AU5431890A – Protein complex
– Google Patents
AU5431890A – Protein complex
– Google Patents
Protein complex
Info
Publication number
AU5431890A
AU5431890A
AU54318/90A
AU5431890A
AU5431890A
AU 5431890 A
AU5431890 A
AU 5431890A
AU 54318/90 A
AU54318/90 A
AU 54318/90A
AU 5431890 A
AU5431890 A
AU 5431890A
AU 5431890 A
AU5431890 A
AU 5431890A
Authority
AU
Australia
Prior art keywords
macromolecule
sperm
sperm motility
motility
daltons
Prior art date
1989-03-31
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
AU54318/90A
Other versions
AU635258B2
(en
Inventor
Eva Akerlof
Ake Pousette
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Applied Research Systems ARS Holding NV
Original Assignee
Applied Research Systems ARS Holding NV
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
1989-03-31
Filing date
1990-04-02
Publication date
1990-11-05
1990-04-02
Application filed by Applied Research Systems ARS Holding NV
filed
Critical
Applied Research Systems ARS Holding NV
1990-11-05
Publication of AU5431890A
publication
Critical
patent/AU5431890A/en
1993-03-18
Application granted
granted
Critical
1993-03-18
Publication of AU635258B2
publication
Critical
patent/AU635258B2/en
2010-04-02
Anticipated expiration
legal-status
Critical
Status
Ceased
legal-status
Critical
Current
Links
Espacenet
Global Dossier
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Classifications
C—CHEMISTRY; METALLURGY
C07—ORGANIC CHEMISTRY
C07K—PEPTIDES
C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
C07K16/42—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins
A—HUMAN NECESSITIES
A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
A61P13/00—Drugs for disorders of the urinary system
A61P13/02—Drugs for disorders of the urinary system of urine or of the urinary tract, e.g. urine acidifiers
A—HUMAN NECESSITIES
A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
A61P15/00—Drugs for genital or sexual disorders; Contraceptives
C—CHEMISTRY; METALLURGY
C07—ORGANIC CHEMISTRY
C07K—PEPTIDES
C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
C07K14/4702—Regulators; Modulating activity
C07K14/4705—Regulators; Modulating activity stimulating, promoting or activating activity
C—CHEMISTRY; METALLURGY
C07—ORGANIC CHEMISTRY
C07K—PEPTIDES
C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
C07K14/76—Albumins
C07K14/765—Serum albumin, e.g. HSA
C—CHEMISTRY; METALLURGY
C07—ORGANIC CHEMISTRY
C07K—PEPTIDES
C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
C07K14/775—Apolipopeptides
C—CHEMISTRY; METALLURGY
C07—ORGANIC CHEMISTRY
C07K—PEPTIDES
C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
C07K16/06—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies from serum
C—CHEMISTRY; METALLURGY
C07—ORGANIC CHEMISTRY
C07K—PEPTIDES
C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
A—HUMAN NECESSITIES
A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
A61K38/00—Medicinal preparations containing peptides
Abstract
The present invention relates to a macromolecule or proteinaceous nature which activates sperm motility, a process for the preparation thereof by purification of the macromolecule from extracellular fluids, pharmaceutical preparations comprising the macromolecule and antibodies directed against determinants specific to said macromolecule and the use thereof as well as a method to assay the potential fertility of sperm.
Description
PROTEIN COMPLEX
Technical field
The present invention relates to a macromolecule of protein¬ aceous nature which activates sperm motility, a process for the preparation thereof by purification of the macromolecule from extracellular fluids, pharmaceutical preparations com¬ prising the macromolecule and antibodies directed against determinants specific to said macromolecule.
Background art
It is previously known that only spermatozoa with a good ability to move and with a relatively straightlined progressive motility have the possibility to achieve fertili¬ zation and that only the spermatozoa with the best morphology enter the cervical mucus (Fredricsson B. and Bjσrk G. (1977): Morphology of postcoital spermatozoa in the cervical secre¬ tion and its clinical significance. Pertil. Steril. 28:841- 845). It is also previously known that serum support sperm motility /Austin R. (1985) «Sperm maturation in the male and female genital tracts.» In: Biology of Fertiliztion, vol. 2 (eds C Metz and A Monroy) pp. 121-147, Academic Press, New York/ and is therefore used in connection with sperm sepa¬ ration for fertilization in vitro.
