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Molecular forms of alkaline phosphatase of ram seminal plasma — Some properties and changes in pathological process of reproductive organs
Animal Reproduction Science, 3 (1980/1981) 307--323 Elsevier Scientific Publishing Company, Amsterdam - - Printed in The Netherlands
307
MOLECULAR FORMS OF ALKALINE PHOSPHATASE OF RAM SEMINAL PLASMA -- SOME PROPERTIES AND CHANGES IN PATHOLOGICAL PROCESS OF REPRODUCTIVE ORGANS J. GLOGOWSKI and J. STRZEZEK
Agricultural-Technical Academy, Department of Animal Biochemistry, Institute of Animal Physiology and Biochemistry, 1 O-718 Olsztyn (Poland) (Accepted 22 April 1980)
ABSTRACT Glogowski, J. and Strze~.ek,J., 1981. Molecular forms of alkaline phosphatase of ram seminal plasma -- some properties and changes in pathological process of reproductive organs. Anita. Reprod. Sci., 3: 307--323.
Three molecular forms of alkaline phosphatase were isolated from ram seminal plasma. These forms, activated with Mg 2+ ions, were characterized by very similar pH optima, K m constant, and molecular weight. They differed in electrophoretic mobility, the latter being most probably determined by the different position of N-aeetylneuraminyl groups in protein structures. Sialic acid also played a protective function for the catalytic centre. Isolated molecular forms possessed antigenic properties. Immunological serum for phosphatase proteins either inhibited or stabilized activity of alkaline phosphatase, depending on the value of the protein ratio. During experimentally induced inflammation of ram reproductive organs, a gradual decrease of the activity of alkaline phosphatase was noted, together with changes in its electrophoretic profile. This phenomenon is most likely caused by intensive synthesis of sialic acid in pathologically changed reproductive organs o f the ram.
INTRODUCTION
Animal semen, and especially ram semen, is characterized by high alkaline phosphatase activity. Catalytic properties of this phosphatase are not fully known as yet. Among others, Strzezek and Glogowski {1979) isolated two molecular forms of alkaline phosphatase from bull seminal plasma, and defined their biochemical and immunological properties. Veselsky (1972) showed, by immunoelectrophoresis, activity of alkaline phosphatase in fluids from the accessory sex glands of the boar. As regards ram alkaline phosphatase, only Zilcov {1974) defined its activity in different reproductive organs. He showed that testicles and epididymis constitute the main source of this enzyme in ram seminal plasma. Heterogeneity of phosphatase proteins of animal semen allows observations to be made not only on physiological phenomena, but also on pathological changes in male reproductive organs. 0378-4320/81/0000--0000/$02.50 © 1981 Elsevier Scientific Publishing Company
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The scope of this work was to isolate molecular forms of alkaline phosphatase from ram seminal plasma, and to define its biochemical and immunological properties. An attempt was also made to define changes in these properties during the inflammation process of the ram reproductive tract. MATERIALS
AND METHODS
Ram semen was collected from animals of the Polish Woolyhaired breed by artificial vagina. In order to separate spermatozoa the sample was centrifuged for 10 min at 10,000 g at 4 ° C. Seminal plasma was stored at - 2 0 ° C until the isolation process. Stages of isolation and purification are presented in Fig. 1. Raw proteins were determined by measuring absorption at 280 nm, and Fig. 1. Purification procedure for the isolation of alkaline phosphatase from ram seminal plasma Step
Procedure and product (temp. 4°C) R a m semen I
10,000 g
II
III
I I
10 min
Spermatozoa (discarded)
Seminal plasma
Supernatant (discarded)
Precipitate
Protein without alkaline phosphatase activity (discarded)
Protein fractions with alkaline phosphatase activity
Protein without alkaline phosphatase activity (discarded)
Three alkaline phosphatase peaks
Ethanol (95%) previously chilled to --10°C and slowly added to the plasma to a concentration of 65%. Centrifuged at 13,000 × g, 15 min.
Dissolved in 0.05 M Tris--HC1 buffer, pH 7.7. Applied to a Sephadex G-200 column (2.5 × 45 cm). The proteins eluted with the same buffer at a flow rate of 1 ml per 20 min and 3 ml fractions were collected for protein and enzyme assays.
DEAE-cellulose column (1.8 x 30 cm) equilibrated with 0.05 M Tris--HCl buffer. All the proteins were absorbed on the column. The column was then treated successively in a batchwise manner with 0.05 M Tris-HCl followed by NaC1 solutions of increasing molarity (from 0.1 M to 0.6 M) in the same buffer. The 4 ml fractions were collected for protein and enzyme assays.
309
quantitatively by the biuret method according to Weichselbaum (1946), and by the method of Lowry et al. (1951) with the Folin phenol reagent, depending on the stage of isolation.Mobility and electrophoretic homogeneity of the isolatedmolecular forms were determined by electrophoresis on polyacrylamide gel according to Davis (1964) and Ornstein (1964) and by electrophoresison agarose gel according to Wieme (1959). Identification of molecular forms of the enzyme was by the histochemical method of Seligman et al. (1951) using sodium/3-naphthylphosphate and Fast Blue R R in 0.1 M veronal buffer at p H 9.1. Alkaline phosphatase activitywas determined by the method of Bessey et al. (1946). A unit of enzyme activityis defined as the quantity of enzyme which liberates1 ~mol of p-nitrophenol during 1 h. Specific activityof the enzyme is equal to the quantity of pmoles of p-nitrophenol liberatedduring 1 h per 1 m g of protein.
Methods for investigating biochemical and immunological properties of isolated molecular forms of alkaline phosphatase The Michaelis constant (K m ) for p-nitrophenylphosphate as a substrate was defined according to the Lineweaver-Burk (1936) method. Molecular weight o f particular forms was determined with thin-layer chromatography in gel Sephadex G-200 Superfine. In order to define the effect of sialic acid on kinetic properties of the enzyme, enzymatic splitting of this c o m p o u n d was made using a neuraminidase preparation from Vibrio cholerae, produced b y Koch Light Ltd, with activity of 500 units/ml. Preparations of particular molecular forms of alkaline phosphatase were incubated b y adding to 0.3 ml of the enzyme, 100 gl of neuraminidase, 10 pl of 0.01 M MgC12,10 pl of 0.01 M CaC12 and 50 pl of 0.4 M acetate buffer with pH 5.3. In control samples neuraminidase was substituted by acetate buffer. After 6 h incubation at 37 ° C, electrophoretic mobility of the molecular forms was studied on agarose gel, and then histochemical staining for alkaline phosphatase was carried out. The effect of the addition of sialic acid, and Mg 2÷ ions, on temperature stability of the enzyme was also defined. The following incubation mixtures were used: (a) 1 ml of the enzyme + 0.6 ml of H 2 0 (b) 1 ml of the enzyme + 0.4 ml of 0.01 M sialic acid + 0.2 ml of H: O (c) 1 ml o f the enzyme + 0.4 ml of H 2 0 + 0.2 ml of 0.1 M MgC12 (d) 1 ml of the enzyme + 0.4 ml of 0.01 M sialic acid + 0.2 ml of 0.01 M MgCI2 Immunological serum toward the isolatedmolecular forms was obtained by rabbit immunization with subcutaneous and intramuscular injections,using Freund's adjuvant. In immunological studies the method of double gel diffusion according to Ouchterlony (1958) was adopted. The effects of immunological serum on phosphatase activityand its catalyticproperties were also observed.
