Acrylamide quenching of apo- and holo-alpha-lactalbumin in guanidine hydrochloride.

We have examined the fluorescence properties and acrylamide quenching of calcium-loaded (holo) and calcium-depleted (apo) alpha-lactalbumin (alpha-LA) as a function of guanidine hydrochloride (GDN/HCl) concentration. The spectral changes accompanying increasing GDN/HCl are consistent with protein unfolding and a release of internal fluorescence quenching, which occurs among the three tryptophan residues located in the region of the so-called "tertiary fold." Values for the intrinsic fluorescence emission, the wavelength maximum of the emission, the Stern/Volmer dynamic quench constant, and the static quench constant are consistent with a significant stabilization effect by calcium against protein unfolding. The dynamic quench constant of apo-alpha-LA increases fourfold to its maximum, in the transition from the native state to protein in 1.5 M GDN/HCl. The dynamic quench constant for holo-alpha-LA remains unchanged until exposed to 2.5 M GDN/HCl, but increases by threefold with addition denaturant to 4 M GDN/HCl. The static quench constant of the apo-protein in the native solvent, approximately 0.2 M(-1), declines to zero in 1 M denaturant, where the molten globule folding intermediate is most populated. A more protracted denaturant-dependent decline in the static quench constant occurs for the holo-protein. Sharp increase in the static quenching occurs for apo-alpha-LA and holo-alpha-LA above 1.5 M GDN/HCl and 3.5 M GDN/HCl, respectively. The results for apo-alpha-LA in dilute GDN/HCl suggest that acrylamide can penetrate the protein molecule (as judged by the collision quenching) but is unable to form a stable complex within the quenching domain for the tryptophans (as judged by the absence of the static quench constant). It seems reasonable to suggest that the protein folding intermediate which occurs in dilute denaturant represents a structure in which the tryptophans are, on average, more accessible to collisional quenching but sufficiently compact to prevent formation of a stable, dark equilibrium complex with acrylamide.

Continuous intravenous dobutamine is associated with an increased risk of death in patients with advanced heart failure: insights from the Flolan International Randomized Survival Trial (FIRST).

OBJECTIVE: To evaluate clinical characteristics and outcomes of patients with advanced heart failure receiving intravenous continuous dobutamine in the FIRST Trial (Flolan International Randomized Survival Trial). METHODS: Four hundred seventy-one patients with class IIIb to IV heart failure who were enrolled in the FIRST trial were included. Eighty patients treated with dobutamine at FIRST randomization were compared with 391 patients not treated with dobutamine at randomization. The occurrence of worsening heart failure, need for vasoactive medications, resuscitated cardiac arrest, myocardial infarction, and total mortality were compared between the 2 groups. RESULTS: The dobutamine group had a higher occurrence of first event (85.3% vs 64. 5%; P =.0006) and a higher mortality rate (70.5% vs 37.1%; P =.0001) compared with the no dobutamine group. Intravenous continuous dobutamine was an independent risk factor for death after adjusting for baseline differences. CONCLUSIONS: Dobutamine use at the time of randomization was associated with a higher 6-month mortality rate. This effect persisted after adjustment for baseline differences. This analysis challenges the concept that continuous intravenous dobutamine is beneficial to advanced heart failure patients with respect to survival.

Subunit conformation and dynamics in a heterodimeric protein: studies of the hybrid isozyme of creatine kinase.

Several physical properties of creatine kinase (EC 2.7.3.2) isozymes MM (CK-MM, muscle-type) and BB (CK-BB, brain-type), both homodimers, and isozyme MB (CK-MB), a heterodimer, were compared to determine how formation of the hybrid modifies subunit conformation and dynamics. Circular dichroic spectra revealed additional alpha-helical content for the hybrid isozyme. Double-beam absorption difference spectra between CK-MB and a stoichiometric mixture of CK-MM and CK-BB revealed decreased exposure of intrinsic chromophores in the hybrid. The relative intensity of the intrinsic fluorescence of CK-MB was between the two homodimers, but was 16% closer to the less fluorescent CK-MM. Steady state anisotropy spectra and decay of the anisotropy of CK derivatized on a single subunit with the fluorescent sulfhydryl reagent 5-[2-(iodoacetyl)amino-ethyl]aminonaphthalene-1-sulfonate indicated that the derivatized sites are more flexible in the heterodimer. The slow component in the anisotropy decay suggests that hybridization results in a small increase in the packing density or contraction of overall conformation of the B-subunit. The KM for MgATP with singly derivatized CK-MB was the same as the KM for the native enzyme. However, derivatization of a single subunit caused the Vmax to decrease by greater than 50%, which indicates that subunit-subunit interactions may modulate the activity of CK. A model for assembly of CK-MB is proposed which includes subunit characteristics more similar to those found in the muscle-type homodimer than in the brain-type homodimer and increased flexibility of the active site domain of both subunits.

