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)     

Rate equation for creatine kinase predicts the in vivo reaction velocity: 31P NMR surface coil studies in brain, heart, and skeletal muscle of the living rat

Brain, heart, and skeletal muscle contain four different creatine kinase isozymes and various concentrations of substrates for the creatine kinase reaction. To identify if the velocity of the creatine kinase reaction under cellular conditions is regulated by enzyme activity and substrate concentrations as predicted by the rate equation, we used 31P NMR and spectrophotometric techniques to measure reaction velocity, enzyme content, isozyme distribution, and concentrations of substrates in brain, heart, and skeletal muscle of living rat under basal or resting conditions. The total tissue activity of creatine kinase in the direction of MgATP synthesis provided an estimate for Vmax (23.4 +/- 2.8, 62.4 +/- 4.5, and 224 +/- 16 mM/s) and exceeded the NMR-determined in vivo reaction velocities by an order of magnitude (4.1 +/- 1.2, 5.1 +/- 1.6, and 18.4 +/- 2.4 mM/s for brain, heart, and skeletal muscle, respectively). The isozyme composition varied among the three tissues: greater than 99% BB for brain; 14% MB, 61% MM, and 25% mitochondrial for heart; and 98% MM and 2% mitochondrial for skeletal muscle. The NMR-determined reaction velocities agreed with predicted values from the creatine kinase rate equation (r2 = 0.98; p less than 0.001). The concentrations of free creatine and cytosolic MgADP, being less than or equal to the dissociation constants for each isozyme, were dominant terms in the creatine kinase rate equation for predicting the in vivo reaction velocity. Thus, we observed that the velocity of the creatine kinase reaction is regulated by total tissue enzyme activity and by the concentrations of creatine and MgADP in a manner that is independent of isozyme distribution.     

Enzyme termini of a phosphocreatine shuttle. Purification and characterization of two creatine kinase isozymes from sea urchin sperm

Two isozymes of creatine kinase have been purified from sperm of the sea urchin, Strongylocentrotus purpuratus. One isozyme was purified from the sperm flagellum, and the other from the head. Both require nonionic detergent for extraction from sperm. The flagellar isozyme is a monomeric species with an Mr of 145,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 126,000 from sucrose density gradient and gel filtration analyses. Creatine kinase from sperm heads was localized to the mitochondrion by an antibody raised against mouse muscle creatine kinase. This purified mitochondrial isozyme is multimeric, with an Mr of 47,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but 240,000 for the native enzyme. Peptide mapping indicates that the two isozymes are not related. The following kinetic characteristics were observed for the purified flagellar and mitochondrial isozymes, respectively. In the direction of ATP formation, at pH 6.6 and 25 degrees C, specific activities were 235 and 180 units/mg; pH optima were 6.7 and 6.9 and Michaelis constants were 0.13 and 0.055 mM for ADP and 5.8 and 2.7 mM for phosphocreatine. In the direction of phosphocreatine formation, at pH 7.5 and 25 degrees C, specific activities were 29 and 47 units/mg; pH optima were 7.5 and 7.7 and Michaelis constants were 0.89 and 0.31 mM for ATP and 39 and 62 mM for creatine. These unique isozymes constitute the termini of the phosphocreatine shuttle of sea urchin sperm that is responsible for energy transport from the mitochondrion to the distal flagellum (Tombes, R. M., and Shapiro, B. M. (1985) Cell 41, 325-334; Tombes, R. M., Brokaw, C. J., and Shapiro, B. M. (1987) Biophys. J., 52, 75-86).     

Purification and cell-free translation of a unique high molecular weight form of the brain isozyme of creatine phosphokinase from mouse

Mouse brain creatine kinase was purified to homogeneity and shown to consist of two polypeptide chains of 50,000 daltons. This protein thus differs in size from all other creatine kinase molecules purified to data including the mouse muscle enzyme which shows a molecular weight between 39,000 and 42,000. The high molecular weight isozyme has been shown to represent the primary translation product of creatine phosphokinase mRNA from mouse brain. The unusual size of this creatine phosphokinase subunit provides unique tools for the study of the differential regulation of creatine kinase gene expression and for the study of subunit interactions in creatine kinase isozymes.     

