Biomedical subjects

W W Wright

Publications and source records attributed to W W Wright.

At least 37 records · Page 2Linked to original sources

PubMed1992

Penetration of analogues of H2O and CO2 in proteins studied by room temperature phosphorescence of tryptophan.

The influence of the protein matrix on the reactivity of external molecules with a species buried within the protein interior is considered in two general ways: (1) there may be structural fluctuations that allow for the diffusive penetration of the small molecules and/or (2) the external molecule may react over a distance. As a means to study the protein matrix, a reactive species within the protein can be formed by exciting tryptophan to the triplet state, and then the reaction of the triplet-state molecule with an external molecule can be monitored by a decrease in phosphorescence. In this work, the quenching ability (i.e., reactivity) was examined for H2S, CS2, and NO2- acting on tryptophan phosphorescence in parvalbumin, azurin, horse liver alcohol dehydrogenase, and alkaline phosphatase. A comparison of charged versus uncharged quenchers (H2S vs SH- and CS2 vs NO2-) reveals that the uncharged molecules are much more effective than charged species in quenching the phosphorescence of fully buried tryptophan, whereas the quenching for exposed tryptophan is relatively independent of the charge of the quencher. This is consistent with the view that uncharged triatomic molecules can penetrate the protein matrix to some extent. The energies of activation of the quenching reaction are low for the charged quenchers and higher for the uncharged CS2. A model is presented in which the quenchability of a buried tryptophan is inversely related to the distance from the surface when diffusion through the protein is the rate-limiting step.(ABSTRACT TRUNCATED AT 250 WORDS)

Carbon Dioxide↗

PubMed1992

Electron transfer from excited tryptophan to cytochrome c: mechanism of phosphorescence quenching?

Parvalbumin, aldolase and liver alcohol dehydrogenase (ADH), proteins exhibiting long-lived phosphorescence lifetimes at room temperature, were examined for their reactivity with ferricytochrome c (cytochrome c Fe3+) as an external electron acceptor. Illumination of a reaction mixture containing protein and cytochrome c in the absence of oxygen brought about reduction of cytochrome c in relation to the duration of light. The largest portion of reduced cytochrome c was found with a sample containing ADH, where a 50% reduction of cytochrome c was reached after 5 min of illumination with a xenon lamp. Parvalbumin and aldolase were about half as effective under the same conditions. Several lines of evidence support the idea that the reaction of cytochrome c occurred by a long-range electron transfer from the excited triplet state of tryptophan. First, cytochrome c quenches the tryptophan phosphorescence and with parvalbumin, its bimolecular quenching rate constant, kq, was 2.9 x 10(6) M-1 s-1. Second, when the illuminated reaction mixture was supplied with 0.2 mM to 1 mM nitrite, a concentration range of nitrite which quenches the tryptophan phosphorescence but not the fluorescence, the amount of reduced cytochrome c on illumination markedly decreased. Finally, for all illuminated protein samples, the extent of cytochrome c reduction occurred parallel to a decrease in tryptophan content as judged from a decrease in fluorescence intensity and/or a decrease in tryptophan absorption at 280 nm.

Alcohol Dehydrogenase↗

PubMed1992

Regulation of the synthesis and secretion of transferrin and cyclic protein-2/cathepsin L by mature rat Sertoli cells in culture.

While numerous studies have examined the response of immature rat Sertoli cells to specific hormones and growth factors, the regulation of mature cells in vitro has not been well examined because highly purified cells have been difficult to isolate. We now describe a detailed method for isolating Sertoli cells from mature (> 60 days of age) rats and generating primary cultures of these cells greater than 90% in purity. We demonstrate that cell density, hormones, and growth factors regulate the synthesis or secretion of two Sertoli cell products, transferrin and Cyclic Protein-2 (CP-2)/cathepsin L. Cell density modulated the response of mature Sertoli cells to some hormones; insulin (at 10 micrograms/ml) and epidermal growth factor (EGF) acted synergistically to stimulate transferrin synthesis by 80% when cells were cultured at a density of 1.65 x 10(5) cells/cm2 but had no effect on transferrin synthesis by cells cultured at 1.46 x 10(5) cells/cm2. A mixture of FSH, retinol, and testosterone increased transferrin synthesis by 30% at both cell densities, and this stimulation was independent of the effect of EGF and insulin. CP-2/cathepsin L synthesis was significantly stimulated by increased cell density. FSH, retinol, and testosterone also stimulated CP-2/cathepsin L synthesis by 30%; however, this stimulation just missed being statistically significant. Finally, we demonstrated that secretion of transferrin and CP-2 was reduced when cells were cultured in the presence of interleukin-1 alpha, a cytokine synthesized by Sertoli cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

Evidence that rodent epididymal sperm contain the Mr approximately 94,000 glucocorticoid receptor but lack the Mr approximately 90,000 heat shock protein.

