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PubMed2005

A novel EB-1/AIDA-1 isoform, AIDA-1c, interacts with the Cajal body protein coilin.

BACKGROUND: Cajal bodies (CBs) are nuclear suborganelles that play a role in the biogenesis of small nuclear ribonucleoproteins (snRNPs), which are crucial for pre-mRNA splicing. Upon nuclear reentry, Sm-class snRNPs localize first to the CB, where the snRNA moiety of the snRNP is modified. It is not clear how snRNPs target to the CB and are released from this structure after their modification. Coilin, the CB marker protein, may participate in snRNP biogenesis given that it can interact with snRNPs and SMN. SMN is crucial for snRNP assembly and is the protein mutated in the neurodegenerative disease Spinal Muscular Atrophy. Coilin knockout mice display significant viability problems and altered CB formation. Thus characterization of the CB and its associated proteins will give insight into snRNP biogenesis and clarify the dynamic organization of the nucleus. RESULTS: In this report, we identify a novel protein isoform of EB-1/AIDA-1, termed AIDA-1c, that interacts with the CB marker protein, coilin. Northern and nested PCR experiments reveal that the AIDA-1c isoform is expressed in brain and several cancer cell lines. Competition binding experiments demonstrate that AIDA-1c competes with SmB' for coilin binding sites, but does not bind SMN. When ectopically expressed, AIDA-1c is predominantly nuclear with no obvious accumulations in CBs. Interestingly, another EB-1/AIDA-1 nuclear isoform, AIDA-1a, does not bind coilin in vivo as efficiently as AIDA-1c. Knockdown of EB-1/AIDA-1 isoforms by siRNA altered Cajal body organization and reduced cell viability. CONCLUSION: These data suggest that specific EB-1/AIDA-1 isoforms, such as AIDA-1c, may participate in the regulation of nucleoplasmic coilin protein interactions in neuronal and transformed cells.

Binding, Competitive↗

PubMed1977

Influence of intravenous nutrients on bilirubin transport. I. Amino acid solutions.

The effects of synthetic amino acids on bilirubin transport were investigated with competitive binding assays, peroxidase assays, isotopic studies of bilirubin uptake by red cells, and difference spectroscopy. Results indicated that amino acids had no significant effect on the distribution of bilirubin at pigment to albumin molar ratios likely to be encountered in clinical situations.

Albumins↗

PubMed1978

Radioimmunoassays that demonstrate type-specific and group-specific antigenic reactivities for the major internal structural protein of murine mammary tumor viruses.

The 28,000-dalton (p28) major structural polypeptide of the mouse mammary tumor virus (MMTV) was isolated and used to develop a highly sensitive and specific radioimmunoassay. Under conditions of limiting antibody in competitive binding assays, as little as 50 pg of purified p28, as well as disrupted MMTV virions and mammary tumor extracts, competed specifically with 125I-labeled MMTV p28. The p28 polypeptide was further shown to contain both group-specific and type-specific antigenic determinants, thus also allowing for further differentiation of various MMTV strains.

Antibodies, Viral↗

PubMed1984

Insulin-like growth factor receptors in rat placental membranes.

The nature and relative quantity of insulin-like growth factor (IGF) receptors in rat placental microsomal membranes was investigated by competitive binding studies and covalent cross-linking followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Binding studies revealed that 100 micrograms membrane protein specifically bound 26.5% of [125I]iodo-IGF-II, 19.6% of [125I]iodomultiplication-stimulating activity III-2 [( 125I]iodo-MSA-III-2), and 11.5% [125I]iodo-IGF-I. IGF-II was equipotent with MSA-III-2, and both were approximately twice as potent as IGF-I in competing with [125I]iodo-IGF-I for binding. In contrast, IGF-I competed for the binding of [125I]iodo-MSA and [125I]iodo-IGF-II with only 5-20% the potency of unlabeled MSA and IGF-II. Insulin competed weakly with [125I] iodo-IGF-I for binding (achieving half-maximal displacement at 20 micrograms/ml), but did not compete with [125I]iodo-MSA for binding. This evidence suggested that while IGF-I binds to both IGF-I and IGF-II receptors, the majority of IGF-I binding is due to an interaction with IGF-II receptors. Studies using [125I]iodo-IGF-I covalently cross-linked to placental membrane receptors followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis confirmed that molecular species characteristic of the subunits of IGF-I receptors are present in rat placenta. It is concluded that rat placenta, like human placenta, contains receptors for both types of IGFs. Unlike human placenta, the majority of the receptors are of the IGF-II type.