Creatine kinase isoenzymes in Torpedo californica: absence of the major brain isoenzyme from nicotinic acetylcholine receptor membranes

Creatine kinase, actin, and nu 1 are three proteins of Mr 43 000 associated with membranes from electric organ highly enriched in nicotinic acetylcholine receptor. High levels of creatine kinase are required to maintain adequate ATP levels, while actin may play a role in maintaining the synaptic cytoskeleton. Previous investigations have prompted the conclusion that postsynaptic specializations at the receptor-enriched membrane domains in electroplax contain the brain form of creatine kinase rather than the form of creatine kinase predominantly found in muscle. We have examined this conclusion by purifying Torpedo brain creatine kinase to virtual homogeneity in order to examine its immunochemical, molecular, and electrophoretic properties. On the basis of immunological cross-reactivity and isozyme analysis, the receptor-associated creatine kinase is identified to be of the muscle type. When the molecular characteristics of Torpedo brain and muscle creatine kinase are compared, the brain enzyme is positioned at a more basic pH during chromatofocusing and on two-dimensional gel electrophoresis (pI = 7.5-7.9). Furthermore, electrophoretic mobilities of the brain and muscle forms of creatine kinase differ in sodium dodecyl sulfate electrophoresis: the brain isozyme of creatine kinase has lower apparent molecular weight (Mr 41 000) when compared with the muscle enzyme (Mr 43 000). On the basis of the results of our current investigations, the hypothesis that the brain isozyme of creatine kinase is a component of the postsynaptic specializations of the Torpedo californica electroplax must be abandoned. Recent sequence data have established close homology between Torpedo and mammalian muscle creatine kinases. On the basis of electrophoretic criteria, our results indicate that a lower degree of homology exists between the brain isozymes.     

Differential distribution of protein kinase C isozymes in the various regions of brain

We have previously identified three types of protein kinase C (a Ca2+-activated phospholipid-dependent kinase) isozymes, designated types I, II, and III, from rat brain (Huang, K.-P., Nakabayashi, H., and Huang, F. L. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 8535-8539). These enzymes are different in their elution profile from hydroxylapatite column, sites of autophosphorylation, and immunoreactivity toward two types of monoclonal antibodies. Now we describe the purification of similar protein kinase C isozymes from monkey brain and their regional distribution in the brain. These primate enzymes all have the same molecular weight of 82,000, and each type of isozyme cross-reacts with the purified monospecific antibodies against its corresponding rat brain counterpart isozyme. These purified antibodies were used to quantify the relative contents of three types of protein kinase C isozymes in various regions of rat and monkey brains. In rat brain, cerebellum contained a high level of the type I isozyme; cerebral cortex, thalamus, and corpus callosum were high in the type II enzyme; and olfactory bulb was highest in the type III enzyme. In monkey brain, the type I isozyme was found to be enriched in cerebellum, hippocampus, and amygdala; the type II enzyme was at very high level in caudate, frontal and motor cerebral cortices, substantia nigra, and thalamus; and the type III enzyme was at the highest level in olfactory bulb. These results indicate that protein kinase C isozymes are differentially distributed in various regions of rat and monkey brains and suggest a unique role for each isozyme in controlling the different neuronal functions in the brain.     