However, there are problems inhereted in the use of a fluid comprising thousands of components and a specific active component would give a safer and more predictible effect than such a fluid. An active factor would also give the effect without dangerous concomittant actions e.g. immunologic fac¬ tors or infection diseases.
Disclosure of invention
During work with fertilization in vitro standardized model systems were developed for e.g. sperm motility test, to obtain more homogeneous populations of motile and viable spermatozoa and to analyze the adenosinetriphosphate (ATP) content of sperm and its specific progressive motility (SPM) : Akerlδf E., Fredricsson B., Gustafson 0., Lundin A., Lunell N-0., Nylund L., Rosenborg L. and Pousette A (1987) Int. J. Androl. 10, 663-669 and Pousette A., Akerlδf E., Lundin A., Rosenborg L. and Fredricsson B. (1986) Int. J. Androl. 9, 331-340.
Said standardized model systems became the tools for the further investigation of serum to find the factor or factors giving the motility activity to the sperm necessary for the fertilization to take place. Through an extensive research work the present inventors have now found that sperm motility is activated by a macromolecule having a molecular wight of about 200.000 Daltons.
Said macromolecule is of proteinaceous nature, essentially pure and activates sperm motility. The molecular weight of said protein is according to a preferred embodiment of the invention about 180.000 Daltons.
A further preferred embodiment of the invention discloses a proteinaceous macromolecule having an isoelectric point of about 5.1 and comprising albumin. The molecular weight of said macromolecule is preferably about 250.000 Daltons.
The macromolecule according to the invention is substantially homogeneous and obtainable from extracellular fluids, particu¬ larly from serum of animals or human beings.
The proteinaceous macromolecule is purified from extracellu¬ lar fluids from animals or human beings, preferably from serum and according to a preferred embodiment from serum of human beings.
According to a further preferred embodiment a proteinaceous macromolecule having an isoelectric point of about 5.1 and comprising apolipoprotein Al, immunoglobulin and albumin is purified through a three step process comprising Ion-exchange Chromatography, Chromatofocusing and Fast Protein Liquid Chromatography.
According to another preferred embodiment a proteinaceous macromolecule having a molecular weight of about 180.000 Daltons and comprising apolipoprotein Al and immunoglobulin is prepared by a four step purification process comprising Ion- exchange Chromatography, Chromatofocusing, Fast Protein Liquid Chromatography and Affinity Chromatography.
According to a yet further preferred embodiment the sperm motility activity is checked after every single purification step.
The invention is also relating to pharmaceutical preparations comprising a macromolecule of proteinaceous nature which is essentially pure, has a molecular weight of about 200.000 Daltons and activates sperm motility together with any suit¬ able excipient. _
Examples of suitable excipients are culture media or other salt solutions.
The pharmaceutical preparations are prepared according to methods known per se. The pharmaceutical preparations accord¬ ing to the invention are used in the treatment of infertility, preferably in-vitro.
The invention further comprises a method to determine the potential fertility of sperm by treating a sample with a macromolecule according to the invention and compare its motility against a known standard.
The invention also comprises antibodies directed against determinants specific to the macromolecule according to the invention, preferably polyclonal antibodies obtainable by immunizing according to methods known per se. Said antibodies are used as a tool in the process and can further be used as a means for the preventing of fertilization by inhibiting the sperm motility activating properties.
The SPAP was purified from serum obtained from male blood donors and incubated at 56°C for 30 minutes before storage at -20°C until use.
According to one embodiment of the invention the purification process was performed in three steps and revealed a macro¬ molecule comprising apolipoprotein Al, Ig and albumin in about equimolar amounts and having a molecular weight of about 200.000 Daltons or more precisely about 250.000 Daltons, and an isoelectric point of about 5.1.
