THE JOURNAL OF B~OLOCICAL CHEMISTRY d 1990 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 265, No. 12, Issue of April 25. pp. 6860-6867. 1990 Prmted in U.S.A.

Bovine Seminal Plasma Constituents Modulate the Activity of Caltrin, the Calcium-transport Regulating Protein of Bovine Spermatozoa*

(Received for publication, August 21, 1989)

Jovenal T. San Agustin and Henry A. LardyS From the Institute for Enzyme Research and the Department of Biochemistry, University of Wisconsin, Madison, Wisconsin 53705

Adsorption of caltrin on a cation exchanger during purification transformed it from inhibitor to enhancer of calcium uptake. Ether extracts of acidified prepa- rations of bovine seminal plasma transformed en- hancer caltrin back to inhibitory caltrin; this capacity of the ether extracts was lost after incubation with an anion exchanger, indicating that anions in the extract could be responsible for the reversal of caltrin activity.

Of the anions identified in the ether extract of acid- ified bovine seminal plasma, phosphatidylserine con- verted enhancer caltrin to the inhibitory form at pH 7.4. Citrate at millimolar concentrations lowered cal- cium uptake of sperm in the presence of enhancer caltrin to near control levels. Cardiolipin at concentra- tions comparable to its natural occurrence in the sem- inal plasma prevented enhancer caltrin from stimulat- ing the sperm cells to take up calcium above their usual capacity.

Dipalmitoylphosphatidylglycerol, phosphatidylcho- line derived from bovine brain, phosphatidylethanol- amine from bovine heart, and other phospholipids with transition temperatures higher than the assay temper- ature had no effect on the activity of enhancer caltrin, while dimyristoylphosphatidylcholine had an effect on enhancer caltrin similar to that of citrate. Phosphati- dylinositol from soybean was also capable of lowering caltrin-stimulated calcium uptake in bovine sperm to control levels.

Data on enhancer caltrin fluorescence in the pres- ence of phosphatidylserine from bovine brain suggest conformational changes in the protein due to binding of the phospholipid. In comparison, the phosphatidyl- choline from bovine brain appeared not to alter en- hancer caltrin.

(1979). Although freshly isolated caltrin initially behaves as an inhibitor of calcium uptake in bovine sperm (Rufo et al., 1983), it subsequently undergoes a remarkable transformation with time to become an enhancer of calcium uptake. This observation led us to speculate that a similar transformation of caltrin might take place when the sperm is about to undergo the acrosome reaction and hyperactivation, processes which are known to be calcium-dependent (Singh et al., 1978; Yan- agimachi and Usui, 1974).

If indeed the initial role of caltrin (as it occurs in bull semen) is the prevention of premature calcium uptake by freshly ejaculated sperm, the seminal plasma should be able to maintain caltrin in a conformation that is inhibitory to this influx. The transformation to enhancer of calcium uptake would then be due to a conformational change in caltrin induced by the appropriate medium, i.e. oviductal fluid or egg outer layers. Bull seminal plasma components range from macromolecules such as glycoproteins, enzymes, and various other proteins, to prostaglandins, phospholipids, and small molecules and ions such as fructose, citrate, L-lactate, and amino acids (Mann, 1964). It is likely that, in semen, caltrin is complexed with specific components of the seminal plasma and that such interaction enables it to maintain its inhibitory conformation. The present paper investigates the possibility that certain small molecule components of bovine seminal plasma may function as inducers of the inhibitory conforma- tion of caltrin.

MATERIALS AND METHODS’

Bovine semen was a gift from American Breeder’s Service, De- Forest, WI and was always kept at ambient temperature during transit and prior to use. Bovine caudal epididymides were collected from local abattoirs and kept on ice until just before extrusion of the spermatozoa, when they were allowed to attain room temperature.

Caltrin is a bovine seminal plasma protein (Afr 5411) of seminal vesicle origin (Singh, 1980; Rufo et al., 1982; Shivaji et al., 1984) which was found to prevent the entry of external calcium into sperm cells (Singh, 1980; Rufo et al., 1982), presumably by binding to calcium porters located in the region of the plasma membrane over the acrosome and the principal tail (San Agustin et al., 1987). Caltrin was shown by Lewis et al., (1985), upon comparison of amino acid residues, to be identical with seminalplasmin, the antimicrobial protein from bovine seminal plasma described by Reddy and Bhargava

* This work was supported by National Institutes of Health Grant AM 10,334. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ To whom correspondence should be addressed: Institute for En- zyme Research, 1710 University Ave., Madison, WI 53705.