310
Methods for studying changes in properties of phosphatase proteins of seminal plasma after experimentally induced inflammation of testicles Inflammation of the reproductive organs of two rams was induced by injection into the intratesticles of 1.5 ml one-day liquid broth culture of Corynebacterium pyogenes, containing 109/ml bacteria cells. For 60 days, indices of semen quality were observed~ as was the clinical state of the reproductive organs. Biochemical analyses of seminal plasma were also made: content of free and bound sialic acid (Warren, 1959), total protein content by the biuret method, and activity of acid and alkaline phosphatase and of its molecular forms. RESULTS
Chromatographic separation of the phosphatase fraction on DEAE-cellulose was achieved after filtration on gel 8ephadex G-200, as presented in Fig. 2. Three components of phosphatase proteins were obtained.
600
500
E280 22
o
d
20
~, 400
18 16
~ 30C
14
5
12 10
2QC
08
q
Q6
10C
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Ol
100
150
200
2 4 0 Fraction number
Fig. 2. DEAE-cellulose column chromatography of protein fraction obtained after filtration on gel Sephadex G-200 ( protein 280 r i m ; - - - - - a c t i v i t y of alkaline phosphatase).
Component I, eluted at concentration of 0.1 M NaC1, showed specific activity toward p-nitrophenylphosphates 10 times higher than the initial activity of phosphatase in ram semen. Component II, the most active one, was eluted at 0.2 M NaC1. Its specific activity was 20 times higher. Component III, eluted at 0.4 M NaC1, was characterized by specific activity 5 times higher than the initial value.
311
i:ii!ii Fig. 3a. Electrophoresis on polyacrylamide gel of fraction obtained after DEAE-cellulose chromatography (staining for proteins). 1. I molecular form; 2. II molecular form; 3. III molecular form. Fig. 3b. Electrophoresis on polyacrylamide gel of fraction obtained after DEAE-cellulose chromatography (staining for alkaline phosphatase). 1. seminal plasma of ram; 2. I molecular form; 3. II molecular form; 4. III molecular form. In electrophoretic studies, both proteinograms and enzymograms (Fig. 3a and 3b), showed homogeneity of all forms, although c o m p o n e n t I was characterized by the highest electrophoretic mobility toward the anode. As in the enzymogram of alkaline phosphatase of ram semen, c o m p o n e n t III was characterized by the lowest electrophoretic mobility. In the immunodiffusion reaction according to Ouchterlony (1958) each specific antiserum gave one precipitation arc with the respective molecular form, both after staining with amide black for proteins, and after histochemical reaction for alkaline phosphatase (Fig. 4).
Fig. 4. Immunodiffusion in the following arrangements: 1. antiserum toward I form (central container) -- I molecular form; 2. antiserum toward II form (central container) -- II molecular form; 3. antiserum toward III form (central container) -- III molecular form. Staining for alkaline phosphatase,
312
Biochemical and immunological properties of molecular forms Molecular weight defined by thin-layer chromatography on gel Sephadex G-200 Superfine, amounted to a b o u t 210,000 for particular components. Fig. 5 presents the effect of pH on the rate of p-nitrophenylphosphate hydrolysis by molecular form II. This form is characterized by :an optimum pH range of 10.6--11.6, both with and without an addition of 2.5 mM Mg 2+ ions. However, in the presence of Mg 2+ ions, the activity of the enzyme is about 16% higher than in their absence.
320 300
.........
without c~dition of Mg2"ions w i t h 2.5 mM Mg2*io
200
~o
J
c~ 10C
.// ;
I
1'o
1'1
~'2
pH
Fig. 5. The effect of pH upon the rate of p-nitrophenylphosphate hydrolysis by molecular form II (50 mM glycine buffer; 7.5 mM p-nitrophenylphosphate). The pH optima for the components I and II did not differ from the values found for the III form. Maximal activities of isolated molecular forms of alkaline phosphatase were noted at pH 11.2. Particular components differed with respect to the rate of p-nitrophenylphosphate hydrolysis, depending on its concentration. In the case of molecular form II, the maximal rate of substrate hydrolysis occurred at a concentration of 8 mM (Fig. 6). For the I and III forms the corresponding concentrations were 7 mM and 3 mM respectively. Above these concentrations substrate inhibition was noted, and was most pronounced for c o m p o n e n t I. Michaelis-Menten constants (Km) for particular forms are presented in Table I. Km values obtained indicate that the isolated forms are characterized by very similar structure of their proteins, and also of the active centre. Electrophoretic heterogeneity of phosphatase proteins of ram seminal plasma is probably determined by the differences in electrostatic charge of particular forms. Studies on functions of sialic acid showed that treatment
313
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32.£ 300
, 20.C
i
~:0
2:0
3'0
4o
go
6'0
7:0
ao
90
~5.0
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Fig. 6. The effect o f p - n i t r o p h e n y l p h o s p h a t e c o n c e n t r a t i o n u p o n the activity of molecular f o r m II. TABLE I Michaelis-Menten constants (Kin) for particular forms toward p - n i t r o p h e n y l p h o s p h a t e (pH 11.2) Molecular forms
Value K m (M)
I II III
0.60 × 10 -3 1.35 × I 0 -s 1.00 × 10 -3
of phosphatase forms with neuraminidase results not only in a decrease of their electrophoretic mobility, but also in lowering of the enzymatic activity (Fig. 7). On the other hand, addition of sialic acid to the incubation mixture results in a slight increase of electrophoretic mobility, especially in the case of the I molecular form (Fig. 8). Simultaneous addition of Mg2÷ ions and sialic acid to the incubation mixture results in an increase of enzymatic activity of component II (Table II), whereas addition of only sialic acid to the incubation mixture decreases the activity of this enzyme. Long-term incubation at 37°C points to the stabilizing and protective role of sialic acid with respect to the active centre of alkaline phosphatase of ram seminal plasma. Immunoelectrophoretic and immunodiffusion observations suggest an antigen character of isolated components of alkaline phosphatase. Interesting results were obtained as regards the effect of specific antiserum on the activity of alkaline phosphatase. As seen in Fig. 9, the activity of
314 m o l e c u l a r f o r m II decreases with an increasing a m o u n t of i m m u n o l o g i c a l s e r u m p r o t e i n s i n t h e i n c u b a t i o n m i x t u r e . T h i s p h e n o m e n o n is e s p e c i a l l y c l e a r i n t h e case o f t h e f o r m I I a n t i s e r u m a n d t h e m o l e c u l a r f o r m II. T h u s , at a ratio of 0.75:1 antiserum proteins to enzyme proteins, activity of the II f o r m w a s 3 4 % l o w e r t h a n t h e a c t i v i t y n o t e d i n t h e p r e s e n c e o f c o n t r o l
®
1
2
2
3
T
4
--
Fig. 7. Electrophoretic mobility of molecular form II after 6 h incubation with neuraminidace: 1. sample with neuraminidase; 2. control sample (electrophoresis on agarose gel). Fig. 8. The effect of sialic acid addition upon electrophoretic mobility of I (1, 2) and II (3, 4) molecular forms of alkaline phosphatase. 1, 3 -- without sialic acid; 2, 4 -- with addition of sialic acid (electrophoresis on agarose gel). TABLE II The effect of Mg2÷ ions, sialic acid and incubation time on the enzymatic activity of molecular form II (initial activity of molecular form II, 100%) Incubation time at 37°C (h)
Content of incubated mixture II form + sialic acid
II form + Mg=÷ ions
II form + sialic acid + Mg2÷ ions
After mixing
85.9
104.7
100.0
Incubation: 6 12 24 48 72 192
42.3 83.3 88.1 83.3 88.1 102.5
140.4 233.3 238.1 200.0 211.9 185.0
153.8 223.8 223.8 195.2 211.9 180.0
315
30C
2QO
rn
~oo Y
Q162/1
Q325:1
075:1
15 1
3:1
6:1
antiserum proteins I ~ J o :. ]]" form. proteins
Fig. 9. T h e effect of various ratios of antiserum proteins toward molecular f o r m II upon its activity. 1. control serum; 2. antiserum toward seminal plasma; 3. antiserum toward molecular f o r m II.