Purification, characterization, and hydrodynamic properties of arginine kinase from gulf shrimp (Penaeus aztecus).

Arginine kinase from the tail muscle of the Gulf shrimp (Penaeus aztecus) was purified to apparent homogeneity, using a rapid, high-yield method. The protein exhibits a molecular weight of 40 kDa according to the methods of gel filtration and gel electrophoresis in sodium dodecyl sulfate, also indicating that arginine kinase from shrimp is a monomer. The amino acid content of arginine kinase from shrimp is similar to arginine kinases from several species and to creatine kinase from rabbit muscle. Arginine kinase derivatized at the reactive sulfhydryl with 2-(4'-(iodoacetamido)anilino)naphthalene-6-sulfonic acid exhibits significant changes in fluorescence anisotropy only in the presence of the guanidino substrate and the so-called "dead-end complex" containing arginine + MgADP. Several compounds structurally similar to arginine, e.g., ornithine do not interact with arginine kinase, suggesting a narrow specificity for substrate binding. The most suitable description of the decay of the fluorescence of arginine kinase derivatized with 5-[(((acetyl)-amino)ethyl)amino]naphthalene-1-sulfonate (AEDANS-AK), from among discrete and distributed models, is a triple exponential discrete decay. The presence of the dead-end complex only marginally increases the rate of decay, but significantly shifts the magnitude of the preexponentials (amplitudes) between the two major decay components. One interpretation of these results is that multiple conformational isomers may occur, in which the relative concentrations are dependent upon the presence of the dead-end complex. Measurement of the time-dependent anisotropy decay of AEDANS-AK reveals a two-term decay law with rotational correlation times of 0.88 and 15.2 ns. The slower component is close to the theoretical value of 16.7 ns for an isotropic rotator of the molecular mass of arginine kinase. This finding suggests that the overall conformation of arginine kinase may differ considerably from the prolate ellipsoidal subunits of the functionally analogous creatine kinase.

Denaturation and urea gradient gel electrophoresis of arginine kinase: evidence for a collapsed-state conformation.

The unfolding transition of monomeric arginine kinase from shrimp was examined in a multiparameter equilibrium approach. Parameters investigated included catalytic activity, circular dichroism, intrinsic fluorescence characteristics including acrylamide quenching, and steady-state anisotropy of arginine kinase derivatized at the reactive cysteine with fluorescent dye 5-[[[(iodoacetyl)amine]ethyl]amino]-naphthalene-1-sulfonic acid. The time course of electrophoretic patterns in urea gradient gels was also determined. Midpoints of the transitions varied considerably depending upon the parameter, indicating the presence of populated intermediates. Significant unfolding began after 2 M urea with most secondary and tertiary structure eliminated in 5 M urea. In dilute denaturant, arginine kinase exhibited a small increase in specific activity and physical properties characteristic of a protein with collapsed structure, including an increase in alpha-helical content, a decrease in intrinsic fluorescence (without a shift in the emission maximum), an increase in anisotropy, and a decrease in fractional accessibility by tryptophan to acrylamide quenching. The electrophoretic pattern of arginine kinase in urea gradient gels is consistent with the presence of a compact conformation in dilute denaturant. The results indicate the existence of a contracted overall conformation in dilute urea. The persistence of catalytic activity suggests this structure may be a functional molecular isoform, but the obvious differences in structure between the native state and the conformation of arginine kinase in 0.5 M urea raise the question of whether such isoforms may also be a type of folding intermediate.

Safety and efficacy of epoprostenol in patients with severe congestive heart failure. Epoprostenol Multicenter Research Group.

