Kinetic analyses of mutations in the glycine-rich loop of cAMP-dependent protein kinase.

The conserved glycines in the glycine-rich loop (Leu-Gly50-Thr-Gly52-Ser-Phe-Gly55-Arg-Val) of the catalytic (C) subunit of cAMP-dependent protein kinase were each mutated to Ser (G50S, G52S, and G55S). The effects of these mutations were assessed here using both steady-state and pre-steady-state kinetic methods. While G50S and G52S reduced the apparent affinity for ATP by approximately 10-fold, substitution at Gly55 had no effect on nucleotide binding. In contrast to ATP, only mutation at position 50 interfered with ADP binding. These three mutations lowered the rate of phosphoryl transfer by 7-300-fold. The combined data indicate that G50 and G52 are the most critical residues in the loop for catalysis, with replacement at position 52 being the most extreme owing to a larger decrease in the rate of phosphoryl transfer (29 vs 1.6 s-1 in contrast to 500 s-1 for wild-type C). Surprisingly, all three mutations lowered the affinity for Kemptide by approximately 10-fold, although none of the loop glycines makes direct contact with the substrate. The inability to correlate the rate constant for net product release with the dissociation constant for ADP implies that other steps may limit the decomposition of the ternary product complex. The observations that G52S (a) selectively affects ATP binding and (b) significantly lowers the rate of phosphoryl transfer without making direct contact with either the nucleotide or the peptide imply that this residue serves a structural role in the loop, most likely by positioning the backbone amide of Ser53 for contacting the gamma-phosphate of ATP. Energy-minimized models of the mutant proteins are consistent with the observed kinetic consequences of each mutation. The models predict that only mutation of Gly52 will interfere with the observed hydrogen bonding between the backbone and ATP.

Catalytic subunit of cAMP-dependent protein kinase: electrostatic features and peptide recognition.

The electrostatic field was calculated for the mammalian cAMP-dependent protein kinase (PKA) catalytic subunit (C-subunit) complexed with a 20-residue peptide from a heat stable protein kinase inhibitor (PKI: 5-24). The electrostatic field was also calculated for the C-subunit complexed with a modeled heptapeptide substrate that has been used extensively in structure/function studies for the C-subunit. Perturbations in the electrostatic free energy were calculated when single ionizable active site residues were mutated to alanine. These perturbations in electrostatic free energy were correlated to changes in the binding energy measured in a charge-to-alanine scan of the homologous yeast C-subunit by M. J. Zoller and C. S. Gibbs [(1991) Journal of Biological Chemistry, Vol. 266, pp. 8923-8931; C. S. Gibbs and M. J. Zoller (1991) Biochemistry, Vol. 30, p. 22]. This analysis indicated that the substrate binding parameters primarily depend on electrostatic interactions between a substrate or inhibitor and the C-subunit. Amino acid replacements that led to large perturbations in the electrostatic field are listed in the text. pKa shifts were also calculated for the substrate's phosphate accepting atom, the serine hydroxyl oxygen, when the active site ionizable residues were changed to structurally similar uncharged amino acids. The theoretical mutation of three active site residues caused large shifts in this parameter: E91Q, D166N, and D184N. The calculated pKa shifts for these mutants indicate that the rate of phosphotransfer should be markedly reduced in these cases. This prediction has been experimentally confirmed for the D166N mutant. The correlation between calculated electrostatic free energy changes and measured binding energy, and pKa shifts with phosphotransfer for C-subunit mutants were within experimental error of the measurements. The calculations of electrostatic energy and delta pKa have identified previously unconsidered active site residues in the mammalian C-subunit that contribute to binding energy and phosphotransfer.

Examination of an active-site electrostatic node in the cAMP-dependent protein kinase catalytic subunit.