Krn = 1.16x10-3 M 1 Krn = 0 9 2 x 1 0 "3 M 2 Krn3= 1 0 2 x 1 0 "3 M
vt
O~ O09
1 3
o
006 OO5
5
Fig. 10. The effect of antiserum toward molecular f o r m II on substrate affinity of this f o r m to p-nitrophenylphosphate. 1. c o n t r o l serum; 2. antiserum toward seminal plasma; 3. antiserum toward molecular f o r m II.
316 serum. Studies on the kinetics of the enzyme incubated with antiserum revealed changes in the maximal rate of p-nitrophenylphosphate hydrolysis by molecular form II. Graphical presentation of Km shows an increase in the substrate affinity of the enzyme (Fig. 10). In the case of seminal plasma (Fig. 11), activity of alkaline phosphatase with antiserum toward form II, with a ratio of 0.5:1 antiserum proteins to plasma proteins, was almost 40% lower than the activity obtained with control serum. On the other hand, an increase of antiserum level in the incubation mixture to a ratio of 2:1 stimulates activity of the enzyme in plasma. ~0
7,0
60
E 40
5
3.0
20
10
Q125:1
Q25:1
0.5:1
1:1
2:1
4:1
antiserum proteins Ratio: ....... seminal plasma proter~
Fig. 11. The effect of various ratios of antiserum proteins toward molecular form II upon activity of alkaline phosphatase of ram seminal plasma. 1. control serum; 2. antiserum toward seminal plasma; 3. antiserum toward molecular form II.
Changes in properties of alkaline phosphatase of ram seminal plasma after experimentally induced inflammation of testicles A culture of the bacterium Corynebacterium pyogenes was injected into the testicles of t w o rams (I and II). Before the experiments both rams possessed healthy reproductive organs and produced semen with normal properties. Injection of bacteria induced an inflammation process in the reproductive organs. The observed symptoms were visible swelling of the scrotum and hardening of the glandular tissue of the testicles. The pathological process was observed for 60 days. Throughout this time both rams retained sexual impulses. Studies of semen properties during this time showed that 10--15 days after the injection, a gradual decrease of spermatozoa concen-
317 tration in the semen commences (aspermy in the case of ram II), together with an increase of pathological forms of spermatozoa. Secondary changes of spermatozoa reached 90% in ram I and 80% in ram II. The activity of acid phosphatase in the seminal plasma decreased as the inflammation process progressed (lowering of activity by 70%), {Fig. 12). U 2500
U
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2000 1500
1500
1000
1000
500
500 i
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20
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i
50
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1200 1000
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c~vs
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Fig. 12. Activity of alkaline and acid phosphatase in ram (I and II) seminal plasma during experimentally induced inflammation o f reproductive organs, a -- alkaline phosphatase; b -- acid phosphatase.
In the case of alkaline phosphatase, changes of enzyme activity were less pronounced. A 10--20% drop of the activity was noted on the average, usually between days 10 and 20 of the disease. Later, however, the alkaline phosphatase activity remained at a high level. Electrophoretic studies on molecular forms of alkaline phosphatase showed changes of both electrophoretic mobility and activity (Fig. 13). It was found that after visible increase in the activity of molecular form I during the first phase of the inflammation process, this form gradually disappeared along with progressing pathogenesis. Molecular forms II and III retained high catalytic activity throughout the whole period of observations. Acute and chronic inflammation processes, similar to neoplasmatic diseases, are usually accompanied by an increase of glycoproteins in blood serum. The level of sialic acid cen constitute an indirect index of the intensity of destructive and proliferation processes in connective tissue of the inflamed organs. Due to the fact that alkaline phosphatase of seminal plasma is of
318
10
3O
60
0
Fig. 13. Changes of the profile of molecular forms of alkaline phosphatase in ram seminal plasma at 10, 30 and 60 days after injection of Corynebacterium pyogenes (electrophoresis on polyacrylamide gel).
sialoproteid character, we have attempted to analyse the content of this c o m p o u n d during the inflammation process of ram reproductive organs. Despite differences in the initial content of free and bound sialic acid in the semen of the two rams, dynamics of changes are similar in both individuals (Fig. 14). Along with progressing inflammation (in our case the critical mnment occurred on the 18th day after injection of Corynebacteriumpyogenes), free and b o u n d sialic acid content in seminal plasma increased. This phenomenon points n o t only to intensively destructive processes in the connective tissue, but also to an intensification of the synthesis of sialoproteins. These results are Supported by microscopic studies of reproductive organs. Apart from some disturbance of spermatogenesis in the testicles and vesicle glands, in both rams there were centres with visibly positive p.a.s, reaction (Fig. 15). High amounts of DNA present in the cells of connective tissue reflect metabolytic stimulation of this tissue.
319 rr~ %
rn9% ] 1 of ram I
61 4
3
i
10
20
30
40
50
,
1
60
i
i
i
J
10
20
30'
40
50
60
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m % 200. 180, 160,
m % 200 2. 180 160. 140" 120. 100. 80' 60. 40' 20
140.
120. 100. 80. 60. 40. 20
~'o
2b
~;o Xo ~5
6'0
days
Fig. 14. Changes in the content of free and bound sialic acid in ram seminal plasma during inflammation of testicles and epididymis. 1. free sialic acid; 2. bound sialic acid.
I ~- I
I= i
Fig. 15. Contents o f positive p.a.s, substance in testicles of rams I and II. Staining according to McManus ( 1 9 6 0 ) ; light microscope, bar = 100 nm.
320
The RNA content in the cells of the spermatogenetic epithelium also increased, especially in places with some morphological damage. Observed microscopic changes suggest that some disturbances in protein and nucleic acids metabolism occurred in the inflamed reproductive organs. Synthesis of proteins with changed biochemical and physical properties is accompanied by changes in their antigenic properties. Our immunodiffusional observations on proteins of seminal plasma during the inflammation process revealed a gradual disappearance of precipitation arcs (Fig. 16). This phenomenon can be explained by the antigen structure being hidden by molecules of sialic acid, as the content of the latter in
£I
Fig. 16. Dynamics of changes in antigenic order of ram seminal plasma I and II with the development of inflammation of the reproductive organs; 1 -- normospermy; 2, 3, 4 ...8 -n u m b e r of days (observations were m a d e every 10 days, from 40th to 90th day) after injection of Corynebacterium pyogenes (central well -- antiserum to normal seminal plasma of ram, negative photography).
