ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS

Vol. 273, No. 1, August 15, pp. 165-169,1989

Identification of Type III Protein Kinase C in Bovine Aortic Tissue

MASATO WATANABE, TAKAHISA HACHIYA, MASATOSHI HAGIWARA, AND HIROYOSHI HIDAKA’

Department of Pharmmologg, Nagoya University School of Medicine, Showa-ku Tsurwnai 65, Nagoya 466, Japan

Received January 3,1989, and in revised form April 21,1989

We identified a subtype of protein kinase C in bovine aortic tissue. In Western blots, both the soluble and the particulate fractions from the aorta reacted only with MC-3a. In the case of hydroxylapatite column chromatography, a single activity peak of protein kinase C from the soluble and the particulate fractions was obtained with about 140 mM of potassium phosphate, a finding similar to that with the Type III protein kinase C from rabbit brain. The sandwich-type enzyme immunoassay for protein kinase C, with which the contents of each protein kinase C isozyme can be determined in the crude extracts, revealed that the Type III bovine aortic protein kinase C included 25.9 ng/mg protein. These results strongly suggest that it is the Type III protein kinase C which is mainly expressed in aortic tissue. Kinetic parameters of the Type III protein kinase C of the soluble and the particulate fractions, with respect to the Km for ATP, were 33 and 15 PM

and the Km values for myosin light chain from chicken gizzard were 6.3 and 4.6 PM, respectively. 0 1989 Academic press, I~C.

When a ligand binds to certain receptors on the cell surface, diacylglycerol is pro- duced by hydrolysis of inositol phospholip- ids. The primary effect of diacylglycerol is to activate protein kinase C which phos- phorylates many endogenous proteins and produces various biological responses (1, 2). Several subtypes of protein kinase C have been identified by cDNA sequence analysis (3-9) and hydroxylapatite column chromatographic techniques (10-X). In many laboratories, including our own, polyclonal antibody or monoclonal anti- body against subtypes of protein kinase C were prepared; some of these exhibited dis- tinct patterns of tissue expression and in- tracellular localization (15-22). These different subtypes probably have distinct roles in the processing and modulation of physiological responses to external sig- nals. We reported that protein kinase C modulated the secretion of serotonin from

r To whom correspondence should be addressed.

human platelets stimulated by TPA’ (23) and that Types II and III were expressed in human platelets (15). We also reported that skinned vascular smooth muscle was relaxed in the presence of protein kinase C purified from the rabbit brain (24). Kariya and co-workers noted the antiproliferative action of protein kinase C in cultured rab- bit aortic smooth muscle cells (25). The subtypes of the protein kinase C expressed in smooth muscle remained to be deter- mined.

We report here the subtype(s) of protein kinase C in aortic tissue, determined using three specific monoclonal antibodies. We also used the sandwich-type enzyme im-

‘Abbreviations used: MLC, myosin light chain from chicken gizzard; TPA, 12-0-tetradecanoyl phor- bol 13-acetate; EGTA, ethylene glycol bis(&amino- ethyl ether) N,N’-tetraacetic acid; DTT, dithiothrei- tol; PS, phosphatidylserine; SDS-PAGE, sodium do- decyl sulfate-polyacrylamide gel electrophoresis; BSA, bovine serum albumin.

165 0003-9861189 $3.00 Copyright 0 1989 by Academic Press, Inc. All rights of reproduction in any form reserved.

166 WATANABE ET AL.

munoassay system to elucidate the three types of protein kinase C and for determi- nation of this kinase in the aorta.

EXPERIMENTAL PROCEDURE

Preparation of protein kinase C Bovine aorta ob- tained at a local slaughterhouse was immediately placed on ice. All experiments were performed at O- 4°C and the supernatant sample centrifuged at 100,000~ was prepared within 3 h after death of the animal. One hundred and fifty grams of the carefully dissected smooth muscle layer was minced and blended with 4 vol of buffer A (25 mM Tris-Cl, pH 7.5/ 2 mM EGTA/SO mM 2-mercaptoethanol/O.OOlW leu- peptin/lO% glycerol) containing 0.25 M sucrose, at low speed in a Waring blender, in two 60-s operations. The homogenate was centrifuged at 10,OOOg for 20 min. The supernatant was filtered through glass wool, then centrifuged at 100,000~ for 60 min (soluble frac- tion). The pellet was homogenized with 4 vol of buffer A containing 0.5% Triton X-100, in a Waring blender. The homogenate was centrifuged at 100,OOOg for 60 min (particulate fraction). Two resultant superna- tants were separately applied to a DE52 column (Whatman, 4 X 12 cm) previously equilibrated with buffer A. The column was washed with 300 ml of buffer A and the enzyme eluted with a linear gradient of NaCl (O-O.4 M; total volume, 1 liter). The fractions containing kinase activity were collected and concen- trated to about 30 ml, using an Amicon concentrating system and UK-10 (TOY0 Roshi Ltd.). NaCl(1 M) was added to this fraction and the preparation applied to a Butyl TOY0 Pearl 650 M column (Tosoh Ltd., 6 X 0.5 cm) previously equilibrated with buffer A containing 1 M NaCl. After the column was washed with 100 ml of the same buffer, the enzyme was eluted from the column with 50 ml of buffer A. The enzyme fraction was concentrated to 10 ml and dialyzed overnight against buffer B (20 mM K2HP04/KHzP04, pH 7.0/2 mM EGTA/O.OOl% leupeptin/2 mM DTT/lO% glyc- erol). The dialyzed fraction was applied to a hydroxyl- apatite column (Bio-Gel HT, Bio-Rad Ltd., 0.5 x 3 cm) and this column was sequentially washed with 10 ml of buffer B and eluted by application of a linear gradi- ent of concentration of potassium phosphate (20- 200 mM).