Patients with advanced heart failure often remain severely symptomatic and have a high mortality rate despite currently available therapy. We studied the safety and efficacy of a new approach to the patient with refractory heart failure: continuous intravenous treatment via a portable infusion pump with epoprostenol (prostacyclin), a potent pulmonary and systemic vasodilator. A group of 33 patients with severe heart failure (64% New York Heart Association class IV and 36% class III) and profound ventricular dysfunction (median left ventricular ejection fraction, 0.15)--despite prior treatment with diuretics (100%), digitalis (91%), angiotensin-converting enzyme inhibitors (85%), and dobutamine (30%)--underwent a baseline 6-minute walk test prior to dose titration with epoprostenol during invasive hemodynamic monitoring. Subjects responding during the dose titration were randomized, on an open basis, to receive either continuous epoprostenol infusion via an indwelling central venous catheter plus conventional therapy or conventional therapy alone for 12 weeks. The initial dose-ranging study with epoprostenol produced a significant decline in systemic and pulmonary vascular resistance and a substantial increase in cardiac index despite a fall in pulmonary capillary wedge pressure. Symptoms related to vasodilation were noted within the first week after randomization to epoprostenol in 9 of 16 patients but resolved with adjustment of the infusion and concomitant medications in all but one subject. Dose adjustments during the chronic epoprostenol infusion were infrequent after the first week and complications related to the drug delivery system were rare. The change in distance walked from baseline to the last available 6-minute walk test was significantly greater in patients who received epoprostenol compared with patients assigned to standard therapy (72 +/- 40 vs -39 +/- 32 m, mean +/- SEM; p = 0.033). Our study suggests that long-term intravenous infusion of epoprostenol is feasible in patients with severe heart failure and our hemodynamic and functional results suggest clinical benefit as well. However, until recent results indicating an adverse effect of epoprostenol on survival are fully evaluated, the role of this drug in the treatment of advanced heart failure will remain uncertain.

An equilibrium study of the dependence of secondary and tertiary structure of creatine kinase on subunit association.

Several physical properties of dimeric creatine kinase in increasing concentrations of guanidine hydrochloride (GDN/HCl) were evaluated and correlated with degree of subunit dissociation, determined by isozyme competitive hybridization. Three distinct stages were observed that correlated with phases before, during and after dissociation. In 0.2 M GDN/HCl, before significant dimer dissociation, creatinine kinase has 75% of its original activity, and exhibits only small decreases in circular dichroism, intrinsic fluorescence and emission maximum. The spectral characteristics of creatine kinase (CK) derivatized with 5-[(((2-iodoacetyl)amino)ethyl)amino]naphthalene-1- sulfonate (AEDANS-CK) at the cysteine near the active site are suggestive of a slightly more non-polar environment. A decrease in steady-state anisotropy (0.140 to 0.132) was characterized by time-resolved methods. The slow component of the time-resolved anisotropy decay law, which reflects global protein rotation, is decreased only from 36.6 to 33.4 ns. The faster component decreases from 1.95 to 0.74 ns which suggests the active-site domain is more sensitive to conformation perturbation than the protein as a whole. Overall these observations suggest the subunits within the dimeric state are rather stable in dilute denaturant, but undergo a minor contraction in conformation. The region of the active site, as reported by the extrinsic fluorophore, is less polar but apparently more flexible in dilute denaturant. Between 0.5 M and 1 M GDN/HCl, most of the dimers dissociate, 63% of helical content is lost and inactivation is complete. The intrinsic fluorescence shifts 8 nm to the red and increases by 35%, indicating exposure of tryptophans to solvent and release of quenching, perhaps between residues on separate subunits. Over the same denaturant range, the spectral characteristics and lifetime of AEDANS-CK suggests less exposure of the active site to solvent. Time-resolved anisotropy measurements show that the sharp decrease in steady-state anisotropy to 0.086 is due to a decrease in macromolecular rotation to 22 ns. This may represent the rotational correlation time of a relatively intact subunit, and suggests limited subunit unfolding accompanying dissociation. Dissociation is complete in 1.5 M GDN/HCl. The subunits still retain 20% helical content in 2 M denaturant and not until 5 M GDN/HCl is all helical structure eliminated. Above 2 M GDN/HCl, AEDANS-CK exhibits sharp decreases in steady-state anisotropy, fluorescence lifetime and the long-lived component in the time-resolved anisotropy decay law. These results reveal a catastrophic loss of tertiary structure by the subunits and may define the physical properties of the random coil.

Effect of oxypurinol on renal reperfusion injury in the rat.