Animals↗

PubMed1985

Selection and characterization of a breast cancer cell line resistant to the antiestrogen LY 117018.

We have selected and cloned a stable variant of the MCF-7 human breast cancer cell line (LY 2) that is resistant to LY 117018 (LY), a potent antiestrogen that inhibits cell growth at concentrations as low as 10(-10) M. The cell line was selected by increasing the concentration of LY in the growth medium in a stepwise manner from 10(-8) to 10(-6) M as the cells become resistant. LY2 has been cloned in soft agar and carried for over 50 passages with no change in resistance. Other antiestrogens, such as tamoxifen and 40-hydroxytamoxifen no longer inhibit cell proliferation of LY 2. The cell line is still responsive to estrogen in a cell proliferation assay, but contains somewhat less estrogen receptors than MCF-7. The cytosolic estrogen receptor sediments to a 4S position on high salt sucrose density gradient centrifugation and is completely shifted to a denser gradient region when the receptor is incubated with a monoclonal antiestrophilin. The nuclear estrogen receptor when covalently labeled with [3H]tamoxifen aziridine has the same mol wt (62,000) in both MCF-7 and LY2 cells, when determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In a competitive binding assay, LY 117018 competes for [3H]estradiol binding to its cytosol receptor with the same Ki in both MCF-7 and LY2 cells. When the induction of estrogen-specific proteins was examined, no detectable progesterone receptor could be detected in either estrogen-induced or control LY2 cells, in contrast to MCF-7 cells. However, both 52,000- and 160,000-dalton proteins were estrogen inducible in the medium of LY2 and MCF-7 cells, as measured by labeling with [35S]methionine. The phenotypic stability of the antiestrogen resistance in LY2 cells coupled with the cross-resistance the antiestrogens of widely different structures make this cell line an ideal model system for the study of hormone resistance in human breast cancer. In addition, while the mechanism of resistance is currently not elucidated, the selective loss of estrogen-inducible functions in this cell line may provide powerful clues for future study.

Binding, Competitive↗

PubMed1988

Glycosylation selectively alters the biological activity of prolactin.

We have undertaken studies to determine the effect of glycosylation on the lactogenic activity of ovine PRL (oPRL). Measuring casein production in the in vitro mouse mammary gland explant assay, we found that glycosylated oPRL had 80% of the activity of oPRL. In competitive binding studies using lactogen receptors from mammary glands of lactating rabbits, glycosylated oPRL had only 20% the potency of oPRL. In the Nb2 assay also, glycosylated oPRL was approximately 24% as potent as oPRL in stimulating mitogenic activity. Thus, these studies show that the glycosylated variant of PRL has less biological activity than the major PRL form and that the alteration of an activity by glycosylation is selective.

Animals↗

PubMed1991

Effect of a highly potent fluoro analog of 1,25-dihydroxyvitamin D3 on human bone-derived cells.