A comparative study of human muscle and brain creatine kinases expressed in Escherichia coli

We report the expression of the human muscle (CK-MM) and brain (CK-BB) creatine kinases in Escherichia coli. The proteins have been purified to apparent homogeneity and several of their physical and kinetic properties investigated. In the process, we have conclusively verified the correct DNA sequence of the genes encoding the respective isozymes, and determined the correct primary structure and mass of the gene products. Alignment of the primary sequences of these two enzymes shows 81% sequence identity with each other, and no obvious gross structural differences. However, Western blot analyses demonstrated the general lack of antigenic cross-reactivity between these isozymes. Preliminary kinetic analyses show the K _m and k _cat values for the creatine and MgATP substrates are similar to values reported for other isozymes from various tissues and organisms. The human muscle and brain CKs do not, however, exhibit the synergism of substrate binding that is observed, for example, in rabbit muscle creatine kinase.     

CREATINE KINASE FROM BRAIN: KINETIC ASPECTS

Abstract— Creatine kinase derived from rabbit brain has been re-examined with respect to its kinetic features. The enzyme from brain has lower Michaelis constants for both ADP and creatine phosphate than does the enzyme from rabbit muscle. Substrate inhibition by excess creatine phosphate occurs at a concentration approximating that found in the tissue. The enzyme from muscle is less sensitive to substrate inhibition.
The crude mitochondrial fraction from rat brain was centrifuged in a sucrose density gradient and the distribution of enzymatic activities among the subfractions was determined. The distribution of creatine kinase resembled that of two glycolytic enzymes; no evidence for a mitochondrial localization was found.     

Purification and characterization of adenylate kinase isozymes from rat muscle and liver

Isozymes of adenylate kinase (ATP:AMP phosphotransferase, EC 2.7.4.3) were purified from skeletal muscle and liver of rats to essentially homogeneous states by acrylamide gel electrophoresis and sodium dodecyl sulfate gel electrophoresis. The isozyme from muscle was purified by acidification to pH 5.0, and column chromatography on phosphocellulose, Sephadex G-75 and Blue Sepharose CL-6B, while that from liver was purified by column chromatography on Blue Sepharose CL-6B, Sephadex G-75 and carboxymethyl cellulose. By these procedures the muscle isozyme was purified about 530-fold in 29% yield, and the liver isozyme about 3600-fold in 27% yield from the respective tissue extracts. The molecular weights of the muscle and liver isozymes were estimated as about 23 500 and 30 500, respectively, by both sodium dodecyl sulfate gel electrophoresis and molecular sieve chromatography, and no subunit of either isozyme was detected. The isoelectric points of the muscle and liver isozymes were 7.0 and 8.1, respectively. The Km values of the respective enzymes for ATP and ADP were similar, but the Km(AMP) of the liver isozyme was about one-fifth of that of the muscle isozyme. Immunological studies with rabbit antiserum against the rat muscle isozyme showed that the muscle isozyme was abundant in muscle, heart and brain, while the liver isozyme was abundant in liver and kidney.     

Studies on adenosine triphosphate transphosphorylases. XVII. A physicochemical comparison of the ATP-creatine transphosphorylase (creatine kinase) isozymes from man,calf, and rabbit

All of the creatine kinase isozymes from human, calf, and rabbit brain and muscle are composed of two noncovalently linked polypeptide chains, based upon sedimentation equilibrium analyses in the presence and absence of disruptive agents. The brain-type isozymes of man, calf, and rabbit proved to be slightly heavier than the muscle types. Various physicochemical properties of the isozymes are recorded. Each group of isozymes, i.e., the muscle, hybrid (muscle-brain), and brain isozymes from man, calf, and rabbit, showed similar electrophoretic behavior, although isoelectric points were not precisely identical for the muscle and hybrid types. Theoretical titration curves constructed from amino acid compositions of the calf isozymes showed reasonable agreement between their calculated and measuredpI ₀ values (isoelectric point extrapolated to zero ionic strength). The three native muscle isozymes and brain isozymes all contain two reactive sulfhydryl groups per mole or one per polypeptide chain of their two-chain proteins, which may be titrated with 5,5′-dithiobis (2-nitrobenzoic acid); and under acidic conditions, quantitative titrations with 4,4′-dithiodipyridine yield a total of ten- SH groups per mole of each brain-type and eight- SH groups per mole of muscle-type isozyme in the case of man, calf, and rabbit. A comparison of their amino acid compositions and tryptic peptide maps shows that there is only a slightly greater degree of homology between the individual isozymes of the same type (muscle type or brain type) than between the muscle- and brain-type isozymes of the same species.     