Assay for protein kinase C. The standard assay con- dition mixture contained, in a final volume of 0.2 ml, 25 mM Tris-Cl (pH 7.0), 10 mM MgClz, 0.8 mM CaCl,, or 2 mM EGTA, 10 pg PS, 40 pg MLC, and aliquot of protein kinase fraction, 10 pM [@*PlATP (5 X lo5 cpm). The reaction was carried out at 30°C for 5 min and terminated by addition of 20% trichloroacetic acid. Radioactivity was counted in a liquid scintilla- tion counter (LKB-1219 type).

Enzyme immunoassay for protein kinase C Homog- enates from bovine aortic tissue, obtained using 0.05

M sodium phosphate buffer, pH 7.4, containing 1% BSA, 0.1 M NaCl, 20 mM EGTA, 1 mM MgCl,, and 0.001% leupeptin, were centrifuged at 100,OOOg for 60 min. The resultant supernatant was passed through a 0.45-pm filter and 100 ~1 of filtrate was added to the three subtype-specific enzyme immunoassay plates for 2 h at 4°C. After repeated washings with PBS, the plates were incubated with 100 ~1 of 1:lOOO diluted peroxidase-conjugated second polyclonal antibody for 2 h at 4°C. The plates were washed with PBS and 100 ~1 of assay solution was added for peroxidase ac- tivity, using 0-phenylenediamine as substrate. After 10 min, the reaction was halted by adding 100 ~1 of 2 N HzSO*. Optical density was read in a spectropho- tometer (Tosoh Ltd.) at 492 nm. Characterization of second polyclonal antibody and methods for prepara- tion of enzyme immunoassay plates will be described elsewhere.3

Other m&hods. Western blotting analysis was car- ried out as described (15). MLC was prepared accord- ing to the method of Perrie and Perry (26). Protein was determined by the method of Bradford (27) using a Bio-Rad protein determination kit and bovine se- rum albumin as a standard.

RESULTS AND DISCUSSION

We reported the preparation of subtype- specific monoclonal antibodies against rabbit brain protein kinase C (18). These three monoclonal antibodies cross-reacted with protein kinase C from various bovine tissues. Therefore, we attempted to iden- tify the subtypes of protein kinase C from bovine aorta, using these monoclonal anti- bodies. The soluble and the particulate fractions concentrated by DE52 column chromatography were analyzed by West- ern blots. Each fraction was electropho- resed by 10% SDS-PAGE (28) and trans- ferred to a nitrocellulose membrane (29). Figure 1 clearly shows that the immunore- active protein contained both fractions, obtained at 80 kDa when the nitrocellulose sheet was probed with MC-3a which recog- nized Type III protein kinase C of rabbit brain. In lanes 2-5 to which the sample from bovine aorta was applied and trans- ferred, a 75kDa immunoreactive band was visualized. However, this band was also ob- tained when normal mouse serum was used as a probe (Fig. 1, lane 5). When the first antibody (monoclonal antibody or

3 M. Hagiwara et al, submitted for publication.

PROTEIN KINASE C IN

A B

FIG. 1. Identification of the subtypes of protein ki- nase C from bovine aortic tissue. Western blotting analysis was carried out as described under Experi- mental Procedure. (A) Soluble fraction; (B) particu- late fraction. Twenty micrograms of sample from rabbit brain was applied in lane 1 as a positive control and 20 pg of sample from bovine aortic tissue was ap- plied in lanes 2 to 5. Lanes 1 and 2 were probed with MC-la, lane 3 with MC-2a, lane 4 with MC-3a, and lane 5 with normal mouse serum.

normal mouse serum) was omitted in Western blots using an ABC kit from Vec- tor Laboratories, or when the sample was extracted from rabbit or rat aorta instead of bovine aorta, this 75kDa band did not appear (data not shown). These results suggest that the components of the ‘75kDa band which nonspecifically bind to mouse

0 10 20 30 40 50 60 70 IO

Fraction number (OSmlltube)

BOVINE AORTIC TISSUE 16r

immunoglobulin are present in bovine aor- tic tissue. Thus, we separated the subtypes of protein kinase C by hydroxylapatite col- umn chromatography. The elution pattern of the soluble protein kinase C from hy- droxylapatite column is shown in Fig. 2A. A single activity peak was obtained at about 145 mM of potassium phosphate. Fig- ure 2B shows the elution pattern of the particulate fraction of protein kinase C. This pattern was similar to that from the soluble fraction. However, the concentra- tion of potassium phosphate at which pro- tein kinase C eluted from the column was about 140 mM. These two patterns of the soluble and the particulate fractions were similar to that of Type III protein kinase C from rabbit brain (15).