The fluorine introduced analog of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3], 26,26,26,27,27,27-hexafluoro-1,25-dihydroxyvitamin D3 [26,27-F6-1,25-(OH)2D3] is 5-10 times more potent than 1,25-(OH)2D3 in vitamin D-deficient rats and chicks. In this study we established cultures of human bone cells in order to elucidate the mechanisms responsible for the higher activity of this compound. The effects of 26,27-F6-1,25-(OH)2D3 and 26,26,26,27,27,27-hexafluoro-1,23(S),25-trihydroxyvitamin D3[26,27-F6-1,23(S),25-(OH)3D3], the postulated main metabolite of 26,27-F6-1,25-(OH)2D3, were assessed by the response of alkaline phosphatase (ALP) activity. 26,27-F6-1,25-(OH)2D3 increased ALP activity in a dose-related fashion, from a concentration of 10(-11) M and caused a 3-fold elevation at a concentration of 10(-9) M. To achieve the same stimulating effect on ALP activity, the required dose of 26,27-F6-1,25-(OH)2D3 was 100 times less than that of 1,25-(OH)2D3. Analysis of the receptors of these cells revealed that they have specific receptors for 1,25-(OH)2D3, which have a dissociation constant of 0.9 x 10(-10) M. The competitive binding assays of 26,27-F6-1,25-(OH)2D3 on these receptors showed that binding ability of 26,27-F6-1,25-(OH)2D3 is almost the same as that of 1,25-(OH)2D3. Therefore, receptor binding affinity does not account for the higher potency of 26,27-F6-1,25-(OH)2D3. The trihydroxylated compound, 26,27-F6-1,23(S),25-(OH)3D3 revealed almost the same stimulatory activity on ALP activity in these cells. The most likely explanation for the higher activity of 26,27-F6-1,25-(OH)2D3 than 1,25-(OH)2D3 is that 26,27-F6-1,25-(OH)2D3 is metabolized to 26,27-F6-1,23(S),25-(OH)3D3, which has almost the same activity as 26,27-F6-1,25-(OH)2D3 in target tissues, whereas 1,25-(OH)2D3 is degraded to less active metabolites such as 1,24,25-(OH)3D3.

Alkaline Phosphatase↗

PubMed1995

Regulation of renal atrial natriuretic peptide receptors in pregnant sheep.

These studies were designed to characterize the atrial natriuretic peptide (ANF) receptor subtypes (guanylyl cyclase GC-A and GC-B and ANF-C) in normal sheep kidneys and to evaluate alterations in receptor kinetics during pregnancy. Kidneys were obtained from 12 nonpregnant and 12 pregnant sheep during late gestation and maintained on a 100 mmol/day salt intake. Competition binding receptor assays using [125I]human ANF showed that inner medullary membranes are exclusively of the GC-A subtype. The maximum binding capacity (Bmax, 109 +/- 12 vs. 89 +/- 18 fmol/mg protein) and dissociation constant (Kd, 240 +/- 70 vs. 324 +/- 99 pM) are not altered by pregnancy. Specific binding of glomerular membranes to [125I]Tyr-C-type natriuretic peptide, which shows the highest affinity toward GC-B receptors, was observed, but this binding was abolished when ANF-C receptors were saturated with excess C-ANF-(101-121), suggesting that [125I]Tyr-C-type natriuretic peptide binding was mediated by ANF-C receptors. Binding of [125I]human ANF to glomerular membranes revealed that glomerular ANF receptor number was reduced during pregnancy (1040 +/- 212 vs. 335 +/- 42 fmol/mg protein; P = 0.001), but binding affinity was not changed. The reduced number was mainly due to a decrease in ANF-C receptor density (832 +/- 213 vs. 260 +/- 31 fmol/mg protein; P = 0.005). Autoradiography of whole kidney frozen sections produced similar findings. These studies demonstrate that GC-B receptors are absent from renal glomeruli and inner medulla, and that ANF receptor subtypes are differentially regulated in the pregnant sheep kidney, suggesting a role for ANF in the altered volume and pressure homeostasis of pregnancy.

Animals↗

PubMed1997

Electrophoretic mobility and glycosylation characteristics of heterogeneously expressed calcitonin receptors.