The active site of the cAMP-dependent protein kinase catalytic subunit harbors a cluster of acidic residues-Asp 127, Glu 170, Glu 203, Glu 230, and Asp 241-that are not conserved throughout the protein kinase family. Based on crystal structures of the catalytic subunit, these amino acids are removed from the site of phosphoryl transfer and are implicated in substrate recognition. Glu 230, the most buried of these acidic residues, was mutated to Ala (rC[E230A]) and Gln (rC[E230Q]) and overexpressed in Escherichia coli. In contrast to the mostly insoluble and destabilized rC[E230A], rC[E230Q] is largely soluble, purifies like wild-type enzyme, and displays wild-type-like thermal stability. The mutation in rC[E230Q] causes an order of magnitude decrease in the affinity for a heptapeptide substrate, Kemptide. In addition, two independent kinetic techniques were used to dissect phosphoryl transfer and product release steps in the reaction pathway. Viscosometric and pre-steady-state quench-flow analyses revealed that the phosphoryl transfer rate constant decreases by an order of magnitude, whereas the product release rate constant remains unperturbed. Electrostatic alterations in the rC[E230Q] active site were assessed using modeling techniques that provide molecular interpretations for the substrate affinity and phosphoryl transfer rate decreases observed experimentally. These observations indicate that subsite recognition elements in the catalytic subunit make electrostatic contributions that are important not only for peptide affinity, but also for catalysis. Protein kinases may, therefore, discriminate substrates by not only binding them tightly, but also by only turning over ones that complement the electrostatic character of the active site.

Pre-steady-state kinetic analysis of cAMP-dependent protein kinase using rapid quench flow techniques.

The phosphorylation of a peptide substrate by the catalytic subunit of cAMP-dependent protein kinase was monitored over short time periods (2-1000 ms) using a rapid quench flow mixing device and a radioactive assay. The production of phosphokemptide [LRRAS(P)LG] as a function of time is characterized by a rapid "burst" phase (250 s-1) followed by a slower, linear phase (L/[E]t = 21 s-1) at 100 microM Kemptide. The amplitude of this "burst" phase varies linearly with the enzyme concentration and represents approximately 100% of the total enzyme concentration, indicating that the "burst" phase is not due to product inhibition. The observed rate constants for the "burst" and linear phases and the "burst" amplitude vary hyperbolically with the substrate concentration. From these dependencies, a maximum "burst" rate constant of 500 +/- 60 s-1 and a Km and Kd for Kemptide of 4.9 +/- 1.4 and 200 +/- 60 microM were determined. The kcat and Km data extracted from the linear portion of the rapid quench flow transients are indistinguishable from those obtained by standard steady-state kinetic analyses using low catalytic subunit concentrations and a spectrophotometric, coupled enzyme assay. Both rate constants for the "burst" and linear phases decreased in the presence of Mn2+. The data imply that the phosphorylation of Kemptide by the catalytic subunit occurs by a mechanism in which the substrate is loosely bound, is rapidly phosphorylated at the active site, and is released at a steady-state rate that is likely controlled by the dissociation rate constant for ADP. The combined pre-steady-state kinetic data establish a comprehensive, kinetic mechanism that predicts all the steady-state kinetic and viscosometric data. This study represents the first chemical observation and characterization of phosphoryl transfer at the active site of a protein kinase and will be useful for further structure-function studies on this and other protein kinases.

Lag screw stabilization of a cervical vertebral fracture by use of computed tomography in a horse.

A traumatic fracture of C2 was diagnosed radiographically in a 1-year-old German Warm-blood stallion. Fracture configuration was difficult to see on survey radiographs. Computed tomography yielded a more accurate assessment of the fracture and facilitated fracture repair with cortical lag screws. Precise screw placement, to avoid spinal cord damage, was obtained by use of computed tomography. Follow-up radiography revealed normal bone healing, and the horse was in dressage schooling 24 months after surgery.

Relationship between locomotor forces, hoof position and joint motion during the support phase of the stride of galloping horses.