Said steps were:
1. Ion-exchange chromatography on DEAE-Sepharose <_§> and eluation with a linear NaCl gradient, 0-0,25 mol/1.
2. Chromatofocusing on PBE TM94 with histidine-HCl as the starting buffer. The pH interval was 6.0-4.0.
3. Fast Protein Liquid Chromatography (FPLC_: , Pharmacia) on two Superose*^ 12 HR 10/30 columns (serially) with molecular weight separation.
According to a yet preferred embodiment of the invention by addition of a fourth step such as Affinity chromatography on
Blue Sepharose (§> a SPAP comprising apolipoprotein Al and immu¬ noglobulin with a molecular weight of about 180.000 Daltons is obtained.
To follow the occurence of the sperm motility activity in serum fraction after every single purification step the preparations were submitted to a specific progressive motility (SPM) test and sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis.
For the preparation of a macromolecule comprising apolipo¬
elimination of albumin) was used. The pure protein, at a concentration of 20-70 nmol/1, activated the motility to the same extent as serum.
SDS polyacrylamide gel electrophoresis under non-reducing con¬ ditions showed one band corresponding to a molecular weight of about 180 kD. In the presence of mercaptoethanol the fraction after Blue-Sepharose gave two bands corresponding to 50 kD and about 25 kD. Without the Blue Sepharose^ step the non-reduced complex eluted as a larger component and after reduction SDS polyacrylamide gel electrophoresis then revealed an additional band at about 67 kD, suggesting that the molecule is then in complex with albumin, the latter being removed by the Blue Sepharosew step. Amino acid sequence analysis of the Blue Sepharose^ eluate, identified three protein chains – those of apolipoprotein Al and immunoglobulin heavy and light chains – suggesting that the preparation was an apolipoprotein Al – immunoglobulin complex. Antiserum, raised in a rabbit, inhi¬ bited sperm motility, when added directly to spermatozoa. Pre- treatment of human serum with rabbit antiserum, significantly reduced its ability to activate sperm motility. The sperm activating capacity of the protein complex was destroyed by heating at 100°C for 5 minutes, suggesting that the activity was dependent on intact protein conformations. Albumin, apo¬ lipoprotein Al and immunoglobulins by themselves had only minor effects on sperm motility. It appears likely that an apolipoprotein Al – immunoglobulin complex, mediates activa¬ tion of sperm motility.
In this context the following abbreviations are used:
ApoAl Apolipoprotein Al
ATP Adenosine triphosphate
D Dalton
FPLC® Fast Protein Liquid Chromatography
HEPES N-2-Hydroxyethylpiperazine-N’-2-ethanesulphonic acid
Ig Immunoglobulin
IVF In Vitro Fertilization
MW Molecular Weight
SDS Sodium Dodecyl Sulphate
SPAP Sperm Activating Protein
SPM Specific Progressive Motility v/v volume/volume
eluted with a linear gradient 0-0.25M NaCl. Fractions were collected and the’ 280 monitored. Aliquots of all fractions were assayed for SPM. The sperm activating capacity was eluted at about 0.2M NaCl.
Fig. 1-B illustrates Chromatofocusing on PBE TM94. After dia¬ lysis against the starting buffer the pooled fractions were added to the PBE 94 chromatofocusing column and Polybuffer^ 74, pH 4.0 was applied. Fractions were collected >and monitored for pH and SPM. The sperm activating capacity was eluted at pH 5.1.
Fig. 1-C illustrates FPLC . The pooled fractions were dia- lysed, lyophilized (dissolved in water) and fractionated on two Superose 12 HR 10/30 columns (serially). The columns were calibrated using ready made standards (Pharmacia Fine Chemi¬ cals, Uppsala, Sweden). A part from every fraction was tested for SPM. The sperm activating capacity was eluted correspond¬ ing to a molecular weight of about 250 kD.
Fig. 2 illustrates sodium dodecyl sulphate (SDS) poly¬ acrylamide gel electrophoresis in the presence of and in the absence of 2-mercaptoethanol and was performed using ready made gels and calibration standards.