Discrete subcellular localization of a cytoplasmic and a mitochondrial isozyme of creatine kinase in intestinal epithelial cells

Two isozymes of creatine kinase have been purified differentially from mitochondrial and cytoplasmic subfractions of intestinal epithelial cells. These intestinal epithelial cell creatine kinases were indistinguishable from the cytoplasmic (B-CK) and mitochondrial (Mi-CK) creatine kinase isozymes of brain when compared by SDS-PAGE, cellulose polyacetate electrophoresis, and peptide mapping. In intestinal epithelial cells, immunolocalization of the Mi-CK isozyme indicates that it is associated with long, thin mitochondria, which are excluded from the brush border at the apical end of each cell. In contrast, immunolocalization of the B-CK isozyme indicates that it is concentrated distinctly in the brush border terminal web domain. Although absent from the microvilli, B-CK also is distributed diffusely throughout the cytoplasm. Terminal web localization of B-CK was maintained in glycerol-permeabilized cells and in isolated brush borders, indicating that B-CK binds to the brush border structure. The abundance and localization of the mitochondrial and cytoplasmic creatine kinase isozymes suggest that they are part of a system that temporally and/or spatially buffers dynamic energy requirements of intestinal epithelial cells.     

N-terminal sequence of creatine kinase from skeletal muscle of rabbit and rhesus monkey

The first 20 amino acids from the N-terminus of skeletal muscle (MM) creatine kinase from both rabbit and rhesus monkey have been identified and these sequences show considerable homology. Contrary to an earlier report, the N-terminus was not found to be blocked. Both of these sequences show much less homology with the N-terminal sequence of heart muscle (MM) creatine kinase and no homology with that of the heart muscle mitochondrial (MiMi) isozyme. No homology was found between the N-terminal sequence of the mitochondrial isozyme and the URF (unidentified reading frame) proteins of the human mitochondrial genome, indicating that the mitochondrial enzyme is encoded by nuclear genes. This suggests the possibility that an N-terminal peptide may be cleaved from the mitochondrial isozyme on its translocation across the mitochondrial membrane.     

Is the subunit the minimal function unit of creatine kinase?

The dimeric rabbit muscle isozyme of creatine kinase (MM) is modified by iodoacetamide to produce the inactive dimer (M'M') and then hybridized with native dimeric brain isozyme (BB). The hybrid enzyme (M'B), as isolated by PAGE, has the same Km for both ATP and creatine but half the specific activity of the brain isozyme (BB). Likewise, the hybrid of the modified brain with the native muscle isozyme (MB') has half the activity of the native muscle enzyme. The M'B, MB' and MB hybrid dimers all have essentially the same electrophoretic properties, and their intrinsic fluorescence and CD spectra in the far-ultraviolet region are very similar to those of the homodimers MM and BB. Similar results were obtained for the hybrid (M"B) containing the muscle enzyme subunit modified at both the thiol group with iodoacetamide and the Trp residue with dimethyl(2-hydroxy-5-nitrobenzyl)sulfonium bromide and the native brain enzyme submit. The above results suggest strongly the independent catalytic function of the subunit of creatine kinase.     

Interaction of Brain-Type Creatine Kinase with its Transition State Analog: Kinetics of Inhibition and Conformational Changes

Abstract

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 K_m 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 theK wasO.16mM; in the presence of the transition state analog, MgADP + NO₃” + creatine, the K 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 nitration 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 ±0.05M^?1min to 6.96 ± 0.11 M^?1min^?1.