Three methods were used simultaneously to determine the relationships between the vertical forces exerted on the hooves and the positions of the limbs and hooves at the times of peak vertical forces from 2 horses galloping on a track straightaway. Vertical forces were recorded from an instrumented shoe, fetlock joint motion was measured with an electrogoniometer and the angles of the carpus, fetlock and hoof were determined from slow-motion films. At hoof contact, the mean angles of the carpus and fetlock were 181-182 degrees and 199-206 degrees, respectively. Peak vertical forces on the heel occurred at or near maximum extension of the carpal and fetlock joints. Peak forces on the toe occurred during flexion of the fetlock joint and at mean hoof angles of 28-31 degrees from the horizontal. The mean angles of the hoof from the horizontal at the time of heel contact were 6-7 degrees. Hoof lift occurred at mean carpal angles of 173-174 degrees and mean fetlock angles of 199-200 degrees.

Ultrasonographic evaluation and surgical treatment of humeral osteitis and bicipital tenosynovitis in a horse.

Ultrasonography and radiography were used to diagnose bicipital tenosynovitis and humeral osteitis in a horse with a history of lameness of 7 weeks' duration. Surgical exploration confirmed the diagnosis.

Testicular feminization syndrome in a mare.

Testicular feminization syndrome was diagnosed in a mare with aggressive, stallion like behavior and a history of infertility. She was found to have a high baseline testosterone concentration suggesting that testicular tissue was present, and ovarian-like structures examined by use of transrectal ultrasonography had the appearance typical of testicular tissue. Although her external female genitalia appeared normal, her vagina ended in a blind sac, and no cervix or uterus were identified. Surgery was performed, and structures removed from the abdominal cavity were determined to be hypoplastic testicles. Removal of the testicular tissue resulted in complete resolution of her aggressive behavior. Chromosomal evaluation revealed that the mare had 64X,Y (normal male) karyotype. Testicular feminization syndrome is a condition characterized by insensitivity of reproductive tissues to androgens during development because of an abnormality in androgen receptors. This androgen insensitivity results in development of normal external female genitalia, with high testosterone concentrations being released from developing testicles. Testicular feminization syndrome has not been commonly diagnosed in horses, but should be considered as a differential diagnosis for overly aggressive mares with a history of infertility.

Suspensory apparatus prosthesis in the horse. Part 1: In vitro mechanical properties.

Mechanical properties of equine suspensory apparatus preparations and three braided synthetic prostheses were evaluated in vitro. Force versus displacement plots and failure modes were recorded from single load-to-failure testing in 18 cadaver limbs before and after replacement of each suspensory apparatus with a prosthesis. Mean load at failure, energy to failure, and stiffness values of polytetrafluoroethylene (PTFE) prostheses were lower than those of the suspensory apparatus and aramid prosthesis. The PTFE prosthesis failed by elongation or rupture of the prosthesis. Mechanical properties of the aramid prosthesis with collar augmentation were not significantly different from the suspensory apparatus. The main site of failure in both aramid prostheses was at the screw fixation to bone.

Suspensory apparatus prosthesis in the horse. Part 2: In vivo evaluation in a suspensory apparatus disruption model.

The suspensory apparatus of one forelimb was surgically disrupted in six adult horses by transecting the distal sesamoidean ligaments. A double-braided prosthetic ligament made of aramid yarn was installed to support the flexor surface of the metacarpophalangeal joint. The prosthesis was routed through tunnels in the third metacarpal bone and proximal phalanx, and secured to bone with screws. Evaluation by radiography, synovial fluid analysis, cinematography, and dynamography was performed before surgery and at weeks 16 and 30. Supracondylar cortical lysis and periosteal proliferation were observed on postoperative radiographs. Synovitis and fragmentation of the prosthesis were apparent from synovial fluid evaluation. Weight bearing and metacarpophalangeal joint motion were decreased and loading was transferred in part to the opposite forelimb. Clinical lameness improved and weight bearing increased during the second half of the 30-week period. At necropsy, there was abrasion of the prosthesis and the articular surfaces in contact with the prosthesis. Diffuse granulomatous synovitis developed in response to aramid fiber fragments within the synovium.

Muscle responses of thoroughbreds to conventional race training and detraining.