321
ram plasma increased. It should be added that among these hidden antigens of seminal plasma there were also antigens with the activity of alkaline phosphatase. From the results it may be stated that despite changes in the secondary structure of protein molecules, alkaline phosphatase of ram seminal plasma retains its catalytic properties due to the protective function of sialic acid, the latter being intensively synthesized during the inflammation of reproductive organs. DISCUSSION
It was shown that alkaline phosphatase of ram seminal plasma is a heterogenic enzyme. Limited amounts of the isolated material, and also difficulties in obtaining a high degree of purification of the enzyme, did not allow broad kinetic studies to be made. Nevertheless, the results indicate that the three components of this alkaline phosphatase possess very similar biochemical properties. They are characterized by: similar molecular weight, similar pH optima, high affinity for synthetic p-nitrophenylphosphate substrate, activating effect of Mg2+ ions, and sialoproteid structure of proteins. These properties point to a certain similarity with alkaline phosphatase of bull seminal plasma (Strzezek and Glogowski, 1979). From results of observations on the function of sialic acid it may be concluded that this compound plays an especially significant role in the enzymatic catalysis of alkaline phosphatase of ram reproductive organs, both in the physiological and pathological states. Desialization of particular molecular forms results both in a drop of their enzymatic activity and in deep changes of protein structure, especially as regards the molecular form I. The II and III forms seem to be characterized by differences in the location of N-acetylneuraminyl groups in their protein molecules. Free sialic acid is a strong inhibitor of the activity of component II, as well as a protective compound against denaturing factors. Although the mechanisms of this reaction are not known, it may be supposed that free sialic acid is directly bound to the active centres of the enzyme. Magnesium ions result in an increase of maximal activity of alkaline phosphatase of ram seminal plasma, as in the case of other tissue phosphatases (Hashimoto et al., 1976). Slight differences in the catalytic activity of molecular form II when incubated for a prolonged period at 37°C, in the presence of Mg2+ ions, and of a complex of Mg2÷ ions and sialic acid, support the idea that different mechanisms are involved in the action of these two compounds at the active centre of alkaline phosphatase of ram seminal plasma. We have observed similar reactions in the case of alkaline phosphatase of bull seminal plasma (StrzeT.ek and Glogowski, 1979). In the pathological state of ram reproductive organs, sialic acid plays a decisive role in maintaining activity of alkaline phosphatase on a sufficiently high level, because it functions as a stabilizer of alkaline phosphatase. It also affects the profile of phosphatase isoenzymes.
322
Immunological studies showed that alkaline phosphatase of ram seminal plasma belongs to the antigenic complex of this fluid. Our studies on kinetic properties of the enzyme in the presence of specific antibodies seem to be of significance in clarifying the biological function of this enzyme in the repro ductive system of the ram. Inhibition of the activity of alkaline phosphatase of ram seminal plasma by antiserum toward form II depended on the protein ratio. At low values of the ratio between antiserum proteins and seminal proteins, antibodies had an inhibiting effect. On the other hand, b e y o n d a certain value of this ratio, maximal activity of the enzyme increased. Inhibition of the enzyme activity by immunological serum is of practical and theoretical significance. Arnon (1974) and Lehmann (1971) showed that the mechanism of this reaction is connected with the effect of antibodies upon the allosteric centre of an enzyme. Conformational changes in the protein molecule under the effect of antibodies may result in an inhibition, or stimulation, of enzyme katalytic activity, depending on the character and location of antigen determinants upon the enzyme molecule. In the case of molecular form II, incubation in the presence of specific antiserum resulted n o t only in an increase of maximal rate of p-nitrophenylphosphate hydrolysis, but also in an increase in substrate affinity. This would indicate an allosteric character of alkaline phosphatase of ram seminal plasma, as well as the existence of some biological mechanism, protecting the enzyme from denaturation, such as may occur during inflammation of the reproductive system. A similar protective effect of antibodies with respect to the active centre of alkaline phosphatase was observed in an enzyme isolated from bull seminal plasma (Strzez.ek and Glogowski, 1978). Results obtained in the present studies lead to the conclusion that alkaline phosphatase of ram seminal plasma is characterized by high plasticity of its molecular structures, favouring rapid adaptation of this enzyme to changeable conditions of enzymatic catalysis, especially in pathologic states of the reproductive system. Observations on biochemical and immunological properties of isoenzymes of alkaline phosphatase in such a state constitute evidence for changes in semen metabolism. Studies of this type should be continued. REFERENCES
Arnon, R., 1974. Enzyme inhibition by antibodies. In: E. Diczfalusy (Editor), Immunological Approaches to Fertility Control. Karolinska Symposia on Research Methods in Reproductive Endocrinology. Karolinska Institutet, Stockholm, pp. 133--15: Bessey, O.A., Lowry, O.H. and Brock, M.J., 1946. A method for the rapid determination of alkaline phosphatase with five cubic millimeters of serum. J. Biol. Chem., 164: 321--325. Davis, B.J., 1964. Disc electrophoresis. II. Method and application to human serum proteins. Ann. N.Y. Acad. Sci., 121: 404--427. Hashimoto, T., Sakurai, T. and Nomoto, S., 1976. Alkaline phosphatase: reaction mechanisms and its physiological significance. Taisha, 13: 279--283. Lehmann, E.G., 1971. Glutamate dehydrogenase from human liver. III. Antibody-binding sites and properties of the antigen--antibody complex. Biochem. Biophys. Acta, 235: 259--275.
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Lineweaver, H. and Burk, D., 1936. The determination of enzyme dissociation constants. J. Am. Chem. Soc., 56: 658--666. Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, K.J., 1951. Protein measurements with the Folin phenol reagent. J. Biol. Chem., 193: 265--275. McManus, J.F.H. and Mowry, R.W., 1960. Staining Methods. Hoeber, N e w York, 351 pp. Ornstein, L., 1964. Disc electrophoresis. I. Background and theory. Ann. N.Y. Acad. Sci., 121: 321--349. Ouchterlony, O., 1958. Diffusion in gel methods for immunological analysis. Progress in Allergy, 5: 1--78. Seligman, A.M., Chauncey, H.H., Nachlas, M.M., Manheimer, L.H. and Ravin, H.A., 1951. Colorimetric determination of phosphatases in human serum. J. Biol. Chem., 190 : 7--16. Strze~ek, J. and Glogowski, J., 1979. Molecular forms of alkaline phosphatase in bull seminal plasma. I. Isolation and characterization of two forms. Int. J. Biochem. 10: 135--146. Strze~ek, J. and Glogowski, J., 1979. Molecular form~of alkaline phosphatase in bull seminal plasma. II. Immunological properties of the two phosphatase forms. Proceedings of the 4th International Symposium: Immunology of Reproduction, Varna, 1978, pp. 849--854. Veselsky, L., 1972. Immunoelectrophoretic study on acid and alkaline phosphatase in blood serum, spermatozoa and genital tract fluids of boars. XIIth Europ. Conf. Anita. Blood Groups Biochem. Polymorphism, Budapest, pp. 387--391. Warren, L., 1959. Sialic acid in human semen and in the male genital tract. J. Clin. Invest., 38: 755--761. Weichselbaum, T.E., 1946. An accurate and rapid method for the determination of protein in small amounts o f blood serum and plasma. Am. J. Clin. Path. Techn. Sect., 10: 40--49. Wieme, A., 1959. Studies on Agar Gel Electrophoresis. Arscia Uitgaven N.Y., Briissel, 531 pp. 7,ilcov, N.Z., 1974. Izmenene aktivnosti transminas i fosfatas w krowi i sekretach polowogo puti barana. Trudy WASHNIIL, cz. I: 39--43.
