Membrane-associated protein kinase activities in seminal plasma from vasectomized men

Membrane-associated protein kinase activities in seminal plasma from vasectomized men M. J.

Wilson*, K. W. Kaye, J. Skalicky* and A. A. Sinha

Research Service, VA Medical Center and Departments of * Laboratory Medicine and Pathology, t Urologie Surgery, and tGenetics and Cell Biology, University of Minnesota, Minneapolis, MN 55417, U.S.A.

Summary. Differential centrifugation was used to prepare heavy and light membrane fractions from the seminal plasma of vasectomized men. The two membrane fractions combined contained half of the phosvitin and histone kinase activities but only 7% of the total protein content in vasectomy semen. These two kinase activities as well as phosphorylation of endogenous membrane proteins were optimally stimulated by Mg2 ; Mn2+ could effectively substitute for Mg2+ only in endogenous phosphorylation reactions. Neither the phosvitin nor histone kinase responded to cAMP or cGMP, but the histone kinase was strongly inhibited by the heat-stable cAMP-dependent protein kinase inhibitor. The phosvitin kinase was not affected by this inhibitor. The phosphorylation of endogenous proteins in the heavy membrane fraction was not affected by the protein kinase inhibitor but protein phosphorylation in the light membrane fraction was partly (45%) inhibited. The differential effects of increased ionic strength, sulphydryl protecting agents, and the protein kinase inhibitor on protein kinase activity towards lysine-rich histones, phosvitin and endogenous proteins, as well as differential extractability and binding to an anion exchange column of histone kinase and phosvitin kinase activities, indicate that more than one kinase activity is present in these +

membrane subfractions. Electron microscopic examination showed that there are several kinds of membranelimited components in vasectomy seminal fluid that vary in size, density, and ultrastructure. The association of type(s) of protein kinase to individual membrane components remains to be established.

Keywords: protein kinase; seminal fluid; prostate; membranes, vasectomy; man Introduction Protein kinase activities in semen are associated with both spermatozoa (Hoskins et al, 1972; Garbers et al, 1973; Majumder, 1978a; Tash & Means, 1982; Horowitz et al, 1984; Atherton et al, 1985) and seminal plasma (Majumder, 1978b; Fabbro et al, 1982; Wilson et al, 1982). Approxi¬ mately 70-80% of the protein kinase activities in seminal plasma are correlated with the number of spermatozoa originally present in the ejaculate (Fabbro et al, 1982; Wilson & Kaye, 1983). The prostate gland is the principal source of the non-sperm-related protein kinase activities in seminal plasma (Wilson & Kaye, 1983). These enzymic activities are found in both the membrane and soluble fractions of prostate secretion (Wilson & Kaye, 1982; Stegmayr et al, 1982b). In view of reports of altered prostatic fluid protein kinase activities in prostatitis (Wilson et al, 1984) and of a seminal plasma membrane fraction stimulating progressive forward motility of washed human spermatozoa with concomitant protein phosphorylation (Stegmayr & Ronquist, 1982a), we

investigated the distribution and enzymic properties seminal plasma (fluid) from vasectomized men.

of membrane-associated

protein kinases

in

Materials and Methods Materials. Lysine-rich histones were obtained from Worthington Diagnostics, Copper Biomedicai, Malvern, PA; rabbit muscle protein kinase inhibitor from Sigma Chemical Co., St Louis, MO; and 32Pi from ICN, Irvine, CA. All other chemicals were of the highest grade available. Preparation of 32P-labelled adenosine triphosphate (ATP) and partly dephosphorylated phosvitin was carried out as previously described (Ahmed et al, 1975). Methods. Fresh sperm-free semen samples from vasectomized men (N 37) were allowed to stand at room temperature for 30-60 min and then maintained at 4°C for all further processing. Semen samples were separated into cellular, heavy membrane, and light membrane fractions by differential centrifugation as follows. The samples were routinely centrifuged at 900 g for 5 min to remove any cells that were present in the semen. The supernatant of this step was centrifuged at 20 000 g for 30 min (20000 g or heavy membrane fraction) and the resultant supernatant was centrifuged further at 105 000g for 60 min (105 000g or light membrane fraction). All pellets and the supernatant after the last centrifugation were saved. The sedimented fractions were resuspended in 0-25 M70°C and stored at sucrose and these membrane fractions and the high speed supernatant were frozen at -20°C. =