Chemicals and materials used were from the designated sources: YM-2 Diaflo membranes. Amicon: Dowex 1 (OH-). Dowex 50 (H’). protein assay reagent concentrate, Bio-Rad; carbo&cyanide-m-chid: rophenylhydrazone, Calbiochem; A23187, Lilly; Acrb I%SA (0.45 pm), Gelman: ultroeel AcA 202. LKB Bromma: %aCl,. DuPont-New England Nuclear; CM Sephadex C-50, DEAE-Sephadex A-50, PBE 94, phenyl-Sepharose CL-4B, Sephadex G-25 F, Sephadex G-50 SF, Sephadex G-75, Pharmacia LKB Biotechnology Inc.; cardiolipin (bo- vine heart), DTT,’ dilauroyl-PE, dimyristoyl-PC, dipalmitoyl-PC,

1 Portions of this work (including part of “Materials and Methods”) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are included in the microfilm edition of the Journal that is available from Waverly Press.

’ The abbreviations used are: DTT, 1,4-dithiothreitol; MOPS, mor- pholinopropanesulfonic acid; PC, phosphatidylcholine; PE, phospha- tidvlethanolamine: PG. uhosphatidvlglvcerol: PI, phosphatidylinosi- tol; PS, phosphatidyl-i-ierine; SDS-PAGE, sod&m dbdecyl-sulfate polyacrylamide gel electrophoresis; DSP, deproteinized seminal plasma.

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Bovine Seminal Plasma Modulates Caltrin Activity 6861

dipalmitoyl-PE, dipalmitoyl-PG, sn-glycerol-3-phosphoryl-L-serine, PC (bovine brain), PE (bovine liver), PI (soybean), PS (bovine brain), Sigma.

Purification of Cc&in-Homogeneous caltrin was obtained from bovine seminal plasma using either the urea/DTT method or the phenyl-Sepharose method.

Preparation of Homogeneous Cultrin by Urea Denaturation and DTT Reduction of Coeluting Bovine Seminal RNase-The first three steps of caltrin purification were as described by Rufo et al. (1982, and see Table I), except that the column buffer of step 3 was 10 mM Tris, pH 8.5, with 50 mM NaCl. After pooling and concentrating (using YM-2 Diaflo membrane), the caltrin-rich fractions were made 50 mM in DTT and 8 M in urea. This treatment causes bovine seminal RNase to uncoil. Nitrogen was bubbled through the denatured and reduced protein solution which was then allowed to stand overnight. The protein sample was fractionated in a Sephadex G-50 SF column (1.6 x SO cm) using as elution buffer 100 mM acetate previously adjusted to pH 3.6 with pyridine. The column flow rate was main- tained at 3.5 cm. h-i. Uncoiled bovine seminal RNase eluted ahead of caltrin (San Agustin, 1988). Fractions from the caltrin peak were then assessed for purity by SDS-PAGE and the fractions containing homogeneous caltrin were pooled and lyophilized.

Calcium Up&e Assay-The preparation of bovine epididymal cells for use in the assay followed the procedure described earlier (San Agustin et al., 1987). The assay medium consisted of 10 mM MOPS, pH 7.4,110 mM NaCl, 5 mM KCl, 0.50 mM NaH2P04, and 10 mM DL- P-hydroxybutyrate as substrate. Caltrin (74 or 37 fiM final concentra- tion) was mixed with the assay medium first. Bovine seminal plasma components were added to the medium 5 min after caltrin and 10 min before the cell suspension (final cell count, 4 x lo7 cells/ml). Ten min after addition of cells to the medium, CaCl* containing 45Ca2+ (specific activity 12,000-15,000 cpm/nmol, 0.5 mM final con- centration) was introduced to start the assay. Caltrin was always kept on ice prior to addition to the assay medium and calcium uptake of the spermat,ozoa was always determined at 30 “C. Removal of cell suspension aliquots (at least 225 ~1) from the assay medium at particular times, vacuum filtration of the aliquots to separate the cells from the medium, washing of the filtered cells, and subsequent counting for radioactivity accumulated in these cells were as described before (San Agustin et al., 1987).

Removal of Proteins from Seminal Plasma and Extraction ofAnionic Components with Ether-Fresh bovine semen (lo-20 ml) was centri- fuged at 12,000 X g for 15 min and the seminal plasma separated from the pelleted spermatozoa. All operations were carried out at 4 “C. The collected seminal plasma was acidified to pH 1.5 with 10% HClO,, with constant stirring, in order to precipitate the seminal plasma proteins, The resulting paste was then centrifuged at 25,000 X g for 30 min and the coagulated proteins discarded. Ether-soluble components were extracted twice with 5 volumes of ether. In other experiments the pH of the recovered plasma was slowly brought back to the pH of the original seminal plasma with concentrated KOH. The precipitated KClO, was removed by centrifugation at 600 X g for about 5 min. The clear supernatant was next treated overnight with dry Dowex 50 (H+ form) to remove residual caltrin and seminal plasma calcium. The deproteinized seminal plasma prepared thus (DSP) was used in the calcium uptake experiments. Further treat- ments to DSP are as described in Table II.