Monoclonal antibodies directed against four different polypeptide epitopes on the Mr approximately 94,000 steroid-binding subunit of the rat liver cytosolic glucocorticoid receptor (GcR) were used to probe Western blots of epididymal spermatozoa from rats and mice. Two sperm polypeptides with apparent molecular weights of 94,000 (indistinguishable in size from the liver GcR subunit) and 150,000 reacted with these antibodies. Other polypeptides that are present in a wide variety of somatic cells [lamin-A, -B, and -C; topoisomerase-I; poly(ADP-ribose) polymerase; the 62-kilodalton internal nuclear matrix protein; the nucleolar protein B23; and histone H1] could not be detected in these preparations of spermatozoa, thus appearing to rule out contamination by somatic cells. Rat and mouse pachytene spermatocytes and round spermatids contained much lower amounts of the Mr approximately 94,000 and 150,000 polypeptides. These results suggested that the steroid-binding subunit of the GcR might be accumulated late in spermatogenesis. Consistent with this view, a 6-kilobase mRNA (identical in size to a mRNA detected in mouse somatic cell lines) was detected when Northern blots of mouse round spermatid RNA were probed with a cDNA to the steroid-binding GcR subunit. Although the results described above suggest the presence of GcR in rodent sperm, high affinity binding of glucocorticoids to epididymal sperm could not be detected in a whole cell binding assay. Further analysis revealed that the Mr approximately 90,000 heat shock protein (hsp90), a component reportedly required for high affinity ligand binding to the GcR, was present in early germ cells, but absent from rodent epididymal sperm. These results suggest that the Mr approximately 94,000 steroid-binding subunit of the GcR and an immunologically related Mr approximately 150,000 polypeptide are specifically accumulated during the later stages of rodent spermatogenesis, but are not assembled into receptor complexes capable of binding steroid. In addition, these results support the view that hsp90 is required for high affinity binding of glucocorticoids to the Mr approximately 94,000 GcR subunit in intact cells.

Adrenalectomy↗

PubMed1991

Cyclic protein-2, a secretory product of rat Sertoli cells, is the proenzyme form of cathepsin L.

Previous studies demonstrated that secretion of Cyclic Protein-2 (CP-2) by mature rat Sertoli cells increased 30-fold from stage II to stages VI-VII of the cycle of the seminiferous epithelium and suggested that this protein was concentrated around compacted spermatids. Analysis of other organs revealed that CP-2 was also detectable in the epithelium of the proximal kidney tubule and in neurons originating from the supraoptic and paraventricular nuclei of the hypothalamus. We now have isolated a partial 1.8-kilobase (kb) cDNA for CP-2 mRNA, and sequence analysis revealed that CP-2 was the proenzyme form of the cysteine protease cathepsin L; this was corroborated by immunoprecipitation of CP-2 by anticathepsin L immunoglobulin G and by enzymatic analysis of purified CP-2. Northern analysis of testis mRNA revealed major (1.7 kb) and minor (2.2 kb) transcripts which differed in the length of their 3'-untranslated sequences. Low levels of CP-2/cathepsin L transcripts were detected in many organs, while high levels were only detected in testis, kidney, and liver. In seminiferous tubules, CP-2/cathepsin L mRNA was undetectable at stage II, increased to maximal levels at stages VI and VIIa,b, and was again undetectable at stage XII. At stages VI-VII, CP-2/cathepsin L mRNA was present in Sertoli but not germ cells. Taken together, these data suggest that CP-2/cathepsin L gene expression is regulated in a cell-specific manner and that in Sertoli cells this expression is influenced by germ cells at specific steps of development. We propose that at stages V-VII, secreted CP-2/cathepsin L degrades adhesion molecules which bind compacted spermatids to Sertoli cells, thereby facilitating movement of these spermatids toward the lumen of the tubule.

Amino Acid Sequence↗

PubMed1990

Oxygen gradients in mitochondria examined with delayed luminescence from excited-state triplet probes.