—

into Protein kinase assays. The measurement of protein kinase-mediated transfer of 32P from -32 phosphoprotein substrates was carried out as detailed previously (Wilson et al, 1982). The reaction medium (0-5 ml final reaction volume) for assaying protein kinase activity toward lysine-rich histones consisted of 30 mM-Tris-HCl (pH 80 at 37°C), 1 mM-dithiothreitol, 6 mM-MgCl2, 0-5 mM-ATP (specific radioactivity about 9000 d.p.m./nmol), and 6 mg lysine-rich histones/ml. The measurement of protein kinase activity towards partly dephosphorylated phosvitin used a standard reaction medium (final volume 0-50ml) consisting of 30mM-Tris-HCl (pH80 at 37°C), 1 mMdithiothreitol, 240mM-NaCl, 6mM-MgCl2, 3 mM-y-32P-ATP (specific radioactivity about 1500 d.p.m./nmol), and 6 mg phosvitin/ml. The phosphorylation of endogenous phosphoproteins was determined using a reaction medium (0-50 ml final volume) consisting of 30 mM-Tris-HCl (pH 80 at 37°C), 1 mM-dithiothreitol, 6 mM-MgCl2, and 50 nM-32 - (specific radioactivity 1-7 IO5 d.p.m./nmol). The duration of phosphorylation reactions was 20 min with lysine-rich histone, 30 min with phosvitin, and 1 min with endogenous proteins as substrates. The reactions were linear with respect to time for each assay system. All assays were performed in duplicate or triplicate and individual assay values were within 7% of one another. The phosphorylation reactions were started by addition of seminal plasma membrane fractions, and terminated by addition of 0-50 ml cold 30% (w/v) trichloroacetic acid containing 2 mM-inorganic phosphate and 3% sodium pyrophosphate. Precipitated phosphoproteins were washed and prepared for liquid scintillation counting (Wilson et al, 1982). Anion exchange chromatography. Membrane fractions were extracted for Mono Q chromatography by fast protein liquid chromatography (FPLC) (Pharmacia, Piscataway, NJ) using the following procedure. Five volumes of extrac¬ tion buffer (005% Lubrol, 50 mM 2-mercaptoethanol, 1 mM EGTA, 50 mM Tris-HCl, pH 7-5 at 4°C) were added to one volume of newly thawed membranes prepared from vasectomy seminal fluid. Phenylmethylsulphonylfluoride was added to a final concentration of 1 mM while homogenizing the suspension with a Teflon-glass tissue grinder (50 strokes). The homogenized suspension was stirred on ice for 1 h and then centrifuged for 1 h at 40000 g. The supernatant was saved and the pellet was re-extracted as above using the same volume of extraction buffer. The supernatants were combined and concentrated using a YM10 Amicon filter and pressure under nitrogen. The extract was then filtered sequentially through silica wool and 0-45 and 0-2 µ fluoropolymer membranes (Gellman, Ann Arbor, MI) before being applied to a Mono Q column equilibrated with 20 mM-Tris-HCl (pH 7-5 at 4°C), 5 mM-2mercaptoethanol, 50 mM-NaCl and 0-2 mM-EDTA. The column was washed extensively with the same buffer and then eluted with a linear gradient of 005 to 0-50 M-NaCl. Fractions of 1-6 ml were collected. Lubrol was chosen as the detergent for the extraction after preliminary studies showed that it and Triton X-100 (005%) were more effective in extracting histone kinase activity than was Tergitol NP-10, Emulgen 911, or Triton N-101 (all detergents were at a concentration of 005%). Lubrol was then chosen because the kinase activity was more stable after storage for 3^1 weeks at 20°C and lubrol has minimal absorption at 280 nm. SDS-polyacrylamide gel electrophoresis of membrane phosphoproteins. The phosphorylation of membrane proteins to be separated by SDS-polyacrylamide gel electrophoresis was carried out as described above for phosphorylation of endogenous proteins except that the -32 - concentration was 25 mM (about 6 IO6 d.p.m./incubation), 01% Triton X-100 was included and the reaction time was 5 min to allow maximal labelling of proteins. The reaction was stopped by addition of an equal volume of SDS sample buffer (3% SDS, 20% glycerol, 62-5 mM-Tris-HCl, pH6-8). The samples were heated for 3 min in a boiling water bath and aliquants were electrophoresed in 10% SDS-polyacrylamide gels (Laemmli, 1970) prepared on Gelbond films (FMC, Rockland, ME). These gels were stained with Coomassie blue R-250, dried, and exposed to Kodak X-Omat AR film for autoradiography. Molecular weight determinations were made in reference to prestained protein standards (Diversified Biotech, Newton Center, MA) electrophoresed in these gels. These standards were: phosphorylase B, Mr 95 500; glutamate dehydrogenase M, 55 000; ovalbumin, Mr 43 000; lactic dehydrogenase M, 36 000; carbonic anhydrase, M, 29 000; lactoglobulin, Mr 18 400; cytochrome C, Mr 12 400. —