Oxygen-based free radicals produced by the enzyme xanthine oxidase may be involved in postischemic reperfusion injury. To determine whether oxypurinol, a xanthine oxidase inhibitor and the major metabolite of allopurinol, attenuates renal ischemic reperfusion injury, and, if so, to determine its most effective dose, oxypurinol 2.5, 5, 10 or 20 mg/kg BW was infused 20 min prior to 20 min of complete renal ischemia in uniephrectomized rats. Animals treated with 5 mg/kg BW oxypurinol had significantly higher creatinine clearances on the first and second days postischemia than did untreated animals. In other animals given either buffered saline or oxypurinol at 5 mg/kg BW i.v. 20 min before ischemia, the inulin clearance (CIn) returned to near-control values within 1 h after ischemia. At 24 h there was a secondary decline in the CIn in animals receiving buffered saline, whereas in the animals treated with oxypurinol, this decline was less evident. In animals given oxypurinol at 5 mg/kg BW 40 min after ischemia, the CIn was significantly greater than in those receiving buffered saline. No changes in renal blood flow or renal vascular resistance were observed, suggesting that the effect of oxypurinol was not hemodynamically mediated. Analysis of plasma hypoxanthine, xanthine, uric acid and oxypurinol levels by high-pressure liquid chromatography revealed that in the absence of oxypurinol, a significant increase in uric acid production occurred between 20 and 170 min after the period of ischemia. In the presence of oxypurinol, there was a marked reduction in the rate of production of uric acid for the first 3 h postischemia.(ABSTRACT TRUNCATED AT 250 WORDS)

Heterogeneous flexibilities of the active site domains of homodimeric creatine kinase: effect of substrate.

(1) A single subunit and both subunits of creatine kinase from rabbit muscle was derivatized at the active site with the thiol-specific reagent 2-(4'-(iodoacetamido)anilino)-naphthalene-6-sulfonic acid. (2) The highly biphasic kinetics of the labelling reaction were characterized from measurements of activity, steady-state fluorescence and anisotropy. Derivatization of one thiol and both thiols resulted in 48 and 100% inhibition, respectively. The dead-end complex (DEC), consisting of creatine, MgADP and protein, inhibited the rate, but not the extent, of derivatization and resulted in a 2-fold increase in fluorescence. (3) The fluorescence of singlylabelled (1AANS/CK) and doublylabelled (2AANS/CK) protein exhibited three discrete lifetime components or a two-term Lorentzian distribution. The decay laws for both preparations were not remarkably different, except that, unlike 1AANS/CK, the longer decay component of 2AANS/CK was distributed, which narrowed in the presence of the DEC. (4) The steady-state anisotropies of 1AANS/CK and 2AANS/CK at 25 degrees C were 0.305 and 0.240, respectively. It was concluded that the fast reacting site was immobile and the slow reacting site was flexible. Kinetics of labelling and anisotropy emission spectra indicated that the DEC immobilized the flexible site. (5) The anisotropy decay of 1AANS/CK with and without the DEC was described by a rotational correlation time of about 50 ns, characteristic of the molecular rotation of the CK dimer. At least two terms were required to fit the data for 2AANS/CK, indicating additional segmental motion which was eliminated upon formation of the DEC. (6) Energy transfer from tryptophans to AANS indicated movement of approx. 3 A accompanying formation of the DEC.

Conformational heterogeneity of creatine kinase determined from phase resolved fluorometry.

