properties of creatine kinase-bb from canine and human brain tissues

Properties of Creatine Kinase-BB From Canine and Human Brain Tissues 1

JOAN MILLER 2 and ROBERT WEI 3

Department of Chemistry, Cleveland State University, Cleveland, Ohio 44115

Creatine kinase (EC 2.7.3.2) BB isoenzyme (CK-BB) was purified to homogeneity from canine and human brain tissues. The purified protein from both sources exhibits Mr of 84,700 daltons. The canine isoenzyme exhibits several properties similar to human isoenzyme with respect to reactive and total thiol groups, UV spectra, isoelectric points and reaction kinetics. While both canine and human CK-BB isoenzymes are unstable compared to other CK isoenzymes, canine CK-BB is even less stable than the human enzyme, losing most of its activity within 20 h at 4°C at pH 5.0. Addition of 2-mercaptoethanol does not prevent rapid loss of the enzyme activity. Increasing the pH to 9.0, however, increases the stability of both CK-BB isoenzymes. Agarose electrophoresis demonstrated the presence of MM as well as BB isoenzyme in various parts of brain tissues. BB was present at an activity of 90.8-93.3 U/mg and MM at 6.7-9.2 U/mg.

KEY WORDS: creatine kinase-BB, properties, distribution

p resent in highest concentration in the brain, the BB isoenzyme (CK-BB) of adenosine triphosphate:

creatine N-transphosphorylase, creatine kinase (E.C. 2.7.3.2) has been used clinically to identify neurologic damage (1). This isoenzyme is found also throughout the smooth muscles of gastrointestinal and genitour- inary tracts, and has been studied as a possible diag- nostic marker in certain neoplastic diseases (2). It has been obtained in various stages of purity from the brain tissues of chickens (3), pigs (4), rabbits (3), and humans (5). Compared to the other isoenzymes, CK-BB has not been well characterized, possibly due to its instability or possibly because the enzyme is present in the brain at much lower activities than CK-MM in skeletal muscle or CK-MB in cardiac tissues. CK-BB, like other isoenzymes, is a dimeric protein with molecular weight in the range of 78,000-85,000 daltons (6). In this work, a simplified procedure developed for preparing homogeneous CK-BB from canine and human brains has facilitated their characterization.

Materials and methods

CK-BB PURIFICATION SCHEME

All procedures were performed in a cold room maintained at 4°C. All buffers contained 2 mmol/L

1Portions of this paper are included in a dissertation sub- mitted to the Graduate College of Cleveland State University by Joan Miller in partial fulfillment of the requirements for the degree of Doctor of Philosophy.

2present address: New England Pathology Services, 330 Cummings Park, Woburn, MA 01801.

aCorrespondence: Dr. Robert Wei. Manuscript received April 28, 1984; revised manuscript

received July 18, 1984; accepted for publication August 17, 1984.

2-mercaptoethanol. Approximately 100 g of brain tissue was homogenized in a Waring single speed blender using 10-15 s bursts in a 2-3 fold volume of 10 mmol/L KC1. The homogenate was gently stirred for 2 h and the enzyme contained in the supernatant recovered by centrifugation at 28,000 × g for 20 min.

A 50-70% (NH4)2SO4 fraction was precipitated from the supernatant by dropwise addition of saturated (NH4)2SO4, pH 7.2. After 1 h, the precipitate was recovered by centrifugation at 28,000 × g for 30 min. The recovered precipitate at 50-70% (NH4)2SO4 was re- dissolved and dialyzed against 50 mmol/L imidazole buffer, pH 6.6, containing 100 mmol/L NaC1.

The 50-70% fraction was applied to a 1.5 x 30 cm column packed with DEAE-Sephacel (Pharmacia Fine Chemicals) previously well equilibrated with 50 mmol/L imidazole buffer, pH 6.6, containing 100 mmol/L NaC1. After elution of a large absorption peak associated with the void volume, a linear gradient (total 240 mL) of 100-300 mmol/L NaC1 in imidazole buffer was eluted. Exactly 1.8 mL fractions were collected and the conductance and CK activity were monitored in all frac{ions. Fractions with the highest enzyme activity were pooled, vacuum-concentrated and dialyzed against 50 mmol/L Tris buffer, pH 7.8, containing 0.5 mmol/L EDTA and 1 mmol/L MgC12.