Caltrin Fluorescence at Various Concentrations of PS and PC- Lyophilized enhancer caltrin was dissolved in buffer (10 mM MOPS, pH 7.4, 0.15 M NaCl) to give a stock solution of 60 PM (0.32 mg/ml) and then filtered (using Acre LC3A, 0.45 pm). PS and PC from bovine brain, supplied as chloroform solutions, were freed of the chloroform by vacuum evaporation and dissolved in filtered buffer by bath sonication under argon to a concentration of 1.4 mM each. The formation of vesicles was monitored at 20-min intervals by measure- ments of the light scatter at 280 nm of a lo-fold dilution of the phospholipid solution. Usually the scatter became constant after 1 h of sonication, and sonication was then discontinued. The final mix- ture had 6 PM enhancer caltrin (0.032 mg/ml) and PS or PC from 0 to 80 pM in buffer with a final volume of 0.70-0.75 ml. All manipu- lations were done at 25 “C. Quartz cuvettes with a path length of 0.30 cm were used to reduce inner filter effects. The fluorescence intensity of enhancer caltrin at the emission maximum (355 nm) was linear up to a concentration of about 10 PM. The fluorescence spectra of caltrin at the chosen PC or PS concentrations were obtained using a photon- counting spectrofluorimeter (SLM 8000, Des Plaines, IL) attached to a Hewlett-Packard 9825A calculator and stored through a calculator program (SLM PR8025). The spectra of control solutions (PS or PC

without caltrin) were also taken and subtracted from the spectra of the corresponding solutions containing caltrin. The contribution of scatter caused by the phospholipid vesicles to the observed fluores- cence of caltrin was negligible as verified through the unchanged fluorescence spectra of the tripeptide Gly-Trp-Gly (Dufourcq and Faucon, 1977) in solutions containing these vesicles. Both Gly-Trp- Gly and Lys-Trp-Lys did not bind to the phospholipid vesicles under the experimental conditions employed. The latter binds to PS vesicles

TABLE I Use of cation exchanger during purification of caltrin transforms it

from inhibitor to enhancer of calcium transport into sperm

Protein cont. in assay Calcium uptake,

medium, nmol/lOR

IIdd cells/l5 min”

Purification method of Rufo et al., 1982

Step 1. Ammonium sulfate pre- cipitation

3.0 2.3

Step 2. Anion exchanger (PBE 94)

1.5 0.63

Step 3. Gel permeation (Sepha- dex G-50 SF)

o.40b 0.14

Step 4. Gel permeation (Ultrogel AcA 202)

Control

Purification method of Reddy and Bhargava, 1979

Step 1. Dialysis Step 2. Anion exchanger (DEAE-

Sephadex A-50)

0.40 0.39

None 7.4

3.0 5.5 1.5 0.81

Step 3. Cation exchanger (CM- 1.0 14.3 Sephadex C-50)

Step 4. Gel permeation (Sepha- 0.40b 15.0 dex G-75)

Control None 8.1

y Assay of caltrin activity for the two methods was done on two different sperm samples.

* Caltrin is about 85% pure after this step.

TABLE II Treating enhancer caltrin with acidified (deproteinized) bovine

seminal plasma results in recovery of inhibitory activity Bovine epididymal spermatozoa (2.0 x lo7 cells) were suspended

in 0.50 ml of assay medium containing 10 mM DL-fl-hydroxybutyrate. Bovine seminal plasma preparations comprised 15% of the volume of sperm suspension during the assay. Dry Dowex 1 (OH-) was used to remove anions from DSP overnight. Enhancer caltrin concentration was 37 +M in all cases. All preparations were obtained from one starting sample of bovine seminal plasma as described under “Mate- rials and Methods.” Calcium uptake is the average of three trials using the same cell population. Inhibition by the ether extract of deproteinized seminal plasma in absence of caltrin results from chelation of calcium by the citrate in the extract. DSPA, DSP through anion exchange; DSPE, DSP ether extract; ClCCP, carbonylcyanide- m-chlorophenylhydrazone.

Assay medium containing Calcium uptake

None Enhancer caltrin

A23187, 30 /LM clccp, 5 PM

DSP DSP + enhancer caltrin

DSPA DSPA + enhancer caltrin

DSPE DSPE + enhancer caltrin

nmol/lOR cells/l5 min

6.6 + 0.4 14.0 k 0.6

16.7 f 0.8 0.2 k 0.2

4.1 + 0.2 1.8 + 0.3

4.8 f 0.2 4.3 -c 0.1

2.8 k 0.2 0.6 + 0.2

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6862 Bovine Seminal Plasma Modulates Caltrin Activity