Phosphorescent probes are described that are quenchable by dioxygen and that partition into membranes. These probes are derivatives of porphyrin, in which the central metal, either zinc or palladium, induces intersystem crossing to enhance the triplet yield. The location of the probe in a suspension of membranes depends upon the charge distribution of side groups on the porphyrins. Probes that partition into the membrane are sensitive to phase transitions in lecithin artificial membranes. In the mitochondria these membrane probes are within transfer distance from tryptophans in membrane proteins. Although absolute concentrations of oxygen in membranes cannot be determined by this method, by comparing the oxygen dependence of a probe in the aqueous phase with that in the membrane phase under actively respiring and inhibited conditions, it is possible to examine the extent of O2 depletion at the mitochondrial surface. We show that at oxygen tensions of 0.2 microM and higher these gradients are insignificant at usual oxygen consumption rates of mitochondria.

Animals↗

PubMed1990

Long-range electron exchange measured in proteins by quenching of tryptophan phosphorescence.

Ten proteins that span a wide range of phosphorescence lifetimes were examined for sensitivity to quenching by four agents of disparate chemical nature. The results show that quenching efficiency is relatively independent of the quencher and is highly correlated with depth of burial of the phosphorescent tryptophan. The bimolecular quenching rate constants (kq) measured for the different proteins, spanning 5 orders of magnitude in kq, are found to decrease exponentially with the distance (r) of the tryptophan in angstroms from the protein surface--i.e., kq = Aexp(-r/rho), where A contains a geometrical factor dependent on tryptophan burial and surface geometry [corrected]. Theoretical analysis shows that this behavior can be expected for an electron-exchange reaction between the buried tryptophans and quenchers in solution in the rapid diffusion limit. Therefore, the results obtained provide evidence for an exponential dependence of electron-transfer rate on distance in a protein environment and evaluate the distance parameter, rho, for electron transfer through the general protein matrix at 1.0 A. For a unimolecular donor-acceptor pair with ket = koexp(-r/rho), ko approximately 10(9) sec-1.

Enzymes↗

PubMed1990

Murine beta 1,4-galactosyltransferase: both the amounts and structure of the mRNA are regulated during spermatogenesis.

Previously we have shown that the gene encoding murine beta 1,4-galactosyltransferase (beta 1,4-GT; UDPgalactose:N-acetyl-D-glucosaminyl-glycopeptide 4-beta-D-galactosyltransferase, EC 2.4.1.38) is unusual in that it specifies two sets of mRNAs of about 3.9 and 4.1 kilobases (kb). Translation of the 3.9- and 4.1-kb mRNAs results in the predicted synthesis of two related membrane-bound forms of the protein of 386 amino acids (short form) and 399 amino acids (long form), respectively. In this study we have examined the expression of beta 1,4-GT during murine spermatogenesis. Spermatogonia contain a 4.1-kb transcript that is comparable in size to the beta 1,4-GT mRNA identified in somatic cells. During differentiation from spermatogonia (2n) to pachytene spermatocytes (4n), the amount of beta 1,4-GT mRNA is reduced to barely detectable levels. Continued differentiation to round spermatids (n) is coincident with a renewed production of beta 1,4-GT mRNA to levels comparable with those detected in spermatogonia. However, the characteristic 4.1-kb mRNA detected in spermatogonia is replaced by two truncated transcripts of 2.9 and 3.1 kb. By S1 nuclease analysis, the 2.9- and 3.1-kb transcripts were shown to encode the same open reading frame as the 4.1-kb transcript found in somatic cells. The shorter round spermatid transcripts arise as a consequence of the use of alternative poly(A) signals. Lastly, we show that, in direct contrast to all somatic tissues and cell lines examined to date, male germ cells synthesize only the long form of the beta 1,4-GT polypeptide.

Animals↗

PubMed1990

Sertoli cells, proximal convoluted tubules in the kidney, and neurons in the brain contain cyclic protein-2.