The dialyzed DEAE-Sephacel pool was applied to a column (1.5 x 30 cm) packed with Blue Sepharose CL-6B (Pharmacia Fine Chemicals) equilibrated with the 50 mmol/L Tris buffer, pH 7.8. After approximately 50 mL of buffer had been eluted, the column was eluted with 50 mmol/L KC1. The support was "purged" of protein by addition of 50 mmol/L Tris buffer, pH 8.0, containing 0.5 mol/L NaC1. Using a flow rate of 8 mL/h for the entire chromatographic procedure, 1.8 mL fractions of eluant were collected. Enzymatically active CK fractions were vacuum-concentrated and dialyzed against 25 mmol/L histidine buffer, pH 6.2, containing 25 mmol/L KC1. The dialyzed solution was applied to chromatofocusing chromatography.

Chromatofocusing Polybuffer 74 and Polybuffer Exchanger (PBE) 93 (Pharmacia, Cat. # 17-073 and 17-071-01) were employed. A 0.9 × 60 cm column was packed at a flow rate of 100 cm/h using a 500 g/L slurry ofdegassed PBE-93. The column was thoroughly equilibrated to the pH and conductivity of the starting 25 mmol/L histidine buffer, pH 6.5, containing 5 mmol/L 2-mercaptoethanol, 1 mmol/L EDTA and 25 mmol/L NaC1.

The pH gradient was chosen to elute the desired protein in 1 /3-1 /2 of the total gradient volume. The starting buffer pH was adjusted to 0.4 above the upper

14 CLINICAL BIOCHEMISTRY, VOLUME 18, FEBRUARY 1985

CREATINE KINASE-BB ISOENZYME

limit of the gradient. Polybuffer was diluted 1:8 as suggested by the supplier, the pH adjusted to 4.0 and 2-mercaptoethanol, EDTA and NaC1 were added to yield a solution of 5, 1, and 25 mmol/L for these reagents, respectively. Exactly 5 mL of this buffer was applied to the column, generating an initial gradient through the upper portion of the support media. Fol- lowing column equilibration with the starting buffer, the pooled CK sample was applied to the top of the bed and elution was then initiated with the Polybuffer at a flow rate of 15 mL/h, generating an internal 6 to 4 pH gradient during the process. The elution pattern was monitored at 280 nm since some of the Polybuffer components gave inherently high absorbance values at 254 nm. All collection tubes, except every tenth one which was used to check the pH gradient, were pre- buffered with 1 mL of 0.2 mol/L imidazole buffer, pH 7.0. CK-active fractions were pooled and immediately dialyzed into 50 mmol/L imidazole buffer, pH 7.2.

PROTEIN DETERMINATION

Protein concentrations were assayed by modification of the Lowry method (7). Since the 2-mercaptoethanol added to all of the chromatographic and storage buffers is known to present sulfhydryl interference in the assay, iodoacetate was added as described by Ross and Schatz (8). Carboxymethylation of the free thiol groups permits accurate determination in the presence of up to 2 mol SH/g protein (8).

CREATINE KINASE ACTIVITY AND REACTION KINETICS

Purified CK-BB isoenzymes were assayed using the Biodynamics/bmc Reagent Set (Cat. # 124184) which measures the production of ATP from creatine phosphate and ADP. The set consisted of two vials. One vial contained all of the components for the assay except creatine phosphate. They were reconstituted according to directions but the second vial containing creatine phosphate alone was further diluted in order to vary its concentration.

The assay mixture when reconstituted as directed contains the following concentrations: 100 mmol/L tri- ethanolamine buffer, pH 7.0; 20 mmol/L D-glucose; 10 mmol/L magnesium acetate; 10 mmol/L ADP; 10 mmol/L AMP and >-1200 U/L glucose-6-phosphate dehydrogenase and hexokinase, respectively, at 25°C. One unit of enzyme activity is defined as that amount of enzyme which converts 1 ~mol of substrate per min at 30°C.