“b.;’ 10 15

Citrate, mM

04 I , , .I 0 25 50 75 100

Cardlclipin, )I M

o-l I I , 0 50 100 150 200

Phosphatidylserine. PM

FIG. 4. Effect of ether-extractable components of bovine seminal plasma on the activity of enhancer caltrin. Citrate at various concentrations was included in the assay medium (A). A computer program was used to calculate the relative amounts of citrate and calcium needed to maintain a free calcium ion concentration of 0.50 mM during the assay. The total Na’ concentration was constant at 127 mM throughout the range of citrate concentrations used, with the Cl- concentrations decreasing correspondingly as the citrate concentrations were increased. All the other components of the assay medium as enumerated under “Materials and Methods” were present. To this assay medium with citrate, caltrin was added to a final concentration of 74 pM, followed by the cell suspension 10 min later. In all cases, the osmolality of the medium just before the addition of calcium was kept at 267 mosmol with sucrose. Addition of the calcium solution dropped the final citrate concentration to about 60% of the original concentration. The values plotted in A are citrate concentrations before calcium addition. In B-D, the order of addition to the assay medium was caltrin at 37 fiM, followed by the phospholipid, and then by the cell suspension. The phospholipid stock solution, at approximately 2 mg/ml, was previously sonicated on ice for 5 min at 4 amp under a stream of argon (model LS-75 Sonifier, Branson Instruments, Stamford, CT, fitted with a standard microtip from Heat Systems Ultrasonics, Long Island, NY). There was no significant difference in the results when ultrasonic irradiation was used instead of the longer bath sonication. The actual phospholipid concentration of the stock was determined using the phosphorus assay of Bartlett (1958). Calcium uptake after 40 min was determined as described under “Materials and Methods.” Control tubes (no caltrin in medium) contain the corresponding concentration of phospholipid. The pattern of results shown were reproducible in three different cell populations. 0, no caltrin in medium; 0, with enhancer caltrin in medium.

at lower ionic strength (Johns and Szabo, 1986). The excitation wavelength was 290 nm, and the temperature of the solutions was maintained at 25 “C by means of a thermostated cell holder. Concen- trations in labels of purchased phospholipids were verified according to the method of Bartlett (1959).

SDS-PAGE and Western Blotting-SDS-PAGE was carried out according to Laemmli (1970) using the mini-Protean II system from Bio-Rad. The stacking and running gels were 7.5 and 15% acrylamide, respectively. The procedure followed for Western blotting was as described earlier (Coronel et al., 1988).

Protein Assay-Protein concentrations were routinely determined according to the method of Bradford (1976).

RESULTS AND DISCUSSION

In early attempts at isolating homogeneous caltrin from bovine seminal plasma complexities were encountered due to several factors. For instance, we found that the activity of caltrin as inhibitor of calcium uptake in bovine epididymal spermatozoa was sensitive to the conditions employed in its isolation (San Agustin et al., 1987) as suggested earlier (Singh, 1980). Moreover, the monomer of the bovine seminal plasma RNase (named “bovine seminal RNase” (D’Alessio et al., 1981)) usually copurified with caltrin under the mild condi- tions (Rufo et al., 1982) necessary for the observation of the inhibitory activity of caltrin. And while the purity of the final caltrin preparation could be improved, it could be done only at the expense of activity. Since then we have developed procedures which yield homogeneous caltrin (see “Materials

and Methods”) but the final caltrin preparation per se usually does not show inhibitory activity.

The loss of inhibitory activity of caltrin is either gradual or fast depending on the purification method employed. This is illustrated in Table I which compares two early purification procedures, both yielding caltrin preparations of about the same purity (85% caltrin by gel densitometer scans). Inhibi- tory activity could be observed after every step of the method of Rufo et al. (1982) provided the purification was done quickly, i.e. within 24 h. Loss of activity inevitably followed, however, during storage (San Agustin et al., 1987). There was inhibitory activity in caltrin fractions in the method of Reddy and Bhargava (1979) until after the third step, which involved binding of caltrin to a cation exchanger. Not only was the inhibitory activity lost; it was replaced by an opposite, en- hancing effect on calcium uptake. The above observations indicated a profound influence on caltrin activity of ionic interactions, which could conceivably induce conformational changes in a small, disulfide-free protein like caltrin (Lewis et al., 1985) with relative ease. Because the seminal plasma is inhibitory to calcium uptake (Babcock et al., 1979), caltrin must initially function as an inhibitor of calcium uptake, most likely with the aid of anionic cofactors in the seminal plasma which stabilize the inhibitory conformation of caltrin. Once removed from the seminal plasma, caltrin eventually becomes an enhancer of calcium uptake either because the cofactors

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Bovine Seminal Plasma Modulates Caltrin Activity

dissociate or are altered and caltrin assumes a new confor- mation and a new biological activity.