Ten healthy sedentary Thoroughbreds with previous race training experience were trained conventionally for 9 weeks. Muscle biopsy samples were obtained before and after training and after 6 weeks of detraining pasture rest. Biopsy samples were obtained from the right deltoid, triceps, vastus lateralis, middle gluteal, biceps femoris, and semitendinosus muscles. The deep-frozen biopsy samples were analyzed for activities of succinate dehydrogenase (SDH), 3-hydroxy-acylcoenzyme A dehydrogenase (HAD), and phosphorylase (PHOS) and for glycogen concentration. The triceps and gluteal muscle samples were also serially sectioned and stained for myofibrillar actomyosin adenosine triphosphatase (ATPase) activity after alkaline (pH 10.3) and sequential acidic (pH 4.34) ATPase inactivation. Fiber types I (alkaline preincubation), IIA1, IIA2, and IIA3 (sequential acidic preincubation over 5 minutes) were identified and were evaluated for fiber-type distribution and fiber areas. Increases in response to training were observed in deltoid and vastus muscle SDH and gluteal muscle HAD activities, and deltoid muscle glycogen concentration (P less than 0.05 to P less than 0.01). Changes in PHOS activity were not observed. Type-IIA1, -IIA2, and -IIA3 fiber areas in triceps muscle were increased in response to training (P less than 0.05 to P less than 0.01). Changes in fiber-type distribution did not occur in response to training. Changes in muscle enzyme activities, glycogen concentration, fiber types, and fiber areas were not seen from posttraining to detraining. Further increases were observed when detraining values were compared with pretraining values in deltoid, triceps, vastus, gluteal, and biceps femoris muscle SDH activities and in gluteal muscle glycogen concentration (P less than 0.05 to P less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Standardized exercise test and daily heart rate responses of thoroughbreds undergoing conventional race training and detraining.

Ten healthy sedentary male Thoroughbreds with previous race training experience were studied for 14 weeks. Horses were trained for 9 weeks, using a program designed after those used commonly in the United States. Horses were trained conventionally by slow trotting (250 m/min) for 2 weeks and galloping (390 to 450 m/min) for 4 weeks, followed by 3 weeks of galloping (440 to 480 m/min) and intermittent sprinting exercises (breezes) at distances between 600 and 1,000 m (900 to 950 m/min). The horses were then pasture rested for 5 weeks. A standardized exercise test (SET) involving an 800-m gallop at 800 m/min was administered before and after the 9-week training period and after the 5-week detraining period. Heart rate (HR) was monitored during exercise and at standardized intervals after exercise for 60 minutes. Venous blood for determination of plasma lactate concentration was obtained at 5 minutes after exercise. Heart rate was monitored daily at rest, during exercise, and through the first 60 minutes of recovery. Venous plasma samples (for lactate determination) were obtained 5 minutes after the sprinting exercises. Horses were observed daily before exercise for signs of lameness and were not allowed to train if lame. Differences after 9 weeks' training were seen in the SET recovery HR at 0.5 through 5 minutes after exercise (P less than 0.05 to P less than 0.01). Differences after detraining were seen in the SET recovery HR at 40 and 60 minutes after exercise (P less than 0.05 to P less than 0.01). Neither training nor detraining resulted in differences in plasma lactate concentration after the SET gallop.(ABSTRACT TRUNCATED AT 250 WORDS)

Melanoma as a cause of spinal cord compression in two horses.

Spinal cord compression by melanomas at C7-T1 is described in 2 horses. Despite similar location of the lesions, the onset of neurologic signs and clinical progression varied. Both horses were gray, but few cutaneous melanomas were found. Internal dissemination, however, was extensive in both horses. Antemortem diagnosis was made in one horse, and surgical decompression of the spinal cord was performed. Neoplasms of the vertebral canal and spinal cord are uncommon in horses. Melanoma in the spinal canal should be included in the differential diagnosis of neurologic disease, especially in mature gray horses.

Injuries to the cranial cruciate ligament and associated structures: summary of clinical, radiographic, arthroscopic and pathological findings from 10 horses.