Lane 1 Phosphorylase b (94,000), Albumin (67,000), Ovalbumin (43,000), Carbonic Anhydrase (30,000), Trypsin Inhibi¬ tor (20,100) and Lactalbumin (14.400) Lane 2 FPLC eluate with protein fraction (in the presence of
2-mercaptoethanol) Lane 3 Thyroglobulin (330,000), Ferritin (220,000), Albumin (67,000), Catalase (60,000), Lactate Dehydrogenase (36,000) and Ferritin (18,500) Lane 4 FPLC eluate with protein fraction in the absence of
2-mercaptoethanol. SDS polyacrylamide gel electrophoresis under non-reducing con¬ ditions showed one band corresponding to a molecular weight of about 180 kD. In the presence of mercaptoethanol this fraction gave two bands corresponding to 50 kD (later identified as immunoglobulin heavy chain) and about 25 kD (later identified as apolipoprotein Al and immunoglobulin light chain) . Without the Blue Sepharose step the non-reduced complex eluted as a larger component and after reduction, SDS polyacrylamide gel electrophoresis revealed an additional band at about 67 kD, suggesting that the molecule is then in complex with albumin, the latter being removed by the Blue Sepharose^ step.
Fig. 3 is illustrating SDS polyacrylamide gel electrophoresis in the presence of 2-mercaptoethanol.
Lane 1 As lane 1 Fig. 2
Lane 2 Normal serum
Lane 3 Blue Sepharose eluate with protein fraction
Lane 4 As lane 3 Fig. 2
Lane 5 FPLC Θ eluate with albumin fraction
Lane 6 FPL ^ eluate with protein fraction.
Fig. 4 illustrates the effect of the purified SPAP on SPM (A) and ATP content (B). Sperm were separated using the Percolr^ technique and transferred to Buffer B. Purified SPAP was added in increasing concentrations to aliquotes of the sperm solution and spermatozoa were assayed for motility, 2.5 and 5 hours later and for ATP content 2 and 3 hours after addition of SPAP.
Best model for carrying out the invention.
The model for sperm motility test is explained in the follow¬ ing.
Semen Samples. Ejaculates and blood sera were collected from six donors and were analysed within 2 hours. /Fredricsson B. (1979) Andrologi 11, 57-61/. Separation of motile spermatozoa was achieved by self migration using a swim-up technique or a Percoi ^ gradient technique followed by translocation of sper¬ matozoa to a defined culture medium. /Akerlδf E., Fredricsson B, Gustafson 0., Lundin A., Lunell N-O., Nylund L., Rosenborg L., and Pousette A (1987) Int. J. Androl. 10, 663-669/.
For the swim-up preparation, 0,3 ml semen was covered with 1 ml tissue culture medium, RPMI-1640 (Flow) supplemented with 13% (v/v) male human serum, 24 mM HEPES (Sigma, USA), 50 IU/ ml Penicillin and 50 μg/ml Streptomycin (GIBCO, Scotland) (Buffet B). After incubation at 37°C for 45 minutes, the uppermost 0.5 ml containing motile sperm was harvested. For the Percolr^ gradient technique, 1 ml of the ejaculate was layered gently on top of a Percolr^ gradient. After 3 hours at 37°C (without centrifugation) the upper part was sucked off and discarded, and the lower 3 ml were suspended. /Pousette A., Akerlδf E., Lundin A., Rosenborg L. and Fredricsson B. (1968) Int. J. Androl. 9, 331-340/.
The fractions from 5-10 tubes (separated by swim-up or Per- coir’ ) were pooled and added to a pump-filter system /Aker¬ lδf E., Fredricsson B., Gustafson 0., Lundin A., Lunell N-0. Nylund L., Rosenborg L., and Pousette A. (1987) Int. J. Androl. 10, 663-669/, in order to translocate motile sperma¬ tozoa to Buffer A (Buffer B without serum) .