307
MOLECULAR FORMS OF ALKALINE PHOSPHATASE OF RAM SEMINAL PLASMA -- SOME PROPERTIES AND CHANGES IN PATHOLOGICAL PROCESS OF REPRODUCTIVE ORGANS J. GLOGOWSKI and J. STRZEZEK
Agricultural-Technical Academy, Department of Animal Biochemistry, Institute of Animal Physiology and Biochemistry, 1 O-718 Olsztyn (Poland) (Accepted 22 April 1980)
ABSTRACT Glogowski, J. and Strze~.ek,J., 1981. Molecular forms of alkaline phosphatase of ram seminal plasma -- some properties and changes in pathological process of reproductive organs. Anita. Reprod. Sci., 3: 307--323.
Three molecular forms of alkaline phosphatase were isolated from ram seminal plasma. These forms, activated with Mg 2+ ions, were characterized by very similar pH optima, K m constant, and molecular weight. They differed in electrophoretic mobility, the latter being most probably determined by the different position of N-aeetylneuraminyl groups in protein structures. Sialic acid also played a protective function for the catalytic centre. Isolated molecular forms possessed antigenic properties. Immunological serum for phosphatase proteins either inhibited or stabilized activity of alkaline phosphatase, depending on the value of the protein ratio. During experimentally induced inflammation of ram reproductive organs, a gradual decrease of the activity of alkaline phosphatase was noted, together with changes in its electrophoretic profile. This phenomenon is most likely caused by intensive synthesis of sialic acid in pathologically changed reproductive organs o f the ram.
INTRODUCTION
Animal semen, and especially ram semen, is characterized by high alkaline phosphatase activity. Catalytic properties of this phosphatase are not fully known as yet. Among others, Strzezek and Glogowski {1979) isolated two molecular forms of alkaline phosphatase from bull seminal plasma, and defined their biochemical and immunological properties. Veselsky (1972) showed, by immunoelectrophoresis, activity of alkaline phosphatase in fluids from the accessory sex glands of the boar. As regards ram alkaline phosphatase, only Zilcov {1974) defined its activity in different reproductive organs. He showed that testicles and epididymis constitute the main source of this enzyme in ram seminal plasma. Heterogeneity of phosphatase proteins of animal semen allows observations to be made not only on physiological phenomena, but also on pathological changes in male reproductive organs. 0378-4320/81/0000--0000/$02.50 © 1981 Elsevier Scientific Publishing Company
308
The scope of this work was to isolate molecular forms of alkaline phosphatase from ram seminal plasma, and to define its biochemical and immunological properties. An attempt was also made to define changes in these properties during the inflammation process of the ram reproductive tract. MATERIALS
AND METHODS
Ram semen was collected from animals of the Polish Woolyhaired breed by artificial vagina. In order to separate spermatozoa the sample was centrifuged for 10 min at 10,000 g at 4 ° C. Seminal plasma was stored at - 2 0 ° C until the isolation process. Stages of isolation and purification are presented in Fig. 1. Raw proteins were determined by measuring absorption at 280 nm, and Fig. 1. Purification procedure for the isolation of alkaline phosphatase from ram seminal plasma Step
Procedure and product (temp. 4°C) R a m semen I
10,000 g
II
III
I I
10 min
Spermatozoa (discarded)
Seminal plasma
Supernatant (discarded)
Precipitate
Protein without alkaline phosphatase activity (discarded)
Protein fractions with alkaline phosphatase activity
Protein without alkaline phosphatase activity (discarded)
Three alkaline phosphatase peaks
Ethanol (95%) previously chilled to --10°C and slowly added to the plasma to a concentration of 65%. Centrifuged at 13,000 × g, 15 min.
Dissolved in 0.05 M Tris--HC1 buffer, pH 7.7. Applied to a Sephadex G-200 column (2.5 × 45 cm). The proteins eluted with the same buffer at a flow rate of 1 ml per 20 min and 3 ml fractions were collected for protein and enzyme assays.
DEAE-cellulose column (1.8 x 30 cm) equilibrated with 0.05 M Tris--HCl buffer. All the proteins were absorbed on the column. The column was then treated successively in a batchwise manner with 0.05 M Tris-HCl followed by NaC1 solutions of increasing molarity (from 0.1 M to 0.6 M) in the same buffer. The 4 ml fractions were collected for protein and enzyme assays.
309
quantitatively by the biuret method according to Weichselbaum (1946), and by the method of Lowry et al. (1951) with the Folin phenol reagent, depending on the stage of isolation.Mobility and electrophoretic homogeneity of the isolatedmolecular forms were determined by electrophoresis on polyacrylamide gel according to Davis (1964) and Ornstein (1964) and by electrophoresison agarose gel according to Wieme (1959). Identification of molecular forms of the enzyme was by the histochemical method of Seligman et al. (1951) using sodium/3-naphthylphosphate and Fast Blue R R in 0.1 M veronal buffer at p H 9.1. Alkaline phosphatase activitywas determined by the method of Bessey et al. (1946). A unit of enzyme activityis defined as the quantity of enzyme which liberates1 ~mol of p-nitrophenol during 1 h. Specific activityof the enzyme is equal to the quantity of pmoles of p-nitrophenol liberatedduring 1 h per 1 m g of protein.
Methods for investigating biochemical and immunological properties of isolated molecular forms of alkaline phosphatase The Michaelis constant (K m ) for p-nitrophenylphosphate as a substrate was defined according to the Lineweaver-Burk (1936) method. Molecular weight o f particular forms was determined with thin-layer chromatography in gel Sephadex G-200 Superfine. In order to define the effect of sialic acid on kinetic properties of the enzyme, enzymatic splitting of this c o m p o u n d was made using a neuraminidase preparation from Vibrio cholerae, produced b y Koch Light Ltd, with activity of 500 units/ml. Preparations of particular molecular forms of alkaline phosphatase were incubated b y adding to 0.3 ml of the enzyme, 100 gl of neuraminidase, 10 pl of 0.01 M MgC12,10 pl of 0.01 M CaC12 and 50 pl of 0.4 M acetate buffer with pH 5.3. In control samples neuraminidase was substituted by acetate buffer. After 6 h incubation at 37 ° C, electrophoretic mobility of the molecular forms was studied on agarose gel, and then histochemical staining for alkaline phosphatase was carried out. The effect of the addition of sialic acid, and Mg 2÷ ions, on temperature stability of the enzyme was also defined. The following incubation mixtures were used: (a) 1 ml of the enzyme + 0.6 ml of H 2 0 (b) 1 ml of the enzyme + 0.4 ml of 0.01 M sialic acid + 0.2 ml of H: O (c) 1 ml o f the enzyme + 0.4 ml of H 2 0 + 0.2 ml of 0.1 M MgC12 (d) 1 ml of the enzyme + 0.4 ml of 0.01 M sialic acid + 0.2 ml of 0.01 M MgCI2 Immunological serum toward the isolatedmolecular forms was obtained by rabbit immunization with subcutaneous and intramuscular injections,using Freund's adjuvant. In immunological studies the method of double gel diffusion according to Ouchterlony (1958) was adopted. The effects of immunological serum on phosphatase activityand its catalyticproperties were also observed.