The translated calcitonin receptor (CTR) complementary DNA sequences contain potential N-linked glycosylation sites within the extracellular N-terminus. We investigated the relative molecular mass (M(r)) and degree of N-linked glycosylation of five cloned CTRs (pig, rat C1a, rat C1b, human I1-ve, and human I1+ve), together with the pig hypothalamic CTR, to analyze the potential contribution of carbohydrate moieties to the molecular identity of these receptors. Receptors were cross-linked to 125I-salmon CT with the homobifunctional reagent bis(sulfosuccinimidyl) suberate. Autoradiographic analysis of the cross-linked receptors, following SDS-PAGE, revealed apparent M(r)S, ranging between 70,000 and 80,000 for the rat, human, and pig hypothalamic receptors. However, the cloned, expressed pig CTR was much smaller (approximately 58,000). The lower M(r) of the cloned pig CTR appeared to be due to absence of N-terminal residues, but this did not impact on ligand-receptor specificity when compared with the hypothalamic pig CTR. Cleavage under nondenaturing conditions of N-linked sugars from the CTRs using endoglycosidase F (Endo F), increased the electrophoretic mobility of all receptors, except the pig CTRs, by approximately 10 kDa. Under denaturing conditions, electrophoretic mobilities increased by approximately 30 kDa for the rat C1a, rat C1b, and humanI1-ve (expressed in human embryonic kidney-293 cells) CTRs and by approximately 20 kDa for the cloned pig, pig hypothalamic, and human CTR isoforms (expressed in baby hamster kidney cells). Competition binding studies using glycosylated and partially deglycosylated (nondenaturing conditions) receptor preparations demonstrated no significant differences in binding affinity or specificity. Thus the CTRs are N-linked glycoproteins whose degree of glycosylation is both cell-type and species dependent.

Amino Acid Sequence↗

PubMed1999

Role of N-linked glycosylation on the function and expression of the human secretin receptor.

Secretin is a 27-amino acid long peptide hormone that regulates pancreatic water, bicarbonate, enzymes, and potassium ion secretion. The human secretin receptor (hSR) is a glycoprotein consisting of 440 amino acids, of which there are 5 putative N-linked glycosylation sites at positions Asn72, Asn100, Asn106, Asn128 (N-terminal ectodomain), and Asn291 (second exoloop). Through functional analysis of the hSR-transfected cells cultured in the presence of various glycosylation inhibitors, it was found that tunicamycin and castanospermine were able to significantly reduce the secretin-stimulated cAMP response. On the other hand, the effects of other inhibitors, swainsonine and deoxymannojirimycin, were much lower, suggesting that the high mannose-type carbohydrate side-chain is essential to the expression of a fully functional hSR. The role of individual N-linked glycosylation sites was studied by mutation analysis (Asn to Leu or Ser to Ala) coupled to measurements of cAMP accumulation and extracellular acidification rate. The ED50 values of the wild-type receptor in these two assay systems were 0.25 and 0.11 nM, respectively, and mutation at position 100, 106, or 291 did not affect either the ED50 values or the maximal responses in the two assays. However, the Asn72Leu and Ser74Ala mutations reduced the maximal responses and increased the ED50 values in both assays, suggesting that this site is a true glycosylation signal. This hypothesis was further supported by competitive binding studies, the same mutants were found to be defective in binding with [125I]secretin. To evaluate whether the change in receptor function of the mutants is caused by the change in the process of presenting the receptor to the cell surface, the mutants and the wild-type receptor were tagged with a c-Myc epitope at the C-termini. Using an anti-c-Myc monoclonal antibody and confocal microscopy, all of the mutant receptors were found to be expressed and delivered to the plasma membrane.

Amino Acid Sequence↗

PubMed2001

Evidence for estrogenic contamination of the MAPK inhibitor PD98059.