After dilution to about 3 million spermatozoa/ml, aliquots of 0.5 ml were transferred and 0.1 ml portions of Buffer A (con¬ trol), serum (control), different additions or eluates from the purification steps were added. During establishment of the:
protocol for purification of the protein complex, progressive motility was assayed at different time intervals. For routine preparations, motility was measured 4-6 hours after addition of the test solutions, as the unspecific effect of albumin is minimized at this time.
Additives. Serum (used as control in sperm motility test as an additive in Buffer B), was incubated at 56°C for 30 minutes and stored at -20°C until use. Normal rabbit sera, rabbit anti-protein complex serum as well as immunoprecipitated sera were added directly or in different combinations to the sperm motility test.
Purified human apolipoprotein Al (A-9284 Sigma, MO., USA) was dissolved in Buffer A to 1.7 g/1 and added to the sperm moti¬ lity test, giving final concentrations of 0.04-0.3 g/1 in the test tubes. Gammonativ*^ (Kabi, Stockholm, Sweden), containing human immunoglobulin and albumin, was submitted to Blue Sepha¬ rose^ chromatography to remove the albumin. The immunoglobulin was freeze-dried, dissolved in water and added to the sperm motility test.
Motility. Progressive motility was analysed as described. /Pousette A., Akerlδf E., Rosenborg L. and Fredricsson B. (1986) Int. j. Androl. 9, 1-13/. The number of spermatozoa passing a specific line of known length in a Bϋrker chamber per unit of time was counted. Knowing the concentration, results have been expressed as passages per minute per million spermatozoa/ml. This value is termed the specific progressive motility (SPM). Sperm motility was assyed using aliquots from every fraction eluted. The motility was usually assayed 4-6 hours after addition of spermatozoa to avoid the initial increase in sperm motility that can be observed after addi¬ tion of e.g. albumin.
ATP content was analysed in the «standardized model system» described in Pousette A., Akerlδf E., Lundin A., Rosenbo-.-g L., and Fredricsson B. (1986) Int. J. androl. 9, 331-340 anc
specific ATP defined as content of ATP (epxressed in moles) per spermatozoon.
For the purification of SPAP sera from male blood donors were incubated at 56°C for 30 minutes before storage at -20°C until used for purifications. Fractions were dialysed and/or lyo- philized before assayed for sperm activating capacity in the «standardized model system».
Ion-exchange Chromato (5graphy, Serum (100) was fractionated on a
100 ml DEAE-Sepharose column (Pharmacia Fine Chemicals, Upp¬ sala, Sweden) equilibrated in 0.01M phosphate buffer, pH 7.4. The serum was diluted two-fold with 0.01M phosphate buffer, pH 7.4, and added to the column. After washing, a linear gradient of 2 x 300 ml 0-0.25M NaCl in 0.01M phosphate buffer, pH 7.4, was applied. Fractions (about 5 ml) were collected and the absorbancy at 280 nm was monitored. Aliquots of all fractions were then dialysed against 0.125M phosphate buffer, pH 7.4, and assayed for ability to stimulate SPM. Fractions able to promote sperm motility were pooled. Usually about 10 fractions (50 ml) recovered at about 0.2M NaCl in the gradient were used for further purification.
Chromatofocusing on PBE TM94. The pooled fractions were lyo- philized, dissolved in 5 ml water (Millipore ) , dialysed against the starting buffer (0,025M histidine-HCl, pH 6.0) and added to a 37 ml PBE TM94 chromatofocusing column. After wash- mg, Polybuffer <§> 74, pH 4.0, was applied and fractions were monitored for pH and sperm activating capacity. Samples (1 ml of each fraction) were dialysed against 0.01M phosphate buffer, lyophilized and dissolved in 250 μl 0.125M phosphate buffer, pH 7.4, before added to the test for sperm motility. Fractions showing sperm activating capacity were pooled. Usually about 3 fractions (15 ml), eluted at about pH 5.1, were obtained.
