g-value of 2.014. Computer simulations of the spectra recorded 3 s after the mixture of metMb and H(2)...
Spontaneous production of nitric oxide (NO), prostaglandin (PGE2) and neutral metalloproteinases (NMPs) in media of explant cultures of equine synovial membrane and articular cartilage from normal and osteoarthritic joints. Nitric oxide (NO), prostaglandin E2 (PGE2), and the activity of neutral metalloproteinases (NMPs) were measured in conditioned media of equine synovial membrane and articular cartilage explant cultures from horses with normal joints (n = 7) and from horses affected with moderate (n = 7) or severe osteoarthritis (n = 14) as judged by macroscopic appearance. Normal articular cartilage appeared glossy and bluish-white, was of normal thickness and showed no evidence of discolouration, fibrillation or other cartilage discontinuity. Slight discolouration and fibrillation or minor clefts of the carti...
Immunolocalization of P450C17 in the mare corpus luteum. Although the mare corpus luteum (CL) is capable of aromatization, the expression of other enzymes involved in estradiol synthesis is not yet clear. This study examined the localization of P450C17 in the mare CL at different stages of its functional development. In ovaries from follicular phase mares P450C17 was localized in the theca cells of ovarian follicles. Following ovulation, no immunostaining for P450C17 was detected in the mature CLs of nonpregnant mares. In pregnant mares, no immunostaining for P450C17 was identified in the corpus luteum prior to secretion of eCG by the feto placental...
Heme orientation affects holo-myoglobin folding and unfolding kinetics. Native myoglobin (Mb) consists of two populations which differ in the orientation of the heme by 180 degrees rotation (as verified by nuclear magnetic resonance) but have identical absorption spectra and equilibrium-thermodynamic stability. Here, we report that these two fractions of native oxidized Mb (from horse) both unfold and refold (chemical denaturant, pH 7, 20 degrees C) in two parallel kinetic reactions with rate constants differing 10-fold. In accord, the oxidized heme remains coordinated to unfolded horse Mb in up to 4 M guanidine hydrochloride (pH 7, 20 degrees C).
Isolation and characterization of a cDNA encoding a horse liver butyrylcholinesterase: evidence for CPT-11 drug activation. Butyrylcholinesterases (BuChEs; acylcholine acylhydrolase; EC 3.1.1.8) have been demonstrated to convert the anticancer agent CPT-11 (irinotecan, 7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxycamptothecin) into its active metabolite SN-38 (7-ethyl-10-hydroxycamptothecin). In addition, significant differences in the extent of drug metabolism have been observed with BuChEs derived from different species. In an attempt to understand these differences, we have isolated the cDNA encoding a horse BuChE. Based upon the NH2-terminal amino acid sequence of a purified horse BuChE, we designed deg...
Hepatic and pulmonary enzyme activities in horses. To determine hepatic and pulmonary phase-I and phase-II enzyme activities in horses. Methods: Pulmonary and hepatic tissues from 22 horses that were 4 months to 32 years old. Methods: Pulmonary and hepatic tissues from horses were used to prepare cytosolic (glutathione S-transferase and soluble epoxide hydrolase) and microsomal (cytochrome P450 monooxygenases) enzymes. Rates of microsomal metabolism of ethoxyresorufin, pentoxyresorufin, and naphthalene were determined by high-performance liquid chromatography. Activities of glutathione S-transferase and soluble epoxide hydrolase were determine...
Influence of development and joint pathology on stromelysin enzyme activity in equine synovial fluid. To investigate the role of stromelysin (MMP-3) activity in synovial fluid (SF) at different stages of development and in common joint disorders in the horse. Methods: Stromelysin activity was determined with a fluorogenic enzyme activity assay in SF of normal joints of fetal, juvenile and adult horses, and in SF of horses suffering from the developmental orthopaedic disease osteochondrosis (OC) or osteoarthritis (OA). Additionally, MMP-3 activity was expressed as a ratio of previously reported general MMP activity in the same SF samples. Results: The levels of active stromelysin were 30-fold t...
Sweat production and localisation of carbonic anhydrase in the equine sweat gland during exercise at two ambient temperatures. The aim of this investigation was to study sweat production during exercise at 2 ambient temperatures (20 degrees C and 35 degrees C) and the concurrent localisation of carbonic anhydrase (CA) in the sweat gland. Horses develop alkalosis during prolonged exercise and the sweat contains HCO3-. Carbonic anhydrase is therefore of interest since it catalyses the reaction CO2 + H2OHCO3- + H+. Four standardbred trotters performed an exercise test. Skin biopsies were taken from the neck, and sweat rate, blood and skin temperatures were measured. There was a close relationship between sweat rate, temp...
Muscular and metabolic responses to moderate-intensity short-term training. The objectives of this study were to determine the effects of 10 consecutive days of moderate-intensity training on 1) the muscular metabolic response to exercise at 100% of the pre-training maximum rate of oxygen consumption (VO2max); and 2) mitochondrial enzyme markers (citrate synthase, CS; succinate dehydrogenase, SDH; 3-hydroxy-acyl-CoA dehydrogenase, HAD) of oxidative capacity in middle gluteal muscle. Six mature, unfit Thoroughbred horses completed both incremental (for determination of VO2max) and high-intensity exercise protocols before (HI1) and after (HI2) training. Training consist...