Effect of Deproteinized Seminal Plasma on the Activity of Homogeneous Enhancer Caltrin-Table II summarizes the results of calcium uptake experiments with bovine seminal plasma preparations added to the assay medium containing enhancer caltrin. In combination with deproteinized seminal plasma (DSP), enhancer caltrin reverted to the inhibitory form. Restorative cofactors were thus shown to be present in DSP. Any possible cofactor for inhibitory caltrin would most likely be anionic since 1) the cofactor was not removed by the cation exchanger Dowex 50 (see “Materials and Methods”), 2) caltrin is a basic protein and hence has a net positive charge at the pH of the seminal plasma, and 3) from Table I, the capacity of caltrin to inhibit is lost after adsorption on a cation exchanger, indicating the possibility of conformational changes in the molecule upon loss of bound anions. After incubating the deproteinized seminal plasma with an anion exchange resin overnight, the restorative capacity of DSP was lost (DSPA in Table II), a confirmation that the cofactor(s) was anionic. Furthermore, it can be extracted into ether at low pH, when presumably it is uncharged (DSPE in Table II). It was recognized at this point that more than one anion could be involved because the seminal plasma of the bull contains several components that are anions at the pH range of the seminal plasma, i.e. organic acids, amino acids, and phospholipids (Mann, 1964).

The organic acids present in semen (Mann, 1964) are quite soluble in ether; amino acids in general are not, and those phospholipids with unsaturated acyl chains are readily soluble (Baer et al., 1956; Baer and Mahadevan, 1959; Baer and Maurukas, 1952; Baer and Buchnea, 1959). High performance liquid chromatography of the ether extract of seminal plasma (using the Aminex HPX column of Bio-Rad) permitted the identification of citric and L-lactic acids in the extract, and thin layer chromatography in silica gel detected the presence of the anionic phospholipids PS, PG, and cardiolipin, but not of PC and PE (not shown). The latter two are also found in bovine seminal plasma (Purse1 and Graham, 1967; Clegg and Foote, 1973).

Noncovalent interactions of phospholipids with various proteins are known. Several cytoskeletal proteins, i.e. vincu- lin, spectrin, and protein 4.1, were shown to associate with specific phospholipids (Ito et al., 1983; Niggli et al., 1986; Cohen et al., 1986; Sato and Ohnishi, 1983); glucagon is an amphiphilic protein which complexes with PC (Epand et al., 1977); the basic and amphiphilic protein mellitin was shown to interact with PS and PC (Dufourcq and Faucon, 1977). Thus, caltrin, an amphiphilic and basic protein, might also be expected to interact with the ether-soluble anionic phos- pholipids. The ether-soluble (and ether-insoluble) phospho- lipids of bovine seminal plasma do bind to Dowex l-acetate (Hammerstedt, 1974) and according to the results presented in Table II (uide, caltrin + DSPA), some or all of them must be able to restore, at least to a certain degree, the inhibitory activity of caltrin.

Restoration of Inhibitory Activity of Caltrin: Action of Spe- cific Components of Bovine Seminal Plasma on the Activity of Enhancer Caltrin-Citrate, at 30-60 mM (Mann, 1964), is a major ionic component of the seminal vesicle secretion. The seminal vesicle of the bull is the principal source of both citrate (Hess et al., 1960) and caltrin (Singh, 1980; Rufo et al., 1982; Shivaji et al., 1984) found in the ejaculate. At pH 7.4 citrate could not restore the inhibitory activity of caltrin in the range of the citrate concentrations used, although it diminished the stimulatory effect on calcium uptake by en-

hancer caltrin (Fig. 4A). In control experiments in which citrate was replaced with three equivalents of Cl- (citrate is mostly trivalent at pH 7.4), the activity of enhancer caltrin was not altered (not shown). At a lower pH citrate restores the inhibitory activity of caltrin (San Agustin, 1988).

The ether-soluble phospholipids were found to have distinct effects on enhancer caltrin activity. Because seminal plasma phospholipids are not available commercially, phospholipids from other sources were used in our experiments. At the caltrin concentration used (37 PM), inhibitory caltrin was expected to lower the calcium uptake of bovine epididymal spermatozoa (at 4.0 X lo7 cells/ml) by 50% (Rufo et al., 1982).

The presence of PS (from bovine brain) in the assay mixture enabled enhancer caltrin to revert to its inhibitory form at PS concentrations higher than about 70 PM, or a 2:l molar ratio of PS to caltrin (Fig. 4B). The reported concentration of PS in the seminal plasma is about 40 PM (Clegg and Foote, 1973).

With cardiolipin, a slightly different effect was observed (Fig. 4C). Increasing the concentration of cardiolipin caused an increase in the calcium uptake of the sperm cells. This is

O-4 0 100 200 300

Phosphatidylcholine, pM

I

50 22 g 40

-0

2 30 c

3 20 d 4 10

s ” Y I I I I t . I

0 50 100 150 200 250

Phosphatidylethanolamine. pM

o! I 0 50 100 150

Phosphatidyilnositol, KM

FIG. 5. Effect of other phospholipids on the activity of en- hancer caltrin. The same protocol for the measurement of calcium uptake by bovine epididymal spermatozoa as described in the legend of Fig. 4 for the ether-extractable phospholipids was employed. Shown are representative results of three or more experiments. 0, no caltrin in medium; 0, 37 fiM enhancer caltrin in medium.