We prepared an enzyme immunoassay system which involved the use of two kinds of antibody: one was the type-specific monoclonal antibody as a solid phase; the other was a polyclonal antibody against 40 residues of the carboxyl-terminal of the (Y- cDNA sequence of the protein kinase C of rabbit brain (Type II), as a second antibody conjugated by horseradish peroxidase. Us- ing these three monoclonal antibody-im- mobilized plates, we quantitated Types I, II, and III of protein kinase C down to 4.1, 0.65, and 2.5 ng per 100 ~1 assay, respec-

fpmot/min)

10 20 30 10 IO LO 10

Fraction number (O.Sml/tube)

FIG. 2. Elution profile of protein kinase C of bovine aortic tissue on a hydroxylapatite column. The soluble fraction (A) and the particulate fraction (B) from bovine aortic tissue were separately ap- plied to a column and eluted. The fractions were collected from 0.5 ml of each tube. Kinase activity was assayed as described under Experimental Procedure in the presence (0) and absence (0) of Ca” PS t .

168 WATANABE ET AL.

tively, which correspond to 510,81, and 310 pmol per liter of protein kinase C. When the sample contained more than 0.05% Triton X-100, the immunoreaction was completely suppressed; therefore, we de- termined the soluble fraction of protein ki- nase C from bovine aorta. Table I shows the results. Types I and II protein kinase C in the extracts from bovine aorta were below detection limits. The contents of Type III protein kinase C in bovine aorta were detected at 25.9 ng/mg protein. These data obtained using immunoassay coincide with data obtained using Western blotting analysis and the enzyme preparation by hydroxylapatite column chromatography.

TABLE II

SUMMARY OF Km VALUES AND SPECIFIC ACTIVITIES OF PROTEIN KINASE C FROM BOVINE AORTIC TISSUE

Sol. Par.

Km values (PM)

ATP MLC

33 6.3 15 4.6

Note. Sol., soluble fraction; Par., particulate frac- tion. The enzyme was assayed as described under Ex- perimental Procedure, except either the ATP concen- tration (up to 50 FM) as a phosphate donor or the MLC concentration (up to 20 PM) as a phosphate acceptor was varied.

Thus, Western blotting, hydroxylapatite column chromatography, and enzyme im- munoassay used to detect protein kinase C activity revealed that it is Type III protein kinase C which is mainly expressed in bo- vine aortic tissue.

data from rabbit brain and human plate- lets (15).

The specific activities of the protein ki- nase C from the soluble and the particulate fractions of aortic tissue, using MLC as a substrate, were 459 and 338 pmol/min/mg, respectively (Table II). We then deter- mined the kinetic constants with respect to the Km for ATP as a phosphate donor and for MLC as a phosphate acceptor. As shown in Table II, the apparent Km values for ATP of the soluble and the particulate protein kinase C were 33 and 15 j&M and the apparent Km values for MLC were 6.3 and 4.6 PM, respectively. Specific activities of both preparations from bovine aorta showed a ratio of about 1.3:1. These K,val- ues for ATP and the ratio of specific activities were in good agreement with

Distinct distributions of protein kinase C were revealed using immunocytochemis- try, immunoblotting analysis (15, 18-22), or hydroxylapatite column chromatogra- phy (10-15). Most of these reports related to a qualitative analysis of protein kinase C. The enzyme immunoassay system we used in the present work was most suitable for a quantitative analysis of a limited amount of samples. Therefore, to identify subtypes of protein kinase C, the pre- cise distribution and determination of amounts of this kinase can be assessed when use is made of these three methods, in combination.

ACKNOWLEDGMENTS

We thank M. Ohara for critical comments. This work was supported in part by a grant-in-aid for Sci- entific Research from the Ministry of Education, Sci- ence and Culture, Japan.

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PROTEIN KINASE C FROM BOVINE AORTIC TISSUE DETERMINED USING THE SANDWICH-TYPE

ENZYME IMMUNOASSAY

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Protein kinase C content (ng/mg protein)

5Pe 1 Type II Type III

<4.1” <0.65” 25.9

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2. NISHIZUKA, Y. (1986) Science 233,305-312. 3. PARKER, P. J., COUSSENS, L., Tom, N., RHEE, L.,

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PROTEIN KINASE C IN BOVINE AORTIC TISSUE 169

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