The clinical, radiographic, arthroscopic and pathological findings of 10 horses with injury to the cranial cruciate ligament are presented. The most consistent clinical signs included moderate to severe distension of the femoropatellar joint and a Grade III to a Grade V out of V lameness. Craniocaudal instability could be elicited in five horses under general anaesthesia and in one conscious horse. Radiographic evaluation of the stifles revealed that avulsion fracture of the medial intercondylar eminence was the most common finding in six out of 10 horses. Arthroscopic examination of the affected femorotibial joints were performed in five horses. This confirmed the presumptive diagnosis of cranial cruciate ligament injury or rupture. Post mortem examinations were performed on two horses which documented partial tears of the cranial cruciate ligament.

Long-term follow-up of partial limb amputation in 13 horses.

Thirteen cases of partial limb amputation in horses were reviewed. Nine horses (69%) survived 18 to 111 months (mean, 41 months). Four unsuccessfully treated horses were euthanatized within 21 days of surgery. The horses' ages at amputation were 4 to 13 years (mean, 7.3 years). There was no predilection for the left or right limb nor for fore or hind limb involvement. Osteomyelitis in six horses (46%) was the most common cause for amputation. Severe, open, comminuted fractures of the third metacarpal bone that were impossible to stabilize by any currently available technique in four horses (31%) were the second most common cause for amputation. One gelding was salvaged because of his sentimental value, two horses were used as breeding stallions, and six were broodmares. One stallion lived 30 months, completed two breeding seasons, and started a third. The other stallion began breeding, but became sterile. Three of the mares have produced five foals. One mare died while in foal. One mare aborted near-term twin fetuses and died of uterine hemorrhage. One mare aborted twice before the femoral head ligament on the contralateral limb ruptured, and she was euthanatized.

A modified Cloward's technique for arthrodesis of the normal metacarpophalangeal joint in the horse.

A modified Cloward's technique was performed for arthrodesis of one metacarpophalangeal (MCP) joint in eight horses. Dorsal arthrotomies were performed medial and lateral to the common digital extensor tendon and two 16 mm holes were drilled through the joint. A perforated cylindrical stainless steel basket filled with cancellous bone was impacted into each hole. The limbs were supported in casts for 8 weeks. The joints were examined and radiographed at 4 weeks, 8 weeks, 6 months, and 10 months. One horse was euthanatized at week 14 to assess the progress of the arthrodesis. In the other seven horses, there was clinical fusion at month 6. Dynamographic evaluations were performed 11 months after surgery at the walk and trot. The maximum vertical forces exerted during weight bearing by treated and control limbs were compared. No difference was detected at the walk; however, a significant difference was present at the trot (p less than 0.05). It was calculated that at the trot the horses placed 90% as much force on the treated limb as on the control limb. Eleven months after surgery, the baskets contained compact and cancellous bone. Ingrowth of bone occurred through all openings, completely filling the baskets and fusing the joints.

Cervical vertebral malformations.

Cervical vertebral malformations are recognized clinically when they result in spinal cord compression causing neurologic deficits. Confirmation of diagnosis is based on radiographic and myelographic evidence of cord compression. Medical therapy is not usually successful. Several successful surgical treatments are described.

Long-term results of desmotomy of the accessory ligament of the deep digital flexor tendon (distal check ligament) in horses.

After desmotomy of the accessory ligament of the deep digital flexor tendon (distal check ligament) had been performed in 40 horses with flexural deformity of the distal interphalangeal joint (contracture of the deep digital flexor tendon), the horses' records were evaluated retrospectively. Information concerning soundness, return to intended purpose, and cosmetic appearance of the limb and hoof was obtained. The condition had been diagnosed and treated in 26 horses before they were one year old (group A, acute) and in 14 horses greater than one year of age (group B, chronic). Nine months to 4 years after surgery, 35 horses (24 from group A, and 11 from group B) were not lame and were being used as athletes. Thirty-three horses were being used for what the owner had intended before the development of the flexural deformity, and 7 were not. Of these 7 horses, 6 had complications related to the deformity, whereas one had complications resulting from surgery.