The molecular weight of CK-BB was determined according to the method of Weber and Osborn (10) using molecular weight-markers in the range of 14,300- 71,500 daltons (BDH Chemical Ltd., Poole, England, Cat. # 44223-U). Sodium dodecyl sulfate-polyacrylamide gels were prepared according to the method of Fairbanks (11). Samples were diluted in 0.2 mol/L phosphate buffer, pH 7.2, containing 20 g/L SDS and 20 mL/L 2-mercaptoethanol, to give a final protein concentration of 0.2-0.6 g/L. Electrophoresis was performed in 100 g/L polyacrylamide gel at 8 mA in 0.2 mol/L phosphate buffer, pH 7.2, containing 20 g/L SDS. The gels were stained in 5 g/L Coomassie Brilliant Blue R in methanol/acetic acid/water (5 : 1 : 5, v/v/v).

TITRATION OF REACTIVE AND TOTAL THIOLS

Procedures are based on the method described by Grassetti et al. (12). Each isoenzyme solution was ex- haustively dialyzed against 25 mmol/L phosphate buffer, pH 7.2, to remove any residual 2-mercaptoethanol. A 0.5-1.5 mL aliquot of the enzyme solution was then added to 0.3 mL of 2,2'-dithiodipyridine (2-PDS) in a quartz cuvette, diluted to 3.0 mL with the dialysis buffer and the absorbance measured immediately at 343 nm against a buffer blank. Total sulfhydryl groups were also determined by incubating 1.2 mL of the sample in the presence of 0.3 mL 2-PDS and 1.5 mL of 2 mol/L guanidine-HC1 for 30 min at 56°C. With the use of 1 mol/L guanidine-HC1 in 20 mmol/L phosphate buffer, pH 7.2 as a blank, the absorbance of the mixture was measured at 343 nm.

DETERMINATION OF ISOELECTRIC POINT

Isoelectric focusing used for the determination of isoelectric point of CK-BB was based on the procedure of O'Farrell (13). In brief, to generate a 4.5-7.0 pH gradient with 50 g/L acrylamide and 30 g/L N,N'- methylene bisacrylamide (BIS), 7 mL of 870 mL/L glycerol, 2.25 mL of Bio-Lyte 5/7 (Bio-Rad, Cat. # 163-1152) and 0.75 mL of Bio-Lyte 7/9 (Bio-Rad, Cat. # 163-1172) were combined. The solution was diluted to 60 mL with deionized water. Exactly 50 ~L of N,N,N,N'-tetra ethylenediamine and 1.5 mL of 10 g/L ammonium persulfate were added to effect poly- merization. A constant power (25 W) was applied (480 V, 57 mA) to generate an initial pH gradient without samples. Assayed samples contained 0.05-0.15 mg of protein in 10-15 ~L. Electrophoresis was performed for 1 h.

POLYACRYLAMIDE GEL ELECTROPHORESIS (PAGE)

Analytical disc PAGE was performed as described by Davis (9) using a 70 g/L acrylamide running gel over- layed with a 30 g/L acrylamide stacking gel. Electro- phoresis was conducted at 3 mA/tube in 50 mmol/L Tris-380 mmol/L glycine buffer, pH 8.3 (I = 0.01). Up to 50 ~L of sample was applied to each tube. Protein bands were stained with 10 g/L Amido Black in 70 g/L acetic acid.

ISOENZYME DISTRIBUTION IN CANINE AND HUMAN BRAIN

TISSUES

Fresh canine and human brain tissues were compared for CK-BB activity and isoenzyme distribution. Specific anatomical areas of duplicate brains were care- fully dissected from autopsy specimens obtained within 2 h of death. Sections were taken from temporal, frontal, occipital and parietal lobes, cerebellum, medulla oblongata and pons. Additionally a section of corpus

CLINICAL BIOCHEMISTRY, VOLUME 18, FEBRUARY 1985 15

MILLER AND WEI

Figure 1 - - Molecular weight determination by SDS-PAGE, From left to right: molecular weight standards (K dalton), canine CK-MM, human CK-MM, canine CK-BB and human CK-BB.

callosum was analyzed from human brain tissue. Each sample was homogenized in 50 mmol /L imidazole buffer, pH 7.2, containing 5 mmol/L 2-mercaptoethanol, 150 mmol /L NaCI and 31 mmol/L NAN3. After 2 h of gentle stirring at 4°C, the supernatant was recovered by centrifugation at 28,500 × g for 25 min. All recovered CK pools were immediately analyzed for CK activity, total protein and isoenzyme distribution.