Fluorescence lifetimes of dimeric rabbit muscle creatine kinase specifically dansylated at both active sites and the homologous monomeric lobster muscle arginine kinase singly dansylated were determined using phase-modulation methods with global analysis of overdetermined data sets. For both proteins, the data is adequately described by three discrete exponential decays or a Lorentzian double distributed decay. Analogue phase resolved spectroscopy also reveals the presence of at least two distinct fluorophore domains for the dansyl moieties of creatine kinase. The model fluorophore, dansyllysine, exhibits a monoexponential decay with a value that is highly solvent dependent. Because the monomeric arginine kinase exhibits essentially the same decay law as doubly derivatized dimeric creatine kinase, it is proposed that the multiple lifetimes of creatine kinase reflect two or more isomeric dimeric states and not subunit asymmetry within a conformationally homogeneous dimeric population. Exposure of arginine kinase to 6 M guanidinium chloride results in a shift to shorter lifetimes and narrowing of the lifetime distributions. Creatine kinase displays a small narrowing of the distribution, but little change in fractional populations or lifetimes. These results suggest the presence of structural elements resistant to denaturation. The longest lifetime component in the triexponential discrete decay law of doubly dansylated creatine kinase is totally unquenched by acrylamide, whereas the two shorter lifetime components exhibit limited dynamic quenching. Steady-state quenching by acrylamide is significant and reveals a sharp distinction between accessible and non accessible dansyl groups. The major mechanism for interaction between the dansyl moieties and acrylamide is, atypically, static quenching. The results are consistent with two dansyl domains, one accessible and hydrophilic according to lifetime values and the other inaccessible and hydrophobic in solvent characteristics.Energy transfer between the dansyl group and the eight tryptophan residues of dimeric creatine kinase give similar results(~ 35%) from measurements of lifetimes, steady-state donor quenching and sensitized acceptor emission. The similarity suggests that the overall flexibility of the dimeric protein is limited. The occurrence of multiple conformers of muscle creatine kinase provides an explanation for several previous observations, most notably the structural origins for compartmentation of the muscle isozyme observed in the myofibril.

A physicochemical comparison of the isozymes of creatine kinase from rabbit brain and muscle.

A comparison of specific structural features of creatine kinase from rabbit muscle and brain was undertaken to determine if the observed isozyme specific differences in catalytic cooperativity are related to conformational differences, particularly differences in packing density. The intrinsic fluorescence of the brain isozyme is 2-fold higher than the muscle isozyme. In the denatured state, both proteins display the characteristic red shift in emission maximum; however, the emission intensity of the brain isozyme increases only 5% upon denaturation compared to nearly 100% increase for the muscle protein. The fluorescence lifetimes are 2.65 ns (67%) and 0.48 ns for native muscle enzyme and 4.38 ns (65%) and 0.80 ns for brain enzyme. Upon denaturation, the lifetimes are 3.98 ns (77%) and 0.99 ns for muscle protein and 3.82 ns (79%) and 0.86 ns for brain protein. Stern-Volmer plots of quenching by acrylamide are essentially the same for both native isozymes indicating that the differences of the intrinsic fluorescence of the native proteins are not due to differences in solvent accessibility. The spectral and lifetime differences in the isozymes in the native state and changes accompanying denaturation are consistent with the occurrence of energy transfer in native muscle isozyme. The rotational correlation times of 5-[2-(iodoacetyl)aminoethyl]aminonaphthalene-1-sulfonate conjugated proteins, derivatized at the active site reactive thiol, are best described by two term decay laws. The slower rotations, 45.1 ns (75%) and 40.6 ns (71%) reflect overall macromolecular rotation for the muscle and brain isozymes, respectively. The faster motions, 2.4 ns for muscle isozyme and 0.4 ns for the brain isozyme, are attributed to the probe or probe associated segmental motions and indicate these motions are more restricted in the muscle protein. Reactivity of creatine kinase (2.5-10 microM) with the amino-specific reagent trinitrobenzene sulfonate (0.4-2 mM) was analyzed by pseudo-first-order and second order models, neither of which was adequate for the entire range of data. However, in every case, the rate constants were faster for brain creatine kinase but the extent of reaction was greater for muscle creatine kinase. The faster initial reactivity of the brain isozyme is consistent with greater accessibility for lysine derivatization.(ABSTRACT TRUNCATED AT 400 WORDS)

Resonance energy transfer between the active sites of creatine kinase from rabbit brain.

Resonance energy transfer was measured between the active site domains of the brain isozyme of creatine kinase (CK-BB). The reactive thiol near the active sites, one on each subunit of the dimeric protein, was derivatized using 5-[2-[iodoacetyl)amino)ethyl]aminonaphthalene-1-sulfonic acid (AED), 2-[4'-iodoacetamidoanilino]naphthalene-6-sulfonic acid (AANS) and 5-iodoacetamidofluorescein (AF). Suitable donor/acceptor protein conjugated hybrids were prepared by controlled kinetics producing CK-BB-AED/AF and CK-BB-AANS/AF. Transfer efficiencies, measured from the quenching of the donor lifetime and steady-state sensitized acceptor emission, ranged from 0.10 to 0.17. From determination of the donor/acceptor overlap integrals, donor quantum yields and attempts to delimit the orientation factor using steady-state and phase-resolved anisotropy measurements, it was found that a suitable estimate of the range between the active sites was between 45 and 57 A. This range is similar to that reported previously for the muscle isozyme of creatine kinase (Grossman, S.H. (1989) Biochemistry 28, 4894-4902) but is a significantly greater distance than detected for the hybrid, myocardial specific isozyme (Grossman, S.H. (1983) Biochemistry 22, 5369-5375).