310
Methods for studying changes in properties of phosphatase proteins of seminal plasma after experimentally induced inflammation of testicles Inflammation of the reproductive organs of two rams was induced by injection into the intratesticles of 1.5 ml one-day liquid broth culture of Corynebacterium pyogenes, containing 109/ml bacteria cells. For 60 days, indices of semen quality were observed~ as was the clinical state of the reproductive organs. Biochemical analyses of seminal plasma were also made: content of free and bound sialic acid (Warren, 1959), total protein content by the biuret method, and activity of acid and alkaline phosphatase and of its molecular forms. RESULTS
Chromatographic separation of the phosphatase fraction on DEAE-cellulose was achieved after filtration on gel 8ephadex G-200, as presented in Fig. 2. Three components of phosphatase proteins were obtained.
600
500
E280 22
o
d
20
~, 400
18 16
~ 30C
14
5
12 10
2QC
08
q
Q6
10C
Q4 Q2
Ol
100
150
200
2 4 0 Fraction number
Fig. 2. DEAE-cellulose column chromatography of protein fraction obtained after filtration on gel Sephadex G-200 ( protein 280 r i m ; - - - - - a c t i v i t y of alkaline phosphatase).
Component I, eluted at concentration of 0.1 M NaC1, showed specific activity toward p-nitrophenylphosphates 10 times higher than the initial activity of phosphatase in ram semen. Component II, the most active one, was eluted at 0.2 M NaC1. Its specific activity was 20 times higher. Component III, eluted at 0.4 M NaC1, was characterized by specific activity 5 times higher than the initial value.
311
i:ii!ii Fig. 3a. Electrophoresis on polyacrylamide gel of fraction obtained after DEAE-cellulose chromatography (staining for proteins). 1. I molecular form; 2. II molecular form; 3. III molecular form. Fig. 3b. Electrophoresis on polyacrylamide gel of fraction obtained after DEAE-cellulose chromatography (staining for alkaline phosphatase). 1. seminal plasma of ram; 2. I molecular form; 3. II molecular form; 4. III molecular form. In electrophoretic studies, both proteinograms and enzymograms (Fig. 3a and 3b), showed homogeneity of all forms, although c o m p o n e n t I was characterized by the highest electrophoretic mobility toward the anode. As in the enzymogram of alkaline phosphatase of ram semen, c o m p o n e n t III was characterized by the lowest electrophoretic mobility. In the immunodiffusion reaction according to Ouchterlony (1958) each specific antiserum gave one precipitation arc with the respective molecular form, both after staining with amide black for proteins, and after histochemical reaction for alkaline phosphatase (Fig. 4).
Fig. 4. Immunodiffusion in the following arrangements: 1. antiserum toward I form (central container) -- I molecular form; 2. antiserum toward II form (central container) -- II molecular form; 3. antiserum toward III form (central container) -- III molecular form. Staining for alkaline phosphatase,
312
Biochemical and immunological properties of molecular forms Molecular weight defined by thin-layer chromatography on gel Sephadex G-200 Superfine, amounted to a b o u t 210,000 for particular components. Fig. 5 presents the effect of pH on the rate of p-nitrophenylphosphate hydrolysis by molecular form II. This form is characterized by :an optimum pH range of 10.6--11.6, both with and without an addition of 2.5 mM Mg 2+ ions. However, in the presence of Mg 2+ ions, the activity of the enzyme is about 16% higher than in their absence.
320 300
.........
without c~dition of Mg2"ions w i t h 2.5 mM Mg2*io
200
~o
J
c~ 10C
.// ;
I
1'o
1'1
~'2
pH
Fig. 5. The effect of pH upon the rate of p-nitrophenylphosphate hydrolysis by molecular form II (50 mM glycine buffer; 7.5 mM p-nitrophenylphosphate). The pH optima for the components I and II did not differ from the values found for the III form. Maximal activities of isolated molecular forms of alkaline phosphatase were noted at pH 11.2. Particular components differed with respect to the rate of p-nitrophenylphosphate hydrolysis, depending on its concentration. In the case of molecular form II, the maximal rate of substrate hydrolysis occurred at a concentration of 8 mM (Fig. 6). For the I and III forms the corresponding concentrations were 7 mM and 3 mM respectively. Above these concentrations substrate inhibition was noted, and was most pronounced for c o m p o n e n t I. Michaelis-Menten constants (Km) for particular forms are presented in Table I. Km values obtained indicate that the isolated forms are characterized by very similar structure of their proteins, and also of the active centre. Electrophoretic heterogeneity of phosphatase proteins of ram seminal plasma is probably determined by the differences in electrostatic charge of particular forms. Studies on functions of sialic acid showed that treatment
313
v
32.£ 300
, 20.C
i
~:0
2:0
3'0
4o
go
6'0
7:0
ao
90
~5.0
~0 ,EmM]
Fig. 6. The effect o f p - n i t r o p h e n y l p h o s p h a t e c o n c e n t r a t i o n u p o n the activity of molecular f o r m II. TABLE I Michaelis-Menten constants (Kin) for particular forms toward p - n i t r o p h e n y l p h o s p h a t e (pH 11.2) Molecular forms
Value K m (M)
I II III
0.60 × 10 -3 1.35 × I 0 -s 1.00 × 10 -3
of phosphatase forms with neuraminidase results not only in a decrease of their electrophoretic mobility, but also in lowering of the enzymatic activity (Fig. 7). On the other hand, addition of sialic acid to the incubation mixture results in a slight increase of electrophoretic mobility, especially in the case of the I molecular form (Fig. 8). Simultaneous addition of Mg2÷ ions and sialic acid to the incubation mixture results in an increase of enzymatic activity of component II (Table II), whereas addition of only sialic acid to the incubation mixture decreases the activity of this enzyme. Long-term incubation at 37°C points to the stabilizing and protective role of sialic acid with respect to the active centre of alkaline phosphatase of ram seminal plasma. Immunoelectrophoretic and immunodiffusion observations suggest an antigen character of isolated components of alkaline phosphatase. Interesting results were obtained as regards the effect of specific antiserum on the activity of alkaline phosphatase. As seen in Fig. 9, the activity of
314 m o l e c u l a r f o r m II decreases with an increasing a m o u n t of i m m u n o l o g i c a l s e r u m p r o t e i n s i n t h e i n c u b a t i o n m i x t u r e . T h i s p h e n o m e n o n is e s p e c i a l l y c l e a r i n t h e case o f t h e f o r m I I a n t i s e r u m a n d t h e m o l e c u l a r f o r m II. T h u s , at a ratio of 0.75:1 antiserum proteins to enzyme proteins, activity of the II f o r m w a s 3 4 % l o w e r t h a n t h e a c t i v i t y n o t e d i n t h e p r e s e n c e o f c o n t r o l
®
1
2
2
3
T
4
--
Fig. 7. Electrophoretic mobility of molecular form II after 6 h incubation with neuraminidace: 1. sample with neuraminidase; 2. control sample (electrophoresis on agarose gel). Fig. 8. The effect of sialic acid addition upon electrophoretic mobility of I (1, 2) and II (3, 4) molecular forms of alkaline phosphatase. 1, 3 -- without sialic acid; 2, 4 -- with addition of sialic acid (electrophoresis on agarose gel). TABLE II The effect of Mg2÷ ions, sialic acid and incubation time on the enzymatic activity of molecular form II (initial activity of molecular form II, 100%) Incubation time at 37°C (h)
Content of incubated mixture II form + sialic acid
II form + Mg=÷ ions
II form + sialic acid + Mg2÷ ions
After mixing
85.9
104.7
100.0
Incubation: 6 12 24 48 72 192
42.3 83.3 88.1 83.3 88.1 102.5
140.4 233.3 238.1 200.0 211.9 185.0
153.8 223.8 223.8 195.2 211.9 180.0
315
30C
2QO
rn
~oo Y
Q162/1
Q325:1
075:1
15 1
3:1
6:1
antiserum proteins I ~ J o :. ]]" form. proteins
Fig. 9. T h e effect of various ratios of antiserum proteins toward molecular f o r m II upon its activity. 1. control serum; 2. antiserum toward seminal plasma; 3. antiserum toward molecular f o r m II.