R e s u l t s a n d d i s c u s s i o n

BIOCHEMICAL PROPERTIES OF CK-BB

The CK-BB purified through chromatofocusing is homogeneous as judged by SDS-PAGE (Figure 1) and isoelectric focusing. A mixture of both CK-MM and CK- BB isoenzymes gave a single band, suggesting that the molecular weights of M and B subunits are nearly iden- tical. Additionally, a mixture of M and B subunits of human and canine origin also gave a single band. Our average Mr of 84,700 -+ 300 daltons for the canine CK-BB closely correlated with the only reported estimate of 84,000 daltons (14). A value of 81,000 for human CK-BB is also close to our average estimate.

Table 1 summarizes additional biochemical properties of canine and human CK-BB. CK-BB from both species exhibited absorbance maxima at 276 and 215 nm. With use of a molecular weight of 84,000 daltons for the enzyme, a molar absorptivity of approximately 73,000 was obtained for CK-BB from both sources.

The number of reactive thiol groups on the enzyme was 2.6 and 2.3 for canine and human CK-BB, respectively, suggesting that 2 moles ofsulfhydryl groups are present per mole of enzyme that are essential for catalytic activity (14). Others have reported values of 1 .4-1.9 from chicken (15), 2.1 from ox (16). Total thiol groups of 6.5 are also close to previously reported values of 7 - 8 (17, 18). In addition to the two residues essential for enzymatic activity, therefore, there appear

TABLE 1 Summary of Biochemical Properties of Canine and Human

CK-BB Isoenzymes

Canine Human Biochemical property CK-BB CK-BB

Molecular weight (daltons) 84700 84600 Extinction coefficient (Ell'J'cm. 2so nm) 8.7 8.7 Km (mmol/L creatine phosphate) 2.35 2.38 Vm~x (Ixmol NADPH/(min'mg)) 291 272 pI value 5.2 5.2 Reactive sulfhydryl groups 2.6 2.2 Total sulfhydryl groups 6.5 6.5

to be four residues protected from many sulfhydryl reagents except under denaturing conditions. The remaining residues are relatively inert (14).

Each purified CK-BB preparation produced a single band, migrat ing to an isoelectric point of 5.2 for both species. These results are consistent with their elution response during purification by anion exchange and chromatofocusing chromatography which suggest the pI to be between 5.0 and 5.5. A pI value of 5.6 has been reported for CK-BB isolated from calf brain (19). The kinetic parameters for CK-BB have been arranged such that its activity is dependent only upon the concen- t rat ion ratio of creatine phosphate to creatine. The Km and Vn~x values for each CK-BB were derived from the Woolfplot. Extreme variation appears in the l i terature with respect to creatine kinase kinetic parameters, i.e., temperature, pH, assay direction and substrate/ coenzyme/cofactor concentrations. The only study which was similar in design to our work obtained V ... . values of 200 and 100 U /mg and Km of 10 and 5 mmol /L creatine phosphate for ox CK-BB and CK-MM, respectively (20).

STABILITY OF CK-BB

Creatine kinase BB has been shown to be much less stable than the other isoenzyme forms (21), particularly under acidic conditions. Both canine and human-CK showed no loss of activity, however, when stored at -70°C for as long as 32 weeks (data not shown).

As indicated in Table 2, storage under acidic conditions at 4°C promoted the loss of activity with total loss occurring within about 4 h at pH 5.0. Increasing pH to 6.0 or above greatly stabilized human CK-BB which demonstrated about 70% residual activity at pH 6.0 after 28 days. In contrast, canine CK-BB lost most of its activity in about a day. Both isoenzymes are stable at least one month at 4°C at pH 7.0-9.0, though canine CK-BB lost some of its activity at pH 9.0.