Pharmacokinetics of oral and transdermal triprolidine.

In this open, nonrandomized, three-way crossover study, six healthy male volunteers received single doses of triprolidine (TPL) hydrochloride syrup orally (2.5 mg) and wore transdermal TPL patches (5 mg and 10 mg doses) to compare the pharmacokinetic profiles and dose tolerance of the two formulations. A washout period of at least 1 week was scheduled between the three dosing periods. Blood samples were collected at defined times, and plasma concentrations were determined using a radioimmunoassay. Maximum plasma drug concentration (Cmax) decreased from 5.6 +/- 2.9 ng/mL (mean +/- SD) with oral dosing to 2.0 +/- 1.0 ng/mL and 4.2 +/- 2.0 ng/mL following 5 mg and 10 mg transdermal doses, respectively. Time to reach peak concentration (tmax) increased from 2.0 +/- 1.2 hours with oral dosing to 12.0 +/- 5.9 and 14.3 +/- 9.9 hours following 5 mg and 10 mg transdermal doses, respectively. The differences between AUC0-alpha values with the oral syrup and the 5 mg and 10 mg transdermal doses were not significant when normalized to 2.09 mg (TPL base). The bioavailabilities of the 5 mg and 10 mg transdermal doses relative to the oral 2.09 mg doses were 0.89 +/- 0.32 and 1.04 +/- 0.33, respectively. Mild erythema and pruritus were the most common adverse effects secondary to TPL transdermal application. Drowsiness observed following oral TPL, was not evident following either transdermal dose. The results of this study, therefore, indicate that TPL can be absorbed transdermally, providing consistent plasma concentrations.

Resonance energy transfer between the active sites of rabbit muscle creatine kinase: analysis by steady-state and time-resolved fluorescence.

Resonance energy transfer between the reactive thiols of rabbit muscle creatine kinase was evaluated. The reactive thiols are located at the active site, one occurring on each subunit of the dimeric protein that is known to be a constituent of the M-line structure of the myofibril. Transfer efficiency was evaluated from energy donor quenching of fluorescence by steady-state and phase-modulation lifetime measurements and determination of sensitized emission of the acceptor. Several sulfhydryl-specific donor fluorophores were used including 5-[[[(iodoacetyl)amino]ethyl]amino]naphthalene-1-sulfonic acid, 7-(dimethylamino)-3-(4-maleimidylphenyl)-4-methylcoumarin, and 2-[4-(iodoacetamido)anilino]naphthalene-6-sulfonic acid (IAANS). Energy transfer acceptors included 5-(iodoacetamido)fluorescein and the nonfluorescent dye [4-[[4-(dimethylamino)phenyl]azo]phenyl]iodoacetamide. In order to prepare the necessary homodimer labeled with both donor and acceptor, advantage was taken of the biphasic reaction between creatine kinase and IAANS. In some instances, donor/acceptor hybrids were prepared by denaturation/renaturation procedures, and possible deviations from expected hybridization stoichiometry were considered. Disproportionation of singly labeled dimers (to unlabeled and doubly labeled dimers) was not observed when the brain isozyme of creatine kinase was used to trap dissociated dye-conjugated or unlabeled muscle-type subunits of creatine kinase. From studies of five different donor/acceptor combinations, the efficiency of energy transfer was found to occur over a range of 5-14%, indicating that the reactive thiols are well separated. Overlap integrals and quantum yields were evaluated, and estimates of the range of orientation factor were obtained to determine a range for the distance between the active sites of creatine kinase. When the ranges are overlapped, a limited distance of 48.6-60.4 A is obtained.(ABSTRACT TRUNCATED AT 250 WORDS)

Protective effect of prostacyclin on postischemic acute renal failure in the rat.