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6864 Bovine Seminal Plasma Modulates Cabin Activity

so

f .A

50- -,

c 40. 0 g 30- i

FIG. 6. The acyl side chains of the 2 20

phospholipid may influence its ef- ; 10-y _

feet on the activity of enhancer cal- 0 o, 0

trin. Calcium uptake bv bovine sper- 0 100 200 300 400 matozoa in the presence of enhancercal- trin and phospholipid was determined as described in the legend of Fig. 4. For each phospholipid a different cell popu- lation was used because the experiments were done on different days. Each point is an average of two determinations. 0, no caltrin in medium; l ,37 pM enhancer caltrin in medium.

Dimyristoylphosphatldylcholine, PM

50 100 150 200 250

Dilauroylphosphalidylethanolamine. p M

TABLE III Phase transition temperatures for some phospholipids in excess water

Data from Silvius (1983).

Phospholipid Tc “C

Dimyristoyl-PC 23 Dilauroyl-PE 29 Dipalmitoyl-PC 41 Dipalmitoyl-PE 60

because cardiolipin causes lysis of the cells.” In the presence of enhancer caltrin, however, the increased calcium uptake caused by cardiolipin was not evident and was replaced by a significant inhibition of calcium uptake. The cardiolipin con- tent of seminal plasma was reported to be about 45 PM (Clegg and Foote, 1973). Cardiolipin at this concentration could prevent enhancer caltrin from stimulating calcium uptake in bull spermatozoa (Fig. 4C).

The occurrence of PG in bovine seminal plasma was not observed (Purse1 and Graham, 1967; Clegg and Foote, 1973; Jain and Anand, 1976). Most likely it was formed in our preparations by the acid hydrolysis of cardiolipin. Dipalmi- toyl-PG does not convert enhancer caltrin back to inhibitory caltrin even at the higher concentrations used (Fig. 40). PS and cardiolipin induced clumping of spermatozoa but in the presence of caltrin did not do so.

PC is found in the seminal plasma at about 260 PM and PE at about 135 PM (Clegg and Foote, 1973). Since they were not extracted into the ether layer, their fatty acyl side chains must be largely saturated (Baer et al., 1956). PC from bovine brain (which is soluble in ether) and PE from bovine liver did not lower the activity of enhancer caltrin (Fig. 5, A and B). It is not clear from the literature whether PI is found in bovine seminal plasma. Previous authors apparently could not distin- guish it from PS (Clegg and Foote, 1973; Jain and Anand, 1976). We did not detect the presence of PI in our prepara- tions. Because it is anionic, its effect on enhancer caltrin was included here for comparison with the other anionic phospho- lipids. PI prevented the calcium uptake stimulation by en- hancer caltrin (Fig. 5C) but did not cause the protein to become an inhibitor.

The influence of the acyl side chains on the interaction of

‘J. T. San Agustin and H. A. Lardy, manuscript in preparation.

Dipalmiloylphosphafidylethanolamine. p M

Wavelength. nm Wavelength, nm

0 3 6 9 12 15

P= Cphosphol,ptJ]/Ccaltr~nl

FIG. 7. Fluorescence of enhancer caltrin in the presence of phosphatidylserine and phosphatidylcholine. Fluorescence spectra of 6.0 pM caltrin, using A., = 290 nm, were recorded in the presence of various concentrations of the phospholipid vesicles. A, PS from bovine brain. B, PC from bovine brain; P = [phospholipid] /[caltrin]. In C, a plot was made of the relative increase in fluorescence intensity at 343 nm uer.su.s P; AI = I - IO, where I is the fluorescence intensity of caltrin in the presence of the phospholipid and IO is the fluorescence intensity of enhancer caltrin alone. Shown are the results of three experiments with PS (0, n , A) and a representative experi- ment with PC (0).

the phospholipid with caltrin was discovered when synthetic PC and PE, which are not soluble in ether (in this respect similar to seminal plasma PC and PE), were used. Dimyris- toyl-PC caused a lowering of the stimulatory effect of en- hancer caltrin to near control levels (Fig. 6A). On the other hand, dipalmitoyl-PC, dilauroyl-PE, and dipalmitoyl-PE did not (Fig. 6, B-D). The above results may be explained by comparing the various gel-to-crystalline-liquid transition