Krn = 1.16x10-3 M 1 Krn = 0 9 2 x 1 0 "3 M 2 Krn3= 1 0 2 x 1 0 "3 M
vt
O~ O09
1 3
o
006 OO5
5
Fig. 10. The effect of antiserum toward molecular f o r m II on substrate affinity of this f o r m to p-nitrophenylphosphate. 1. c o n t r o l serum; 2. antiserum toward seminal plasma; 3. antiserum toward molecular f o r m II.
316 serum. Studies on the kinetics of the enzyme incubated with antiserum revealed changes in the maximal rate of p-nitrophenylphosphate hydrolysis by molecular form II. Graphical presentation of Km shows an increase in the substrate affinity of the enzyme (Fig. 10). In the case of seminal plasma (Fig. 11), activity of alkaline phosphatase with antiserum toward form II, with a ratio of 0.5:1 antiserum proteins to plasma proteins, was almost 40% lower than the activity obtained with control serum. On the other hand, an increase of antiserum level in the incubation mixture to a ratio of 2:1 stimulates activity of the enzyme in plasma. ~0
7,0
60
E 40
5
3.0
20
10
Q125:1
Q25:1
0.5:1
1:1
2:1
4:1
antiserum proteins Ratio: ....... seminal plasma proter~
Fig. 11. The effect of various ratios of antiserum proteins toward molecular form II upon activity of alkaline phosphatase of ram seminal plasma. 1. control serum; 2. antiserum toward seminal plasma; 3. antiserum toward molecular form II.
Changes in properties of alkaline phosphatase of ram seminal plasma after experimentally induced inflammation of testicles A culture of the bacterium Corynebacterium pyogenes was injected into the testicles of t w o rams (I and II). Before the experiments both rams possessed healthy reproductive organs and produced semen with normal properties. Injection of bacteria induced an inflammation process in the reproductive organs. The observed symptoms were visible swelling of the scrotum and hardening of the glandular tissue of the testicles. The pathological process was observed for 60 days. Throughout this time both rams retained sexual impulses. Studies of semen properties during this time showed that 10--15 days after the injection, a gradual decrease of spermatozoa concen-
317 tration in the semen commences (aspermy in the case of ram II), together with an increase of pathological forms of spermatozoa. Secondary changes of spermatozoa reached 90% in ram I and 80% in ram II. The activity of acid phosphatase in the seminal plasma decreased as the inflammation process progressed (lowering of activity by 70%), {Fig. 12). U 2500
U
la
2000 1500
1500
1000
1000
500
500 i
~'o
~b
i
3'o
~o
~'o
i
i
6o
10
days
U
i
i
20
30
i
40 doys
i
i
50
60
do
~o
1200 1000
I
50C
c~vs
~o ~o ~o /o
days
Fig. 12. Activity of alkaline and acid phosphatase in ram (I and II) seminal plasma during experimentally induced inflammation o f reproductive organs, a -- alkaline phosphatase; b -- acid phosphatase.
In the case of alkaline phosphatase, changes of enzyme activity were less pronounced. A 10--20% drop of the activity was noted on the average, usually between days 10 and 20 of the disease. Later, however, the alkaline phosphatase activity remained at a high level. Electrophoretic studies on molecular forms of alkaline phosphatase showed changes of both electrophoretic mobility and activity (Fig. 13). It was found that after visible increase in the activity of molecular form I during the first phase of the inflammation process, this form gradually disappeared along with progressing pathogenesis. Molecular forms II and III retained high catalytic activity throughout the whole period of observations. Acute and chronic inflammation processes, similar to neoplasmatic diseases, are usually accompanied by an increase of glycoproteins in blood serum. The level of sialic acid cen constitute an indirect index of the intensity of destructive and proliferation processes in connective tissue of the inflamed organs. Due to the fact that alkaline phosphatase of seminal plasma is of
318
10
3O
60
0
Fig. 13. Changes of the profile of molecular forms of alkaline phosphatase in ram seminal plasma at 10, 30 and 60 days after injection of Corynebacterium pyogenes (electrophoresis on polyacrylamide gel).
sialoproteid character, we have attempted to analyse the content of this c o m p o u n d during the inflammation process of ram reproductive organs. Despite differences in the initial content of free and bound sialic acid in the semen of the two rams, dynamics of changes are similar in both individuals (Fig. 14). Along with progressing inflammation (in our case the critical mnment occurred on the 18th day after injection of Corynebacteriumpyogenes), free and b o u n d sialic acid content in seminal plasma increased. This phenomenon points n o t only to intensively destructive processes in the connective tissue, but also to an intensification of the synthesis of sialoproteins. These results are Supported by microscopic studies of reproductive organs. Apart from some disturbance of spermatogenesis in the testicles and vesicle glands, in both rams there were centres with visibly positive p.a.s, reaction (Fig. 15). High amounts of DNA present in the cells of connective tissue reflect metabolytic stimulation of this tissue.
319 rr~ %
rn9% ] 1 of ram I
61 4
3
i
10
20
30
40
50
,
1
60
i
i
i
J
10
20
30'
40
50
60
40
~o
~o
4'o
~'o
m % 200. 180, 160,
m % 200 2. 180 160. 140" 120. 100. 80' 60. 40' 20
140.
120. 100. 80. 60. 40. 20
~'o
2b
~;o Xo ~5
6'0
days
Fig. 14. Changes in the content of free and bound sialic acid in ram seminal plasma during inflammation of testicles and epididymis. 1. free sialic acid; 2. bound sialic acid.
I ~- I
I= i
Fig. 15. Contents o f positive p.a.s, substance in testicles of rams I and II. Staining according to McManus ( 1 9 6 0 ) ; light microscope, bar = 100 nm.
320
The RNA content in the cells of the spermatogenetic epithelium also increased, especially in places with some morphological damage. Observed microscopic changes suggest that some disturbances in protein and nucleic acids metabolism occurred in the inflamed reproductive organs. Synthesis of proteins with changed biochemical and physical properties is accompanied by changes in their antigenic properties. Our immunodiffusional observations on proteins of seminal plasma during the inflammation process revealed a gradual disappearance of precipitation arcs (Fig. 16). This phenomenon can be explained by the antigen structure being hidden by molecules of sialic acid, as the content of the latter in
£I
Fig. 16. Dynamics of changes in antigenic order of ram seminal plasma I and II with the development of inflammation of the reproductive organs; 1 -- normospermy; 2, 3, 4 ...8 -n u m b e r of days (observations were m a d e every 10 days, from 40th to 90th day) after injection of Corynebacterium pyogenes (central well -- antiserum to normal seminal plasma of ram, negative photography).