PD98059 blocks phosphorylation and activation of MAPK proteins, ERK1 and ERK2. In the course of examining the effect of PD98059 on estrogen-induced transcription of reporter genes in a human breast cancer cell line and in yeast, we found that two of four different batches of PD98059 produced estrogenic effects in a dose-dependent manner. In a competitive binding assay, these preparations of PD98059 displaced radiolabeled estradiol from ER alpha. Furthermore, in the yeast assay, addition of a coactivator protein, AIB1, enhanced the transcriptional effect of PD98059, indicating that it induces receptor-coactivator interactions. Although concentrations of PD98059 required to activate ER alpha in these experimental systems are 10(4)- to 10(5) higher than the concentration of estradiol required to do the same, the concentrations required to block MAPK activation are well above those which would produce maximal estrogenic effects. Thus, when PD98059 is used in estrogen-responsive cells, contaminating estrogenic activity may confound interpretation of experimental results.

Binding, Competitive↗

PubMed1979

Regulation of receptor by homologous hormone enhances sensitivity and broadens scope of radioreceptor assay for human growth hormone.

In standard competitive binding assays (including radioreceptor assays) unlabeled ligand (hormone) competes with labeled ligand (hormone) for binding to a fixed number of binding (receptor) sites. Detection of the unlabeled ligand occurs when the occupancy of binding sites by the unlabeled ligand is sufficient to reduce the binding of labeled ligand. A common feature of the hormone-receptor interaction is the ability of the hormone to regulate the affinity and/or the concentration of its homologous receptor. In the present study, by exploiting the ability of human GH to regulate by negative feedback the concentration of its own receptors, we have enhanced the sensitivity of the human GH radioreceptor assay 5-fold. The ability of hormone to regulate receptor concentration and affinity affords wide opportunities to broaden the scope as well as to enhance the sensitivity of radioreceptor assays.

Binding, Competitive↗

PubMed1982

Presence of human epidermal growth factor in human cerebrospinal fluid.

Human epidermal growth factor (gEGF), a potent stimulator of the growth of many tissues in culture, has been isolated from human urine and subsequently identified in many human biological fluids. We have partially purified and characterized hEGF-like substance(s) from human cerebrospinal fluid (CSF) and determined the concentrations of immunoreactive (IR) hEGF in CSF obtained from patients with a variety of neurological diseases. Competitive binding curves generated by the CSF samples appeared to be parallel to standard hEGF both in RIA and radioreceptor assays using human placental membrane. Sephadex G-50 gel exclusion chromatography of the CSF extract revealed a single peak of IR-hEGF which coeluted with standard hEGF. The apparent mol wt of the CSF hEGF-like substances(s) was estimated to be 8000 by sodium dodecyl sulfate poplyacrylamide gel electrophoresis, and its approximate pI was 4.5 as determined by isoelectric focusing. The concentrations of IR-hEGF in CSF from patients with pituitary and brain tumors and radiculomyelopathy were significantly higher than those from control subjects, while neither patients with hydrocephalus nor encephalomeningitis had CSF IR-hEGF levels statistically different from the control subjects. The presence of hEGF-like substance(s) in human CSF suggests that it may play an important physiological role in the function of the human nervous tissues but does not provide any evidence of its source.

Adolescent↗

PubMed1986

Detection of autoantibodies to thyroid peroxidase in autoimmune thyroid diseases by micro-ELISA and immunoblotting.

Serum autoantibodies to thyroid peroxidase (TPO) in patients with thyroid autoimmune diseases were studied by micro-ELISA and immunoblotting. Twenty-four patients, 15 with Graves' disease and 9 with Hashimoto's thyroiditis, whose serum titers were greater than 3200 on the microsomal hemagglutination test (except for 1 patient with a titer of 800) had autoantibodies to TPO. Both immunoglobulin G and M classes of autoantibodies were detected, with the former being more prominent. When TPO and thyroid microsomes were used as a target in a competitive binding inhibition test, the results suggested that TPO was a major thyroid microsomal antigen. On the other hand, immunoblotting analysis showed 3-4 bands in the 45-60K region stained by patients' sera in addition to human TPO with mol wt of 100K and 107K; only the latter 2 bands stained with antiporcine TPO antibody. In the majority of sera, TPO bands were clearer than others, although some sera showed the clearest band with a mol wt of 55K. These results indicate that patients with autoimmune thyroid disease often have autoantibodies to TPO that can be detected by micro-ELISA and immunoblotting, and that TPO is a major component of the thyroid microsomal antigen.