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Bovine Seminal Plasma Modulates Caltrin Activity

temperatures of the PC and PE vesicles used (Table III). Dimyristoyl-PC vesicles, with a transition temperature of 23 “C, have sufficient fluidity at the assay temperature (30 “C) to be able to interact with caltrin. In the case of dilauroyl-PE (transition temperature, 29 “C), either the vesicles are not fluid enough or the unshielded charge on the primary nitrogen of the headgroup prevents the interaction with enhancer caltrin. Dipalmitoyl-PG has a transition temperature of 41 “C and this could also explain why it had no effect on the activity of enhancer caltrin. Such dependence of phospholipid-protein interaction on the temperature was also observed with melli- tin (Dufourcq and Faucon, 1977). Thus, although caltrin is an amphiphilic protein, its nonspecific hydrophobic interac- tion with the acyl groups of phospholipids is not strong enough to disrupt the acyl hydrophobic bonds in the vesicles below the transition temperature. It would appear that for the caltrin-phospholipid complex to exert an inhibitory effect on calcium uptake, an additional and specific interaction through the phospholipid headgroup is needed. Zwitterionic phospho- lipids, because they cannot favorably interact with cationic sites on caltrin, are unable to produce an inhibitory complex above the transition temperature. PI, although anionic like PS, is not as effective as PS in producing this complex. It is possible that the observed difference in the effects of PI and PS may be due only to the difference in their transition temperatures. Another question that needs to be answered is whether the PS-caltrin interaction does in fact take place below the PS transition temperature. No data on the transi- tion temperatures of PS and PI used in our experiments are available, and so these important considerations need further investigation. We know, however, that extensive clumping of spermatozoa takes place when PS, PI, and cardiolipin vesicles are added to the cells in our calcium uptake experiments. This could be an indication that the vesicles have considerable fluidity, and hence that their transition temperatures are below the incubation temperature (fatty acid analysis of PS and PI showed about 55% unsaturation for both).4 It is valid to propose that a stronger headgroup interaction with caltrin, found in PS and possibly PI but not in the other phospho- lipids, accounts for the differences in their effects on enhancer caltrin. The acyl groups are important in the reversal of caltrin activity, since sn-glycero-3-phosphoryl-L-serine cannot con- vert enhancer caltrin back to inhibitory caltrin (data not shown).

Caltrin Fluorescence in the Presence of PS and PC-Caltrin has only 1 tryptophanyl residue and 1 tyrosyl residue in its total of 47 residues (Lewis et al., 1985). Interpretation of changes in its intrinsic fluorescence is therefore relatively simple and straightforward since this fluorescence would be predominantly due to the single tryptophanyl residue in the molecule. No tyrosine emission fluorescence of caltrin is ob- served (Theil and Scheit, 1983; San Agustin, 1988).

The fluorescence spectra of caltrin in the presence of PS and PC (both from bovine brain) were collected (Fig. 7). Increasing the concentration of PS from 0 to 77.3 pM caused a blue shift in A,,,, from 355 to 343 nm and a progressive increase in fluorescence of a 6.0 pM solution of enhancer caltrin (Fig. 7A), i.e. in the presence of PS the tryptophanyl residue in caltrin moved to a hydrophobic environment. There was actually a decrease in the fluorescence intensity of caltrin when the PS vesicles were formed by ultrasonic irradiation in air, hence this hydrophobic environment must be due to the fatty acyl chains of PS. The quenching of fluorescence is attributed to exposure of the tryptophanyl residue of caltrin to portions of the fatty acyl chains oxidized during the soni-

’ Sigma Chemical Co., personal communication.

A

a bcdefg

C

6865

B

h abcdefgh

D

a bcdefgh abcdefgh

FIG. 8. SDS-PAGE and Western blot of bovine epididymal spermatozoa proteins showing caltrin binding to the cells even in the presence of PS and PC. Bovine epididymal spermatozoa (1 X 10’ cells) were incubated for 25 min with enhancer caltrin (37 pM) plus the desired concentration of PS or PC (both from bovine brain) from 0 to 240 pM (total volume, 0.250 ml), and then spun down (16.000 X 8. 2 min). The incubation medium was identical to that used in thecalcium uptake experiments. The cell pellet was washed twice. each time with two volumes of NKM buffer (125 mM NaCI, 5 rni KCI, 10 mM MOPS, pH 7.4). The final pellet was then treated with 30 PI of SDS-PAGE sample buffer, heated for 5 min, centrifuged (16,000 x g, 2 min), and the clear supernatant separated from the undissolved cell material. SDS-PAGE was carried out on 7 PI of the supernatant. Two gels were prepared for each set of phospholipid concentrations, one for Coomassie staining (A and C) and the other for Western blotting (B and II). A and H: lane a, sperm + caltrin; lane b, sperm + caltrin + 32 pM PS; lane c, sperm + caltrin + 64 gM PS; lane d, sperm + caltrin + 96 PM PS; lane e, sperm + caltrin + 128 uM PS: lane f. sperm + caltrin + 160 MM PS; lane R, sperm + caltrin + 192 pM kS;‘lane h, 0.5 pg of caltrin. C and II: lane a, 0.5 gg of caltrin; lane b, sperm only; lane c, sperm + caltrin + 40 pM PC; lane d, sperm + caltrin + 80 GM PC; lane e, sperm + caltrin + 122 pM PC; lane f, sperm + caltrin + 160 pM PC; lane g, sperm + caltrin + 200 pM PC; lane h, sperm + caltrinf 240 FM PC.