We analyze by immunocytochemistry the in vivo distribution in rat Sertoli cells of Cyclic Protein-2 (CP-2), which is maximally synthesized and secreted in vitro at stages VI and VII of the cycle of the seminiferous epithelium. This analysis demonstrates that CP-2 staining is strongest in Sertoli cells in stage VI and VII tubules. Additionally, we demonstrate that the staining for CP-2 within a stage VII tubule differs from the staining of another Sertoli cell secretory product, androgen-binding protein. CP-2 is not detected by immunocytochemistry in any other tissues of the reproductive tract, though immunoblot analysis demonstrates the presence of CP-2 in rete testis and epididymal fluids. CP-2 was immunocytochemically detected in only three other organs: the kidney, the brain (with greatest concentration in the supraoptic and paraventricular nuclei), and the posterior pituitary. The presence of CP-2 in the kidney was confirmed by metabolic radiolabeling, immunoprecipitation, and peptide analysis. The presence of CP-2 in the brain was confirmed by immunoblot analysis of radioinert protein immunoprecipitated from the anterior hypothalamus.

Androgen-Binding Protein↗

PubMed1990

Germ cell-Sertoli cell interactions: the effect of testicular maturation on the synthesis of cyclic protein-2 by rat Sertoli cells.

Cyclic Protein-2 (CP-2) is synthesized in a stage-specific manner by mature rat Sertoli cells within stage VI and VII seminiferous tubules. To determine how testicular maturation affects CP-2 synthesis, we cultured 20 cm of tubules encompassing all stages of the cycle from rats 17, 35, 45, and 75 days old. The greatest increase in CP-2 synthesis was found to occur between 35 and 45 days and exceeded that observed for transferrin and sulfated glycoprotein (SGP)-2. Additionally, two-dimensional gel analysis indicated that secretion of CP-2 increased from 35 to 45 days to a greater extent than the secretion of SGP-1 and SGP-2 and transferrin. Biochemical analysis also demonstrated that CP-2 synthesis was stage-specific by 45 days. Immunocytochemistry expanded these observations; CP-2 was not detected in 7-35-day-old Sertoli cells. However, at 36 days, CP-2 was detected in Sertoli cells in stage VI and VII tubules but not at any other stage. CP-2 concentration in stage VI-VII tubules was increased by 38 days, but was unchanged thereafter. Finally, we immunocytochemically examined age-related changes in CP-2 concentration of the proximal convoluted kidney tubule. This analysis revealed that, at 1 wk, CP-2 was present in all proximal tubules except those in the subcapsular area; however, by 14 days, CP-2 was detected in all proximal tubules. This comparison of Sertoli cells and proximal tubule cells indicates that CP-2 content is determined by the maturity of a cell and not by the age of the animal.

Animals↗

PubMed1989

The equivocal presence of nuclear androgen binding proteins in mammalian spermatids and spermatozoa.

The presence of nuclear androgen binding proteins measured by nuclear androgen exchange in rat spermatids and spermatozoa was re-examined. Specific binding was observed to be related to less dense contaminating particles when sonicated testes were fractionated by isopcynic centrifugation through a 40-61% Nycodenz linear gradient. No specific binding was observed in a pure preparation of epididymal spermatozoa collected by retrograde perfusion of the cauda epididymidis, even when a nuclear exchange assay of superior sensitivity was used. Contamination could easily be induced by adding prostatic tissue to epididymal spermatozoa prior to sonication. Despite this strong evidence that the measure of nuclear androgen receptors by nuclear exchange in germ cells is artefactual, the persistence of high endogenous concentrations of 5 alpha-dihydrotestosterone and 5 alpha-diols (but not testosterone) in spermatozoa of castrated rats argues for the opposite conclusion.

Androgen-Binding Protein↗

PubMed1989

Characterization and localization of in vivo phospholipid methylation in the hamster testis.

Although previous studies have demonstrated that phospholipid methylation occurs in the testis and may be involved in Leydig cell function, phospholipid methylation in spermatogenic cells has not been characterized. In this study we describe the occurrence, time course, and localization of phospholipid methylation in the hamster testis following intratesticular injection of radioactive methyl precursor. Adult and pubertal (seven day old) hamsters were injected intratesticularly with [3H-methyl]-methionine and sacrificed 10 min. to 31 hours thereafter. The testes were then removed and homogenized or dispersed into cell suspensions. Spermatogenic cell and Leydig cell enriched preparations were isolated from the dispersed cell preparations using elutriation and Percoll gradient centrifugation and assayed for methylated phospholipids and proteins. These experiments demonstrated that 1) phospholipid methylation occurs in the hamster testis at a level seven-fold greater than protein methylation, 2) the incorporation of radioactivity associated with phospholipid methylation is progressive over time, and 3) in vivo, spermatogenic cell preparations enriched with pachytene spermatocytes have an almost four-fold higher level of measurable phospholipid methylation when compared to whole testis preparations. Taken together, these results suggest that phospholipid methylation may play an important stage-specific role in spermatogenesis.