This study provides evidence that, like the muscle isozyme of creatine kinase, the brain form is effectively inhibited by the transition state analog. However, the inhibition is accompanied by small changes in the overall conformation of the protein. This adds to the evidence that the functional differences of the isozymic forms of creatine kinase cannot be attributed to differences in kinetic properties.     

Inactivation of G1ucose-6-Phosphate Dehydrogenase Isozymes from Human and Pig Brain by Aluminum

Prolonged intake of low levels of aluminum from the drinking water has been found to increase the aluminum content in rat brain homogenates and to reduce the activity of hexokinase and glucose-6-phosphate dehydrogenase (G6PD). To determine the interaction of G6PD with aluminum in the brain, we have recently purified two isozymes of G6PD (isozymes I and II) from human and pig brain. Unlike isozyme I, isozyme II also had 6-phosphogluconate dehydrogenase (6-PGD) activity. We report here that G6PD isozymes I and II from human and pig brain purified to apparent homogeneity are inactivated by aluminum. Aluminum did not affect the 6-PGD activity of isozyme II. The aluminum-inactivated enzyme contained 1 mol of aluminum/mol of enzyme subunit. The protein-bound metal ion was not dissociated by exhaustive dialysis at 4 degrees C against 10 mM Tris-HCl (pH 7.0) containing 0.2 mM EDTA. Preincubation of aluminum with citrate, NADP+, EDTA, NaF, ATP, and apotransferrin protected the G6PD isozymes against aluminum inactivation. However, when the G6PD isozymes were completely inactivated by aluminum, only citrate, NaF, and apotransferrin restored the enzyme activity. The dissociation constants for the enzyme-aluminum complex of the isozymes varied from 2 to 4 microM, as measured by using NaF, a known chelator for aluminum. Inhibition of G6PD by low levels of aluminum further strengthens the suggested role of aluminum toxicity in the energy metabolism of the brain.     

Amino-terminal sequence analysis of rat heart and muscle glycogen synthase: homology to the rabbit enzyme and the implications for hormonal control

Glycogen synthase was purified from rat heart and muscle and electroblotted from sodium dodecyl sulfate polyacrylamide gels to polyvinylidene difluoride, and the NH2-terminal amino acid sequence was determined. The NH2-terminal amino acid sequence of the enzymes was identical. Further, phosphorylation site 2, a major cyclic AMP-dependent protein kinase recognition site in the rabbit muscle isozyme, is conserved in the rat isozymes suggesting that it serves an important function in hormonal regulation. However, two potentially important differences were observed. Threonine-5 and valine-8 of the rabbit muscle enzyme are serine and methionine residues, respectively, in the rat isozyme, the latter being critical in the analysis of phosphopeptides produced by cyanogen bromide cleavage. These variations may provide a partial explanation for previously observed differences in rat and rabbit phosphopeptide maps.     

The synthesis of 8-(6-aminohexyl)-amino-GMP and its applications as a general ligand in affinity chromatography.

The steady-state kinetic behaviors of the five rabbit adrenal norepinephrine N-methyl transferase isozymes have been compared with particular reference to substrate inhibition patterns. Four distinct substrate inhibition patterns were observed. The E-1 isozyme was not subject to inhibition by either substrate, while the E-2 isozyme was inhibited by both substrates. The E-3 and E-4 isozymes were inhibited by norepinephrine only, while E-5 is inhibited only by S-adenosylmethionine. The substrate inhibition constants were sufficiently small in relation to the Michaelis constants to make substrate inhibition an important factor in regulation of activities of the isozymes.     