Fast Protein Liquid Chromatography^ (FPLC) . The fractions pooled were dialysed against 0.001M phosphate buffer, pH 7.4, and lyophilized, dissolved in 200 μl distilled water (Milli-
(5) T3J pore ) and fractionated on two Superose 12 HR 10/30 columns (serially) equilibrated in 0.125M phosphate buffer, pH 7.4. Calibrations were performed using ready made standards (Phar¬ macia Fine Chemicals, Uppsala, Sweden). A part (10 μl) from every fraction (600 μl) was tested for sperm activating capa¬ city. Usually the activity was found in two fractions (1.2 ml) corresponding to a molecular weight of about 250 kD.
Blue Sepharose Chromatography. The pooled fractions after FPLC were added to a Blue Sepharose column (10 x 20 mm) equilibrated in 0.125M phosphate buffer, pH 7.4. The sperm activating capacity did not stick to the gel and was re¬ covered in the fractions corresponding to the void volume and the wash with 0.125M phosphate buffer, pH 7.4 (total: 30 ml). This material was lyophilized and dissolved in 1.0 ml water; ten μl was routinely used for the sperm motility test.
Analytical disc electrophoresis in the presence of sodium dodecyl sulphate (SDS) (NaDodSO.), in the presence of (reduc¬ ing) and in absence of 2-mercaptoethanol was performed using the GE 2/4 Vertical System (Pharmacia, Sweden) with ready made Gel Casette Kits (80 x 80 mm) in addition to standard for calibration (Pharmacia Fine Chemicals, Uppsala, Sweden) . Pro¬ teins were visualised using Coomassie Blue. Samples for amino acid sequence analysis were degraded in an Applied Biosystems 470 A gas phase sequencer equipped with an on-line phenylthio- hydantoin analyser 120 (Applied Biosystems Inc., Forster City, CA) .
Quantitation of total protein was performed with the BIO-RAD protein assay (BIO-RAD Laboratories, M nchen, GFR) using bovine serum albumin as standards. Apolipoprotein Al was ana¬ lyzed with nephelometry using reagents from Be.iringwerke (Mar¬ burg, GFR) .
Immunization and Immunoprecipitation were performed on two 1-year-old male New Zealand White rabbits as described /Vaitu- kaitis I. Robbins J B, Nieschlag E and Ross G T. (1971), J. Clin. Endocrinol. Metab. 33, 988-991/ tfith the purified pro¬ tein complex. The protein, 20 μg for one animal and 80 μg for the other, in 1 ml water was mixed with 1 ml Freund’s complete adjuvant and the mixture was injected subcutaneously on the back of the animals. After 10 weeks, antiserum was detected in the rabbit given 20 μg SPAP.
Normal inactivated male serum and rabbit anti-SPAP serum were mixed in proportions ranging between 1:19 to 19:1 (v/v) . The mixture was incubated at 25°C for 20 hours before the tubes were centrifuged at 1400 x g for 25 minutes. The supernatants removed by sucktion and the pellets formed were stored at -20°C until used. The supernatants were tested for sperm motility and analyzed for apolipoprotein Al.
Amino acid sequence analysis were performed with two different pools each of three preparations of SPAP from the Blue Sepha- rose (____} step were analyzed by amino acid sequence degradation.
Results obtained were highly similar, establishing reproduci- bility of preparations, and revealed the presence of three major N-terminal sequences, which were followed for about 25 cycles before all were difficult to trace against a fairly high background (cf. Table I).
Table I
Amino acid sequence analysis of SPAP. Degradation of two pre¬ parations revealed three major N-terminal sequences in average ratios of about 1.4:1.1:1.0 that could be interpreted for 26, 20 and 15 cycles, respectively, before high background, carry¬ over and residue coincidences in the different structures pre¬ vented safe assignments. For the triplets identified at each cycle, the assignment as to sequence I, IT and III respec¬ tively, was frequently ambiguous from rec veries only, but listing as belonging to I, II, III was eεsily assigned once
the various combinations were found to fit into three known structures. I corresponds to apolipoprotein Al, II to immu¬ noglobulin light chains (k) , and III to immunoglobulin heavy chains (g, m and a) . Residues identified at each cycle are listed in that order. At a few positions (1, 7, 11) only two major residues were identified (one in higher yield) , re¬ flecting positions where two of the structures appeared to have identical residues. Recoveries are split accordingly in their listing. Assignments considered tentative, mainly be¬ cause of considerable background, are given within parenthe¬ sis. Two preparations gave almost identical results, except for slight variation in yields of individual residues, ten¬ tative assignments, and lengths of safe sequence tracings.