Infusion of prostacyclin (PGI2) reportedly attenuates renal ischemic injury in the dog and the rat. In the dog, PGI2 is a potent renal vasodilator; in the rat a direct action on the renal vasculature is not always apparent. To determine whether or not the protective effect of PGI2 on postischemic ARF was hemodynamically mediated, studies were performed in uninephrectomized Sprague-Dawley rats before and after a 40 minute period of complete renal artery occlusion. In response to the preischemic infusion of PGI2 for 30 minutes at 160 ng/kg body wt/min i.v. (N = 7), MAP and RBF fell to 86 +/- 7% (P less than 0.0001) and 84 +/- 9% (P less than 0.05) of baseline values, respectively. RVR initially declined to 81 +/- 9% of baseline values (P less than 0.025) but returned to 102 +/- 13% of baseline values prior to the period of ischemia. Following the period of ischemia, reflow of blood in the rats receiving PGI2 was delayed when compared to rats not receiving PGI2 (N = 7). RBF returned to only 76 +/- 19% of the initial values in PGI2-treated rats (P less than 0.01) but to 90 +/- 12% of the initial values in rats receiving buffer alone (NS).(ABSTRACT TRUNCATED AT 250 WORDS)

Interaction of brain-type creatine kinase with its transition state analog: kinetics of inhibition and conformational changes.

The effects of components of the transition state analog (creatine, MgADP, planar anion) on the kinetics and conformation of creatine kinase isozyme BB from monkey brain was studied. From analysis of the reaction time course using the pH stat assay, it was shown that during accumulation of the reaction products (ADP and creatine phosphate), among several anions added, nitrate proved the most effective in inhibiting catalytic activity. Maximum inhibition (77%) was achieved with 50 mM nitrate. The Km for ATP was 0.48 mM and in the presence of 2.5 mM nitrate, 2.2 mM; for ATP in the presence of the dead-end complex, creatine and ADP, the apparent Km was 2.0 mM and the Ki was 0.16 mM; in the presence of the transition state analog, MgADP + NO3- + creatine, the Ki was estimated to be 0.04 mM. Ultraviolet difference spectra of creatine kinase revealed significant differences only in the presence of the complete mixture of the components of the transition state analog. Comparison of gel filtration elution profiles for creatine kinase in the absence and presence of the complete mixture of components of the transition state analog did not reveal any differences in elution volume. Addition of components of the transition state analog to creatine kinase resulted in only a marginal change in intrinsic fluorescence. The presence of the components of the transition state analog increased the rate of reactivity of the enzyme with trinitrobenzenesulfonic acid from k = 6.06 +/- 0.05 M-1 min-1 to 6.96 +/- 0.11 M-1 min-1.(ABSTRACT TRUNCATED AT 250 WORDS)

An analysis of the reassembly of denatured creatine kinase from monkey brain.

Creatine kinase isolated from monkey brain was characterized with respect to denaturation/inactivation and renaturation/reactivation/reassociation in order to determine the mechanism of reassembly. Enzyme unfolded in 8 M urea exhibits several characteristics of denatured protein: complete loss of enzymatic activity, decrease in intrinsic fluorescence with a red shift in the emission maximum and loss of circular dichroism at 220 nm. The renatured protein reassembles to its apparently native condition as judged by these criteria, but small differences of uncertain origin persist. Dependence of activity and fluorescence on denaturant concentration indicate that inactivation is more sensitive to urea than is unfolding; spectral changes at the intermediate urea concentrations suggest formation of aggregated protein. Upon dilution, enzyme previously exposed to 8 M urea for 40 min regains 70-80% native activity, independent of protein concentration over the range of 0.56-160 nM. Reactivation kinetics, measured using the assay mixture with and without trypsin, are independent of protein concentration, and are adequately described by a single rate constant, 3.2 X 10(-3) s-1 and 4.2 X 10(-3) s-1, respectively. Reactivation is completed 20-30 min after initiation of renaturation. Fluorescence changes during refolding are at least biphasic, exhibiting a rapid increase, then a slow decrease completed at approximately 15-20 min after initiating refolding. Reassociation is measured by competitive hybridization between dimerizing B subunits and M subunits to form MB heterodimer. The time dependent decay in heterodimer formation during competitive dimerization shows that reassociation is completed between 60 and 90 min after initiation of reassembly. These results indicate that the brain isozyme of creatine kinase, like the muscle form, is composed of subunits which do not require association for expression of catalytic activity. Furthermore, a comparison of spectral data and susceptibility to trypsin inactivation between the muscle and brain isozymes supports previous suggestions that in the native state, the brain isozyme is a conformationally looser, more open protein.