cation process (Hauser, 1971; Dufourcq and Faucon, 1977). Although argon streaming prevented this oxidation to a cer- tain extent, bath sonication was used in the fluorescence experiments reported here because the results were more reproducible. From studies on the binding of phospholipids to plasma lipoproteins, it is known that such binding is usually accompanied by changes in the circular dichroism spectrum of the lipoprotein that indicate an increase in its a-helical content (Segrest et al., 1974). With PS present in the range of concentrations employed in our experiments here, caltrin in fact shows a large increase in its n-helical content.”

In contrast to the effect of PS, PC caused no blue shift in X,,, and only a slight increase in fluorescence intensity, most likely due to light scattering by the vesicles (Fig. 7B). Binding of PC to caltrin under the conditions employed appears to be practically nil.

’ Unpublished experiments conducted in collaboration with Michael Reily.

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6866 Bovine Seminal Plasma Modulates Caltrin Activity

The above results are consistent with the observed calcium uptake of bovine epididymal spermatozoa in the presence of enhancer caltrin and PC or PS (see Fig. 5A and Fig. 4B). Thus PC does not affect the calcium uptake stimulation of enhancer caltrin because there is no physical interaction between them. On the other hand, PS causes a reversal in the activity of enhancer caltrin because the formation of a caltrin- PS complex results in a caltrin conformation which is inhib- itory to calcium uptake. A plot of the relative increase in fluorescence of caltrin, (AI/Io)343, versus the molar ratio of phospholipid to caltrin shows that maximum enhancement of fluorescence is attained at a PS/caltrin molar ratio of about 6 (Fig. 7C).

To test the possibility that the PS vesicles simply compete with the spermatozoa to make caltrin unavailable, bovine spermatozoa incubated with caltrin and PS (Fig. 8, A and B) and with caltrin and PC (Fig. 8, C and D) were subjected to SDS-PAGE and Western blotting. Caltrin was bound to the cells even at the highest concentrations of PS and PC used, where the phospholipid/caltrin molar ratio was about 6.

The results presented in this paper indicate regulation of caltrin activity by anions in bovine seminal plasma, with PS promoting the inhibitory function of caltrin to a greater extent that the others. We earlier proposed that caltrin bound to the sperm surface changes conformation (and hence function from inhibitor to enhancer of calcium uptake) at some point during the movement of the sperm through the female repro- ductive tract (San Agustin et al., 1987). Removal of PS from caltrin, for example by the action of phospholipases or by hydrophobic competition, then allows this transformation to take place on the sperm surface. When caltrin becomes an enhancer of calcium uptake it would be expected to promote the acrosome reaction and thus facilitate fertilization. We have evidence which shows that enhancer caltrin causes the release of hyaluronidase, an acrosomal enzyme, from bovine spermatozoa.3 At this point then, enhancer caltrin may be considered a significant factor in the development of the acrosome reaction in bovine spermatozoa.

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J T San Agustin and H A Lardycalcium-transport regulating protein of bovine spermatozoa.

Bovine seminal plasma constituents modulate the activity of caltrin, the

1990, 265:6860-6867.J. Biol. Chem. 

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Additions and Corrections

Vol. 265 (1990) 5361-5363 Vol. 265 (1990) 6860-6867

Mutagenesis of a single hydrogen bond in cytochrome Bovine seminal plasma constituents modulate the ac- P-450 alters cation binding and heme solvation. tivity of caltrin, the calcium-transport regulating pro-

tein of bovine spermatozoa. Carmelo Di Primo, Gaston Hui Bon Hoa, Pierre Douzou,

and Stephen Sligar Jovenal T. San Agustin and Henry A. Lardy

Page 5362, right column, line 17 should read: “Hydro- Page 6867, Fig. 3: The fraction numbers in the figure static pressure is also known to force heme solvation favoring legend are incorrectly labeled. In lanes d-n, 86 should be the low-spin form (21, 22) with dissociation of cation (23).” added to each fraction number to correspond to their numbers

in Fig. 2. Page 5363, Ref. 23 should be: Hui Bon Hoa, G., Di Primo, C., Geze, M., Douzou, P., Kornblatt, J., and Sligar, S. (1990) Biochemistry 29,6810-6815

We suggest that subscribers photocopy these corrections and insert the photocopies at the appropriate places where the article to be corrected originally appeared. Authors are urged to introduce these corrections into any reprints they distribute. Secondary (abstract) services are urged to carry notice of these corrections as prominently as they carried the original abstracts.

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