Cycle I II III
1 Asp 250 (Asp 200) Glu 200
2 Glu 240 He 190 Val 180
3 Pro 200 Val 170 Gin 200
4 Pro 210 Met 170 Leu 200
5 Gin 190 Thr 150 Val 140
6 Ser 150 Gin 130 Glu 130
7 Pro 160 Ser 110 Ser 110
8 Trp 50 Pro 140 Gly 130
9 Asp 150 Val 140 Gly 120
10 Arg 100 Thr 110 (Glu 100)
11 Val 210 Leu 90 Leu 90
12 Lys 170 Ser 100 Val 110
13 Asp 140 (Val 100) Gin 80
14 Leu 170 Ser 80 Pro 100
15 Ala 150 (Pro 90) Gly 90
16 Thr 100 (Gly 80)
17 Val 140 Glu 60
18 Tyr 130 Arg 50
19 Val 130 Ala 70
20 Asp 100 Thr 60
21 Val 130
22 Leu 110
23 Lys 80
Table I ( cont . )
24 Asp 90
25 Ser 70
26 Gly 80
The triplets for each position were screened against known structures, revealing that the three protein structures were known before. Thus one, corresponded exactly to human apolipo¬ protein Al /Baker H.N., Gotto A.M.JR. and Jackson R.L. (1975) J. Biol. Chem. 7, 2725-2738/, while the other two were identi¬ cal to the major alternatives known for immunoglobulin light and heavy chains. The light chain corresponds to a typical k chain and the heavy chain, as judged from coincidences with reports of heavy chain structures show maximal fits with several g, m or a chains, and could well be a typical g chain. Although the native of the immunoglobulin chains should not be considered final from a sequence analysis in mixture. The results, nevertheless clearly identify SPAP as composed of three types of protein chain, apolipoprotein Al, and immuno¬ globulin heavy and light chains without obvious signs of multiplicity. Stoichiometries are difficult to judge since initial yields in the degradation may be slightly different for the chains but a complex between the immunoglobulin and the apolipoprotein molecules yielding equimolar amounts of the chains appears possible.
The sperm activating capacity of SPAP is demonstrated as follows:
The eluates from the different purification steps activate spermatozoa to the same extent as 16.7% (v/v) serum. The sperm activating capacity of purified SPAP was found to be dependent on the SPAP concentration. The activation could be detected using SPM as well as ATP (Fig. 4). In the «standardized model system» with 2.5 million spermatozoa/ml the maximum effect was obtained at a concentration of 20-70 nmol/1. Purified SPAP heated to 100°C for 5 minutes ά d not activate sperm motility. Purified albumin, apolipoprotein Al and immunoglobulins had
only minor and short termed (a couple of hours) effects on sperm motility.
The immunization and characterization of anti-SPAP serum was performed by immunizing rabbits with purified SPAP in Freund’s complete adjuvant. Ten weeks after immunization antiserum of sufficient titer was detected in one rabbit. When this antiserum was incubated with normal male sera in proportions of about 9:1 (v/v) a precipitate was formed. Analyzes of the supernatant for apolipoprotein Al showed a low apolipoprotein Al content compared with that of untreated sera, suggesting that most of the apolipoprotein Al had been precipitated by the rabbit serum. Compared with untreated human serum, the precipitated human serum showed less capacity (about 25%) to support sperm motility. When rabbit anti-SPAP serum was added directly to spermatozoa in the «standardized model system» the motility was reduced as compared to that seen with the addi¬ tion of pre-immunized serum.
SPAP antiserum added to spermatozoa was found to inhibit sperm motility. The most likely explanation for this is that SPAP is earlier bound to the spermatozoa and then recognized by the antibodies.
After gel filtration by FPL ,