Relationship between running speed, isoenzymes of serum creatine kinase and lactate dehydrogenase and left ventricular function in stallions. The purpose of this study was to assess the possible relationship between maximal running speed, serum isoenzyme patterns of creatine kinase (CK) and lactate dehydrogenase (LDH) and echocardiographic indices of left ventricular function. A group of 15 healthy, 3-year-old Maremmano stallions were given a 100 day training programme. At the end of this the animals carried out a maximum speed test and were divided into 2 groups (A and B) according to whether or not they had attained a speed of 15 m/s. Venous blood samples were taken from each horse before exercise (T0), 2 min (T1) and 24 h (T2) af...
Zymographic analysis of equine laminitis. To investigate the role of matrix metalloproteinase (MMP) activity in the pathophysiology of equine laminitis, the techniques of in situ zymography and quantitative SDS-PAGE zymography were used to analyse the lamellae and plasma and serum of horses with carbohydrate overload-induced laminitis. The gelatinase activity localised within the epidermal lamellae of laminitic hooves did not differ significantly from normal hooves. In laminitis sections there was an increase in vascular gelatinase activity, possibly associated with the perivascular cuffing of polymorphonucleocytes. Both plasma and se...
Determination of testosterone:epitestosterone ratio after pentafluorophenyldimethylsilyl-trimethylsilyl derivatisation using gas chromatography-mass spectrometry in equine urine. A highly specific method is described for measuring the testosterone:epitestosterone ratio in equine urine by gas chromatography-mass spectrometry (GC-MS) with stable isotope internal standards. The procedure was based on Serdolit Pad-1 resin extraction, enzymatic hydrolysis, and chemical derivatisation prior to instrumental analysis. The mixed derivatives, 3-trimethylsilyl-17-pentafluorophenyldimethylsilyl ether (3-TMS-17-flophemesyl) testosterone and epitestosterone, were found to have excellent analytical properties. The specificity of the derivatisation method exploits a unique feature of ...
Differential expression of steroidogenic enzymes by the primary corpora lutea of pregnant mares during equine chorionic gonadotrophin secretion. At the onset of equine chorionic gonadotrophin (eCG) secretion, eCG stimulates luteal androgen and oestrogen production. Although eCG concentrations increase exponentially from day 37 to day 60 of gestation and eCG is detectable in maternal serum until about day 120-150 of gestation, luteal androgen and oestrogen production peaks between 5 and 10 days after initial exposure to eCG and then decreases gradually. It is not clear how eCG regulates luteal androgen and oestrogen production. In the present study, the steady-state mRNA expression of 3beta-hydroxysteroid dehydrogenase (3beta-HSD), cyto...
Localization and cellular distribution of a unique hyaluronidase in stallion spermatozoa during epididymidal transit. Three protein bands with hyaluronidase activity and molecular masses of 87, 48 and 43 kDa were isolated from purified equine sperm plasma membranes. Indirect immunofluorescence was used to assess sperm labelling patterns using a polyclonal antibody to sperm hyaluronidase. In ejaculated spermatozoa, surface-associated hyaluronidase was localized to the posterior head region of 98 +/- 2% of spermatozoa (n=10). Epididymides were isolated from mature stallions (n=5) and divided into caput, corpus and cauda epididymides in separate Petri dishes. The epididymidal tubules were dissected and washed us...
Indirect determination of stallion sperm capacitation based on esterase release from spermatozoa challenged with lysophosphatidylcholine. A spectrophotometric assay was developed to measure the amount of esterase released from stallion spermatozoa. This assay was used to determine the percentages of capacitated stallion spermatozoa, determined by the ability of spermatozoa to undergo an acrosome reaction and release esterase in response to a lysophosphatidylcholine challenge, for spermatozoa incubated under conditions to increase intracellular calcium and cAMP. Incubation with 100 nmol calcium ionophore A23187 l(-1) induced 66% of stallion spermatozoa to capacitate after 60 min of incubation at 37 degrees C. Subsequent experimen...
Relationship between the degree of endometrial periglandular fibrosis and the presence of angiotensin-converting enzyme in the equine endometrium. Endometrial periglandular fibrosis (EPF) has been proposed as a possible aetiology for equine embryonic and fetal loss. However, the pathophysiology of EPF is not well understood. Angiotensin-converting enzyme (ACE) is found in macrophages, endothelium (during angiogenesis) and myofibroblasts at sites of fibrosis in the heart, kidneys, liver and skin in several species. An increase in local tissue ACE-binding activity appears to be a critical event in the initiation and progression of fibrosis in these tissues. The aim of this study was to investigate the correlation between ACE activity in th...
Comparison of different diluents and chromophores for spectrophotometric determination of livestock blood cholinesterase activity. Effects of seven different blood diluents (distilled water, Triton X-100, saponin, isotonic saline solution, pH 7.5 and 8 phosphate buffers and bovine serum albumin) and two chromophores: 5, 5'-dithiobis 2-nitrobenzoic acid (DTNB) and 2,2'-dithiodipyridine (2- PDS) on blood cholinesterase determination in four domestic species (cow, sheep, goat and horse) are described and compared. Haemolytic diluents (distilled water, Triton X-100 and saponin) gave the best precision results when fresh blood was assayed. However, Triton X-100 induced lower ChE activity values in horses, and saponin yielded v...
Species specificity of plasminogen activation and acquisition of surface-associated proteolytic activity by group C streptococci grown in plasma. Our laboratory previously demonstrated that group C streptococcal isolates from humans and horses secrete streptokinases that preferentially activate plasminogens reflecting the origin of the isolates. To analyze the significance of these findings, series of streptokinase-producing Streptococcus equisimilis isolates recovered from humans and horses were examined. Southern blot analysis revealed that chromosomal DNA of the streptococcal isolates from humans reacted exclusively with a skc(hu) probe and that chromosomal DNA of streptococcal isolates from horses reacted preferentially with an skc(...