These results are comparable to other studies on human CK isoenzymes stabilized with thiol agents at 4°C, which report the optimum storage pH at 4°C to be 6 -7 .5 (22). A comparison of activity with and without 2-mercaptoethanol has suggested that thiol compounds give little or no protection at a pH above 7.5, and, in fact, contribute to isoenzyme instability at a pH of 8.5 (23). Since our study did not include buffers without 2-mercaptoethanol it cannot be estimated how much



TABLE 2 Stability of CK-BB on Storage at 4°C and Under Varying pH

pH 5.0 pH 6.0 pH 7.0 pH 8.0 pH 9.0

Time HuBB b CaBB HuBB CaBB HuBB CaBB HuBB CaBB HuBB CaBB

518 a 299 568 229 810 229 810 230 810 229 0.5 h 470 200 . . . . . . 712 220 1.0 h 217 175 . . . . . . . . 2.0 h 182 162 588 262 . . . . 760 241 4.0 h 27 26 556 280 . . . . 800 - - 8.0 h 0 0 - - 217 . . . . 864 226 20.0 h 0 0 - - 142 . . . . 736 227 2 days - - - - - - 0 . . . . 800 242 3 days - - - - - - 0 . . . . 870 234 7 days - - - - - - 0 810 195 815 - - 808 215 10 days . . . . 880 - - - - - - 866 215 14 days - - - - 460 - - 876 209 834 239 880 218 21 days - - - - 446 0 888 192 896 240 850 176 28 days 0 0 402 0 931 213 882 238 840 212

aln U/L. (--) implies no data point taken. bHuBB (human CK-BB), CaBB (canine CK-BB). Buffer solutions in increments of 1 pH unit were made using 50 mmol/L succinate for pH 5 and 6 and

50 mmol/L Tris for pH 7 to 9. All buffers contained 15 mmol/L 2-mercaptoethanol. Samples were diluted in each of these solutions to a final CK activity of 200-800 mU/mL. Appropriate mounts of heat- inactivated serum were added to compensate for protein concentration differences before the dilutions. All solutions were stored in a cold room maintained at 4°C and the residual CK activity monitored.

this reducing agent contr ibuted to the s tab i l i ty of the CK isoenzymes.

Although the addition of 2 -mercaptoe thanol effected an init ial stabilization of both enzymes, af ter several hours the rate of loss of act ivi ty accelerated regardless of presence of the reducing agent . The h u m a n CK-BB diminished to 50% of its or iginal act ivi ty after 2.2 h; the canine isoenzyme to 50% after 3.2 h (Figure 2). Wi th or without 2-mercaptoethanol, the enzymes showed min- imal residual activity after 6 h. This s tudy is cons is ten t with the stabil i ty study of Cho and Meltzer (21) who describe a total loss of CK-BB act ivi ty af ter 6 h. Our results indicate, however, t ha t our p r epa ra t i on of th is isoenzyme is much more stable t h a n the i r p r epa ra t i on because they observed loss of 50% act iv i ty w i thou t 2-mercaptoethanol in 15 min. Thus, a l though in i t i a l l y providing some stabi l izat ion, 2-mercaptoethanol does little to prevent rapid loss of CK-BB act ivi ty a t 37°C.

CK DISTRIBUTION IN BRAIN TISSUES

A summary of the CK specific act ivi ty and isoenzyme distribution in canine and h u m a n b r a i n t i ssue is g iven in Table 3.

The overall specific act ivi t ies (U /mg ext rac table protein) were 2.51 -+ 0.43 (mean -+ SD) and 1.19 + 0.41 for canine and h u m a n b r a in t i ssue extracts, respectively. Statistical comparison, us ing the t-test, ind ica ted significantly greater specific act ivi ty in can ine t i ssue (p < 0.001). Due to the l imited n u m b e r of ac t iv i ty values wi th in individual areas, no s ta t i s t ica l ana lys i s between b ra in sections was performed.