Animals↗

PubMed1988

Affinity purification of orbital membrane antigens for the study of the pathogenesis of Graves' ophthalmopathy.

We characterized 22 human monoclonal antibodies (MCAB) reactive with human orbital antigens. Most of these monoclonal antibodies had variable degrees of reactivity with eye muscle, orbital connective tissue, thyroid, and extra-thyroid tissue preparations when tested in an enzyme-linked immunosorbent assay (ELISA). Eight of the MCABs, selected on the basis of their reactivity with human eye muscle or orbital connective tissue antigens, were used to affinity purify orbital membrane antigens. All purified fractions were tested in the ELISA for reactivity with the MCAB used for their purification. Four of the purified antigens with a high reactivity were not proteins. To increase the specificity of the reactivity of serum autoantibodies with affinity-purified antigens, we developed a competitive binding ELISA in which the MCAB-immunoglobulin was directly labeled with alkaline phosphatase. We tested the serum of 24 patients with Graves' ophthalmopathy, 10 patients with a history of Graves' hyperthyroidism and no eye disease, 14 patients with Hashimoto's thyroiditis without eye disease, 8 patients with other nonautoimmune thyroid disorders, and 20 normal subjects for reactivity with an affinity-purified nonprotein orbital antigen. The levels of serum autoantibodies against this antigen in patients with Graves' ophthalmopathy were significantly higher than those in normal subjects or patients with Graves' hyperthyroidism with no eye disease. Although unlikely to be of primary pathogenetic significance, it is possible that levels of this antibody in patients with Graves' ophthalmopathy may reflect the extent of orbital inflammation and, therefore, be useful as a clinical marker.

Adult↗

PubMed1997

In vitro expression of endothelin-1 (ET-1) and the ETA and ETB ET receptors by the prostatic epithelium and stroma.

RT-PCR analysis of total RNA prepared from the prostate cancer cell lines DU145 and PC3 and from primary epithelial cells indicated the presence of endothelin-1 (ET-1) messenger RNA (mRNA). Neither the LNCaP cell line nor primary prostatic stromal cells possess ET-1 mRNA transcripts. Seventy-two-hour-conditioned media derived from DU145, PC3, and primary epithelia contain immunoreactive ET concentrations equivalent to 0.814 +/- 0.048, 0.330 +/- 0.050, and 0.856 +/- 0.055 fmol/mL/10(6) cells after 72 h, respectively. Basal immunoreactive ET secretion was exhibited by LNCaP (0.029 +/- 0.009 fmol/mL/10(6) cells after 72 h) and stromal cells (0.067 +/- 0.007 fmol/ mL/10(6) cells after 72 h). Examination of ETA and ETB gene expression by RT-PCR demonstrates that ET receptor mRNA is almost completely undetectable in the prostate cancer cell lines. Both ETA and ETB mRNAs are detectable in primary cultures of prostatic epithelia and stroma. Competitive binding studies demonstrate a single class of binding site in both primary benign epithelia (dissociation constant = 1.85 x 10(-10) mol/L; maximal binding capacity = 2.7 x 10(4) binding sites/cell), and stroma (dissociation constant = 1.93 x 10(-10) mol/L; maximal binding capacity = 3.7 x 10(5) binding sites/cell). Use of selective ET receptor antagonists confirmed that the predominant stromal receptor subtype expressed in vitro is ETB. This receptor seems not to be coupled to mitogenic pathways because no growth response to exogenous ET-1 or cooperation between ET-1 and bFGF could be observed. Similarly, no effect of ET-1 or the ET-converting enzyme inhibitor, phosphoramidon, on benign epithelial cells could be observed over a 4-day period.