Expression of muscle-gene-specific isozymes of phosphorylase and creatine kinase in innervated cultured human muscle

Isozymes of creatine kinase and glycogen phosphorylase are excellent markers of skeletal muscle maturation. In adult innervated muscle only the muscle-gene-specific isozymes are present, whereas aneurally cultured human muscle has predominantly the fetal pattern of isozymes. We have studied the isozyme pattern of human muscle cultured in monolayer and innervated by rat embryo spinal cord explants for 20-42 d. In this culture system, large groups of innervated muscle fibers close to the ventral part of the spinal cord explant continuously contracted. The contractions were reversibly blocked by 1 mM d-tubocurarine. In those innervated fibers, the total activity and the muscle-gene-specific isozymes of both enzymes increased significantly. The amount of muscle-gene-specific isozymes directly correlated with the duration of innervation. Control noninnervated muscle fibers from the same dishes as the innervated fibers remained biochemically immature. This study demonstrated that de novo innervation of human muscle cultured in monolayer exerts a time-related maturational influence that is not mediated by a diffusable neural factor.     

Purification and characterization of cytoplasmic creatine kinase isozymes ofXenopus laevis

The soluble creatine kinase isozymes CK-II, CK-III, and CK-IV fromXenopus laevis have been purified to apparent homogeneity and their subunits characterized by means of molecular weight, peptide pattern, and dissociation-reassociation experiments. CK-III and CK-IV are homodimeric isozymes whose subunits are distinct in both molecular weight (42,000 and 41,000, respectively) andStaphylococcus aureus V₈ peptide pattern. In dissociation-reassociation experiments, those two subunits do form active heterodimeric isozymes with one another or with rabbit M-CK subunits. Hybrid CK-III/IV isozymes occur also during embryonic differentiation and in adult heart muscle, whereas most other adult tissues contain only homodimeric CK-III or CK-IV isozymes. The CK-II isozyme is a heterodimer composed of one CK-III subunit and another subunit specific to CK-II (M _r =41,000). Neitherin vivo norin vitro does this subunit seem able to form homodimers or heterodimers with CK-IV and rabbit M-CK subunits. If we take into account the apparent association of CK-I isozyme with cellular organelles, these results corroborate earlier statements and suggest that the CK isozyme system ofX. laevis is encoded by at least four differentially regulated genomic loci.     

Interaction of Creatine Kinase from Monkey Brain with Substrate: Analysis of Kinetics and Fluorescence Polarization

Abstract: Titrimetric determination of the dissociation constants for the binding of substrates to creatine kinase from monkey brain reveals 13-fold and 4-fold synergism in the forward and reverse directions, respectively. This synergism is expressed as a decrease in the K_D for a given substrate in the ternary complex compared with the binary complex and may be a reflection of substrate-induced conformational change. Creatine kinase labeled with two molecules of 5′-iodoacetamidofluorescein displays a blue shift and a decrease in fluorescence intensity upon binding of MgADP, indicative of movement of the dye into a more hydrophobic environment and quenching of the extrinsic fluorescense. Rotational relaxation times determined from analysis of fluorescence polarization of dansylated brain creatine kinase decrease from 212 ± 7 ns to 189 ± 6 ns upon MgADP binding. Dansylated creatine kinase in 0.5% sodium dodecyl sulfate has a rotational relaxation time of 135 ± 6 ns. The rotational relaxation time of dansylated muscle-type isoenzyme is unaffected by MgADP and has the same value as the brain isoenzyme-MgADP complex. Polarization values at 25°C for muscle and brain enzyme labeled with 3 - (4 - maleimidylphenyl) - 7 - diethylamino - 4 - methylcoumarin compared with limiting polarization and polarization of the free dye suggest that the dye rotation is severely restricted in the muscle form, but possesses freedom of rotation in the brain form. These results support the conclusion that compared with the muscle isoenzyme, the brain isoenzyme is more open at the active site and more flexible overall. Binding of MgADP by brain creatine kinase produces a protein more compact across one or both of its rotational axes, thus resembling the conformation of the muscle isoenzyme. It is probable that creatine kinase in the brain, unlike that from muscle, is subject to kinetic regulation accompanied by conformational modification. This suggests that the neurobiochemical role of the brain isoenzyme is distinct from the metabolic function of the muscle isoenzyme.