Agarose electrophoresis demons t ra ted the presence

4 t 0

k

"~ 1.(

4 6

Tim~, h

Figure 2 - - Thermal lability of CK-BB indicated at 37°C in heat-inactivated sera with and without 15 mmol/L 2-mercaptoethanol. Canine CK-BB (O--O), Canine CK-BB with 15 mmol/L 2-mercaptoethanol (A--A), Human CK-BB ( . - - ~ ) , Human CK-BB with 15 mmol/L 2-mercaptoethanol (O--Q). Normal human and canine serum was heat-inactivated at 56°C for 30 min. CK activities were measured to verify complete loss of activity. Purified isoenzymes were added to the appropriate serum with and without the addition of 15 mmol/L 2-mercaptoethanol. All samples were incubated at 37°C and the residual CK activity monitored to study the time course of inactivation in human and canine serum, respectively.


MILLER AND WEI

TABLE 3 CK Activity and Isoenzyme Distribution in Brain Tissues a

CK Specific % CK isoenzyme activity activity U/mL b U/mg MM MB BB

Canine brain sections Pons 22.45 2.97 8.5 n.d. 91.5 Medulla oblongata 17.13 2.76 8.5 n.d. 91.5 Parietal 42.78 2.67 7.1 n.d. 92.9 Cerebellum 52.34 2.39 9.5 n.d. 90.5 Temporal 29.24 1.99 9.9 n.d. 90.1 Frontal 21.25 1.96 14.5 0.7 84.8 Occipital 24.45 2.83 7.6 n.d, 92.4

Human brain sections Pons 5.84 0~92 6.2 n.d. 93.8 Medulla oblongata 7.83 1.07 7.1 n.d. 92.9 Parietal 11.35 1.04 6.0 n.d. 94.0 Corpus callosum 8.00 0.94 3.8 n.d. 96.2 Cerebellum 9.85 1.13 8.7 n.d. 91.3 Temporal 14.61 1.20 4.9 n.d. 95.1 Frontal 19.37 2.00 13.2 n.d. 86.8 Occipital 10.61 1.20 3.7 n.d. 96.3

aThe results are the mean value of duplicate brains, n.d. = not detectable.

bRepresent values corrected for substrate control (no creatine phosphate) and enzyme control (heat-inactivated at 56°C for 30 rain).

of CK-MM as well as CK-BB isoenzymes in brain tissues of both species. Isoenzyme distributions (%) from canine and human sections.were 9.2 -+ 2.8 (mean + SD) and 6.7 --- 3.1 for CK-MM, and 90.8 -+ 3.1 and 93.3 -+ 3.1 for CK-BB. Canine brain tissue contained a significantly greater percentage of CK-MM than human brain tissue (p < 0.05). Within individual area, the frontal lobe characteristically showed a higher percentage of CK-MM. A small amount of CK-MB isoenzyme was detected in this lobe in canine tissue. Due to the instability of the isoenzymes and possible recombi- nations, it cannot be stated definitively whether this MB was truly present within frontal tissue or was an artifact of the higher CK-MM concentrations. Pub- lished data on the distribution of total CK activity and of CK isoenzymes in human brains vary for several reasons. Tissue autolysis with a decrease in CK activity may affect the individual isoenzymes differently. The method of tissue extraction leads to variable recoveries. Lastly, the isoenzyme pattern depends to a certain degree on the method used for separation and detection of the fraction.

The reported CK specific activities in human brain tissue range from 0.01 to 100 U/g tissue wet weight (24-27). Several alternative extraction procedures recovered 3.3 and 3.6 U / m g extractable protein (28, 29). No studies on canine tissue specific activities are available. Isoenzyme distributions of 100% CK-BB have been reported using methods other than agarose electrophoresis (24-27). A more recent publication (30) confirmed the presence of CK-MM. Other authors reported trace amounts of MM isoenzyme in rat brain extracts (31) and small amounts in canine brain (32).

Acknowledgements

The authors gratefully acknowledge the valuable contribution made by Dr. Mark Johnson. They als0 thank Mrs. Richelle Emery for assistance in the preparation of this manuscript. This research was supported in part by the Research and Creative Activities Grant of the Graduate College, Cleveland State University.

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properties of creatine kinase-bb from canine and human brain tissues

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