Binding, Competitive↗

PubMed2001

Phytochemical glyceollins, isolated from soy, mediate antihormonal effects through estrogen receptor alpha and beta.

The flavonoid family of phytochemicals, particularly those derived from soy, has received attention regarding their estrogenic activity as well as their effects on human health and disease. In addition to these flavonoids other phytochemicals, including phytostilbene, enterolactone, and lignans, possess endocrine activity. The types and amounts of these compounds in soy and other plants are controlled by both constitutive expression and stress-induced biosynthesis. The health benefits of soy-based foods may, therefore, be dependent upon the amounts of the various hormonally active phytochemicals within these foods. The aim was to identify unique soy phytochemicals that had not been previously assessed for estrogenic or antiestrogenic activity. Here we describe increased biosynthesis of the isoflavonoid phytoalexin compounds, glyceollins, in soy plants grown under stressed conditions. In contrast to the observed estrogenic effects of coumestrol, daidzein, and genistein, we observed a marked antiestrogenic effect of glyceollins on ER signaling, which correlated with a comparable suppression of 17 beta-estradiol-induced proliferation in MCF-7 cells. Further evaluation revealed greater antagonism toward ER alpha than ER beta in transiently transfected HEK 293 cells. Competition binding assays revealed a greater affinity of glyceollins for ER alpha vs. ER beta, which correlated to greater suppression of ER alpha signaling with higher concentrations of glyceollins. In conclusion, we describe the phytoalexin compounds known as glyceollins, which exhibit unique antagonistic effects on ER in both HEK 293 and MCF-7 cells. The glyceollins as well as other phytoalexin compounds may represent an important component of the health effects of soy-based foods.

Benzopyrans↗

PubMed1990

Temporal expression, polar distribution and transition of an epitope domain in the perinuclear theca during mouse spermatogenesis.

A novel domain of epitopes is expressed by a family of high-Mr proteins at the anterior pole of the germ cell nucleus during spermiogenesis, and later by two low-Mr proteins at the anterior and posterior poles of the nucleus during sperm maturation in the epididymis. Initially, monoclonal antibodies (mAbs) PNT-1 (IgG2b) and PNT-2 (IgG2a) bound to antigens present in a cap-like configuration at the apical pole of the spermatid nucleus at step 5 of spermiogenesis. The distribution of epitopes on the nucleus expanded posteriorly until, in testicular sperm they covered the anterior pole down to the distal limits of the subacrosomal perforatorium. By contrast, sperm from the epididymis and vas deferens bound both mAbs in two distinct regions on the nucleus, one on the dorsal margin of the anterior pole, and the other in a ventral zone at the posterior pole. On SDS-PAGE and isoelectric focusing (IEF) immunoblots, both mAbs bound three major proteins with Mr of approximately 80,000, 77,000 and 75,000 from spermatids and testicular sperm, and proteins of Mr 50,000 and 48,000 in epididymal and vas deferens sperm. Both the high- and low-Mr protein families were recovered in germ cell nuclear/perinuclear matrices. Their mobilities on SDS-PAGE were not altered by exo- or endoglycosidases or by aminoethylation in denaturing conditions. mAb PNT-1 bound to the sperm proteins with a Ka of 3.53 x 10(12) M-1 and mAb PNT-2 with a Ka of 2.08 x 10(12) M-1. From competition binding data, mAbs PNT-1 to -10 appeared to recognize six adjacent or overlapping epitopes on the same proteins. These data suggest the high-Mr proteins, the thecins, present at the anterior pole of haploid germ cells are processed at the onset of sperm maturation to yield two low-Mr proteins that then occupy two distinct domains at the anterior and posterior poles of the nucleus.

Animals↗