Electron microscopy. The ultrastructural characteristics of the membrane subfractions were examined by transmission electron microscopy. The cellular, heavy and light membrane fraction pellets were fixed for 2 h in 2% paraformaldehyde and 2% glutaraldehyde in OT M-phosphate buffer at pH 74. Specimens were washed several times with buffer and post-fixed for 1 h with 1 % osmium tetroxide in phosphate buffer and dehydrated in graded ethanol, propylene oxide and embedded in Epon 812 (Luft, 1961). Thin sections were stained with a combination of uranyl acetate and lead citrate and examined with an RCA EMU-4 or Zeiss IOC electron

microscope.

Other methods. The protein content of seminal standard (Lowry et al, 1951).

plasma

fractions

was

measured

using bovine

serum

albumin

as

Results

Distribution

ofprotein kinase activities in subfractions of seminal plasma The distribution of protein kinase activities towards partly dephosphorylated phosvitin, lysinerich histones, and endogenous membrane proteins was examined in membrane subfractions from sperm-free semen of vasectomized men. Protein kinase activities towards all three substrates were found in the cellular pellet, the heavy and light membrane fractions, and in the supernatant of the 105 000 g centrifugation (Table 1, representative subfraction preparation). Addition of Triton X-100 to the reaction medium stimulated the protein kinase activities substantially in the mem¬ brane fractions but with little or no effect upon activities in the soluble portions of seminal plasma. The percentage stimulation of protein kinase activity by 01% Triton X-100 was greater in the light membrane fraction than other fractions (Table 1). Varying the concentration of Triton X-100 from 002 to 0-40% did not affect the percentage stimulation of protein kinase activity towards phosvitin or lysine-rich histones (data not shown). About 87% of seminal plasma protein was found in the supernatant of the 105 000 g centrifugation whereas this fraction accounted for about 40% of the phosvitin and histone kinase activities (Table 1). On the other hand, the two membrane fractions combined accounted for nearly 50% of these two kinase activities but only for 7% of seminal plasma protein. The distribution of endogenous phosphory¬ lation reactions was different from that of the phosvitin and histone kinases; about 60% of endogenous phosphorylating activity was found in the low speed, large vesicle/cellular fraction. The endogenous phosphorylation of membrane proteins was sensitive to heat as it was completely destroyed by heating at 60°C for 5 min (data not shown). The protein kinase activities described here appear to be bound within the granules/vesicles and/or are integral membrane proteins and not simply adhering to membrane components since they could not be removed from the mem¬ brane fractions by washing them with 10 mM-Tris-HCl (pH 7-5) containing 0-25 M-sucrose and 0-17M-KC1.

Properties of membrane associated protein kinase activities Some factors which affect protein kinase activities towards phosvitin and lysine-rich histones in prostatic fluid (Wilson et al, 1984) and seminal fluid (Wilson et al, 1982) were examined with regard to membrane-associated protein kinase activities (Table 2). Protein kinase activities toward the three types of protein substrates required a divalent cation for activity and they were stimulated

to some extent by sulphydryl protecting agents such as dithiothreitol. Mg2+ was the most effective of divalent cations tested for stimulating the protein kinase activities in either membrane subfrac¬ tion. Mn2+ substituted effectively for Mg2+ in the endogenous phosphorylation reactions, but not in those phosphorylating phosvitin or lysine-rich histones. An increase in the reaction concen¬ tration of MgCl2 from 6 mM to 20 mM inhibited all three types of protein kinase activities except for the histone kinase of the 105 000g membrane fraction. Increasing the ionic strength of reaction media by the addition of NaCl produced a small stimulation of phosvitin kinase activities (40% at 240 mM) but a further increase in NaCl concentration above that for optimal activity (i.e. addition

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