321
ram plasma increased. It should be added that among these hidden antigens of seminal plasma there were also antigens with the activity of alkaline phosphatase. From the results it may be stated that despite changes in the secondary structure of protein molecules, alkaline phosphatase of ram seminal plasma retains its catalytic properties due to the protective function of sialic acid, the latter being intensively synthesized during the inflammation of reproductive organs. DISCUSSION
It was shown that alkaline phosphatase of ram seminal plasma is a heterogenic enzyme. Limited amounts of the isolated material, and also difficulties in obtaining a high degree of purification of the enzyme, did not allow broad kinetic studies to be made. Nevertheless, the results indicate that the three components of this alkaline phosphatase possess very similar biochemical properties. They are characterized by: similar molecular weight, similar pH optima, high affinity for synthetic p-nitrophenylphosphate substrate, activating effect of Mg2+ ions, and sialoproteid structure of proteins. These properties point to a certain similarity with alkaline phosphatase of bull seminal plasma (Strzezek and Glogowski, 1979). From results of observations on the function of sialic acid it may be concluded that this compound plays an especially significant role in the enzymatic catalysis of alkaline phosphatase of ram reproductive organs, both in the physiological and pathological states. Desialization of particular molecular forms results both in a drop of their enzymatic activity and in deep changes of protein structure, especially as regards the molecular form I. The II and III forms seem to be characterized by differences in the location of N-acetylneuraminyl groups in their protein molecules. Free sialic acid is a strong inhibitor of the activity of component II, as well as a protective compound against denaturing factors. Although the mechanisms of this reaction are not known, it may be supposed that free sialic acid is directly bound to the active centres of the enzyme. Magnesium ions result in an increase of maximal activity of alkaline phosphatase of ram seminal plasma, as in the case of other tissue phosphatases (Hashimoto et al., 1976). Slight differences in the catalytic activity of molecular form II when incubated for a prolonged period at 37°C, in the presence of Mg2+ ions, and of a complex of Mg2÷ ions and sialic acid, support the idea that different mechanisms are involved in the action of these two compounds at the active centre of alkaline phosphatase of ram seminal plasma. We have observed similar reactions in the case of alkaline phosphatase of bull seminal plasma (StrzeT.ek and Glogowski, 1979). In the pathological state of ram reproductive organs, sialic acid plays a decisive role in maintaining activity of alkaline phosphatase on a sufficiently high level, because it functions as a stabilizer of alkaline phosphatase. It also affects the profile of phosphatase isoenzymes.
322
Immunological studies showed that alkaline phosphatase of ram seminal plasma belongs to the antigenic complex of this fluid. Our studies on kinetic properties of the enzyme in the presence of specific antibodies seem to be of significance in clarifying the biological function of this enzyme in the repro ductive system of the ram. Inhibition of the activity of alkaline phosphatase of ram seminal plasma by antiserum toward form II depended on the protein ratio. At low values of the ratio between antiserum proteins and seminal proteins, antibodies had an inhibiting effect. On the other hand, b e y o n d a certain value of this ratio, maximal activity of the enzyme increased. Inhibition of the enzyme activity by immunological serum is of practical and theoretical significance. Arnon (1974) and Lehmann (1971) showed that the mechanism of this reaction is connected with the effect of antibodies upon the allosteric centre of an enzyme. Conformational changes in the protein molecule under the effect of antibodies may result in an inhibition, or stimulation, of enzyme katalytic activity, depending on the character and location of antigen determinants upon the enzyme molecule. In the case of molecular form II, incubation in the presence of specific antiserum resulted n o t only in an increase of maximal rate of p-nitrophenylphosphate hydrolysis, but also in an increase in substrate affinity. This would indicate an allosteric character of alkaline phosphatase of ram seminal plasma, as well as the existence of some biological mechanism, protecting the enzyme from denaturation, such as may occur during inflammation of the reproductive system. A similar protective effect of antibodies with respect to the active centre of alkaline phosphatase was observed in an enzyme isolated from bull seminal plasma (Strzez.ek and Glogowski, 1978). Results obtained in the present studies lead to the conclusion that alkaline phosphatase of ram seminal plasma is characterized by high plasticity of its molecular structures, favouring rapid adaptation of this enzyme to changeable conditions of enzymatic catalysis, especially in pathologic states of the reproductive system. Observations on biochemical and immunological properties of isoenzymes of alkaline phosphatase in such a state constitute evidence for changes in semen metabolism. Studies of this type should be continued. REFERENCES
Arnon, R., 1974. Enzyme inhibition by antibodies. In: E. Diczfalusy (Editor), Immunological Approaches to Fertility Control. Karolinska Symposia on Research Methods in Reproductive Endocrinology. Karolinska Institutet, Stockholm, pp. 133--15: Bessey, O.A., Lowry, O.H. and Brock, M.J., 1946. A method for the rapid determination of alkaline phosphatase with five cubic millimeters of serum. J. Biol. Chem., 164: 321--325. Davis, B.J., 1964. Disc electrophoresis. II. Method and application to human serum proteins. Ann. N.Y. Acad. Sci., 121: 404--427. Hashimoto, T., Sakurai, T. and Nomoto, S., 1976. Alkaline phosphatase: reaction mechanisms and its physiological significance. Taisha, 13: 279--283. Lehmann, E.G., 1971. Glutamate dehydrogenase from human liver. III. Antibody-binding sites and properties of the antigen--antibody complex. Biochem. Biophys. Acta, 235: 259--275.
323
Lineweaver, H. and Burk, D., 1936. The determination of enzyme dissociation constants. J. Am. Chem. Soc., 56: 658--666. Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, K.J., 1951. Protein measurements with the Folin phenol reagent. J. Biol. Chem., 193: 265--275. McManus, J.F.H. and Mowry, R.W., 1960. Staining Methods. Hoeber, N e w York, 351 pp. Ornstein, L., 1964. Disc electrophoresis. I. Background and theory. Ann. N.Y. Acad. Sci., 121: 321--349. Ouchterlony, O., 1958. Diffusion in gel methods for immunological analysis. Progress in Allergy, 5: 1--78. Seligman, A.M., Chauncey, H.H., Nachlas, M.M., Manheimer, L.H. and Ravin, H.A., 1951. Colorimetric determination of phosphatases in human serum. J. Biol. Chem., 190 : 7--16. Strze~ek, J. and Glogowski, J., 1979. Molecular forms of alkaline phosphatase in bull seminal plasma. I. Isolation and characterization of two forms. Int. J. Biochem. 10: 135--146. Strze~ek, J. and Glogowski, J., 1979. Molecular form~of alkaline phosphatase in bull seminal plasma. II. Immunological properties of the two phosphatase forms. Proceedings of the 4th International Symposium: Immunology of Reproduction, Varna, 1978, pp. 849--854. Veselsky, L., 1972. Immunoelectrophoretic study on acid and alkaline phosphatase in blood serum, spermatozoa and genital tract fluids of boars. XIIth Europ. Conf. Anita. Blood Groups Biochem. Polymorphism, Budapest, pp. 387--391. Warren, L., 1959. Sialic acid in human semen and in the male genital tract. J. Clin. Invest., 38: 755--761. Weichselbaum, T.E., 1946. An accurate and rapid method for the determination of protein in small amounts o f blood serum and plasma. Am. J. Clin. Path. Techn. Sect., 10: 40--49. Wieme, A., 1959. Studies on Agar Gel Electrophoresis. Arscia Uitgaven N.Y., Briissel, 531 pp. 7,ilcov, N.Z., 1974. Izmenene aktivnosti transminas i fosfatas w krowi i sekretach polowogo puti barana. Trudy WASHNIIL, cz. I: 39--43.