Purification and characterization of creatine kinase isozymes from the nurse shark Ginglymostoma cirratum.

Creatine kinase from nurse shark brain and muscle has been purified to apparent homogeneity. In contrast to creatine kinases from most other vertebrate species, the muscle isozyme and the brain isozyme from nurse shark migrate closely in electrophoresis and, unusually, the muscle isozyme is anodal to the brain isozyme. The isoelectric points are 5.3 and 6.2 for the muscle and brain isozymes, respectively. The purified brain preparation also contains a second active protein with pI 6.0. The amino acid content of the muscle isozyme is compared with other isozymes of creatine kinase using the Metzger Difference Index as an estimation of compositional relatedness. All comparisons show a high degree of compositional similarity including arginine kinase from lobster muscle. The muscle isozyme is marginally more resistant to temperature inactivation than the brain isozyme; the muscle protein does not exhibit unusual stability towards high concentrations of urea. Kinetic analysis of the muscle isozyme reveals Michaelis constants of 1.6 mM MgATP, 12 mM creatine, 1.2 mM MgADP and 50 mM creatine phosphate. Dissociation constants for the same substrate from the binary and ternary enzyme-substrate complex do not differ significantly, indicating limited cooperatively in substrate binding. Enzyme activity is inhibited by small planar anions, most severely by nitrate. Shark muscle creatine kinase hybridizes in vitro with rabbit muscle or monkey brain creatine kinase; shark brain isozyme hybridizes with monkey brain or rabbit brain creatine kinase. Shark muscle and shark brain isozymes, under a wide range of conditions, failed to produce a detectable hybrid.

Further characterization of the reassembly of creatine kinase and effect of substrate.

Upon exposure to 8 M urea, creatine kinase from rabbit muscle exhibited a rapid increase in intrinsic fluorescence and a rapid decrease in fluorescence polarization. Polarization changes were complete after 5 min, while fluorescence changes continued for at least 15 min. Fluorescence polarization changes accompanying reassembly were complex, and appeared to involve a concentration dependent reaction. Enzyme sampled at intervals during denaturation exhibited refolding kinetics displaying two first-order rate constants, the first dependent and the second independent of the duration of exposure to urea. There was evidence for an additional renaturation step, occurring within the mixing phase of the denatured protein with solvent. Reactivation kinetics and yield of reactivated enzyme exhibited a dependency upon length of exposure to denaturant. The exposure of renaturing creatine kinase to trypsin was shown to prevent further reactivation, and provided use of a method to determine reactivation rates at discrete intervals after initiation of reassembly. The presence of 2 mM MgADP during reactivation enhanced the rate of reactivation immediately after initiation of reactivation. Reactivation was not accelerated if nucleotide substrate was added after reactivation was initiated nor did nucleotide substrate increase the overall reactivation yield. The presence of MgADP also enhanced the rate of refolding at an early stage as judged by changes in intrinsic fluorescence and resistance to tryptic hydrolysis. While in addition to MgADP, creatine phosphate accelerated resistance by refolding creatine kinase to trypsin, according to the other criteria measured, the phosphagen substrates did not promote reactivation or renaturation. The unfolding-refolding studies and role of substrate in reassembly were consistent with a mechanism involving at least two steps, possibly involving cis-trans isomerization of proline. These data also supported the suggestion that the formation of the nucleotide binding region is an early event in the refolding of creatine kinase in vitro.

The effects of age and smoking on the plasma protein binding of lignocaine and diazepam.

In 63 healthy ambulant subjects 18 to 88 years of age, the plasma protein binding of diazepam (principally bound to albumin) decreased with age. Diazepam binding in plasma correlated positively with plasma albumin concentration which also decreased with age. In contrast, the plasma protein binding of the basic drug, lignocaine (predominantly bound to alpha 1-acid glycoprotein [AAG]), tended to increase slightly with age. Lignocaine binding in plasma correlated positively with plasma AAG concentration which also increased slightly with age. Smoking did not affect the plasma protein binding of diazepam or lignocaine or the plasma concentrations of albumin, AAG or nonesterified fatty acids. These results suggest that age-related changes in plasma protein binding of lignocaine and diazepam are determined in part by age-related changes in the concentrations of the binding proteins in plasma. The ageing process alone causes only small changes in the plasma protein binding of these drugs compared with the effect of disease states, however.