Animals↗

PubMed1989

Germ cell-Sertoli cell interactions. Studies of cyclic protein-2 in the seminiferous tubule.

This review briefly describes the discovery and isolation of a novel Sertoli cell product, cyclic protein-2, (CP-2) and the generation of an antiserum against this protein. Using this antiserum, we demonstrated a stage-specific change in the synthesis of CP-2 by Sertoli cells within intact seminiferous tubules; synthesis is maximal at stages VI and VIIa,b of the cycle and minimal at stage XII. That CP-2 is a product of Sertoli cells was confirmed by immunohistochemical analysis. Comparison of CP-2 and transferrin synthesis by immature (17-day) and mature (75-day) Sertoli cells within intact seminiferous tubules has documented a significant increase in the synthesis of both proteins during testicular maturation. It was noteworthy, however, that the increase in CP-2 synthesis was much greater than the increase in transferrin synthesis. These data in conjunction with previous comparisons of the stage-specific changes in CP-2 and transferrin synthesis and secretion led to the hypothesis that the synthesis of these two proteins is regulated by different cellular interactions. Examination of cultured Sertoli cells obtained from mature rats demonstrated that transferrin synthesis and secretion were stimulated by hormones and vitamins, whereas CP-2 synthesis and secretion were not significantly affected by the same factors. Therefore, these data demonstrate that hormonal regulation of transferrin synthesis by Sertoli cells differs from hormonal regulation of CP-2 synthesis. Indeed, our data suggest that CP-2 synthesis is not directly regulated by hormones and vitamins. Finally, we demonstrated that when Sertoli cells are separated from germ cells and the Sertoli cells placed in culture, the age-dependent increase in CP-2 synthesis, noted with cultured tubules, is lost. In contrast, significantly more transferrin is synthesized by primary cultures of Sertoli cells obtained from old animals than from young animals. Taken together, all of these data indicate that the regulation of CP-2 synthesis and secretion by the Sertoli cell is unique and is primarily stimulated by paracrine signals or direct cell contact with the germ cells. Which of these mechanisms of cell-cell communication in the testis is important to regulation of CP-2 synthesis by Sertoli cells is unknown. Neither do we know which spermatogenic cell type provides this stimulus. These issues can now be addressed, however, because we have developed the protocols for isolating and culturing Sertoli cells from mature rat testes.

Age Factors↗

PubMed1989

Clinical evaluation of a posterior composite resin containing a semiporous filler particle.

Bowen recently developed a new filler particle for composite resin restorations. The surface particles of a diphasic strontium glass are superficially etched and after silanating the surface, the microscopic defects are filled with prepolymerized resin. The filler particles are then bonded chemically and mechanically to the resin matrix. This paper discusses the clinical performance of this material as a posterior composite resin over a two year period.

Bicuspid↗

PubMed1988

Quenching of room temperature protein phosphorescence by added small molecules.

A number of molecular agents that can efficiently quench the room temperature phosphorescence of tryptophan were identified, and their ability to quench the phosphorescence lifetime of tryptophan in nine proteins was examined. For all quenchers, the quenching efficiency generally follows the same sequence, namely, N-acetyltryptophanamide (NATA) greater than parvalbumin approximately lactoglobulin approximately ribonuclease T1 greater than liver alcohol dehydrogenase greater than aldolase greater than Pronase approximately edestin greater than azurin greater than alkaline phosphatase. Quenching rate constants for O2 and CO are relatively insensitive to protein differences, while H2S and CS2 are somewhat more sensitive. These small molecule agents appear to act by penetrating into the proteins. However, penetration to truly buried tryptophans is less favorable than previously suggested; in five proteins studied, quenching efficiency by O2 is 20-1000 times lower than for NATA, and up to 10(5) lower for H2S and CS2. Larger and more polar quenchers--including organic thiols, conjugated ketones and amides, and anionic species--were also studied. The efficiency of these quenchers does not correlate with quencher size or polarity, the quenching reaction has low energy of activation, and quenching rates are insensitive to solvent viscosity. These results indicate that the larger quenchers do not approach the buried tryptophans by penetrating into the proteins, even on the long phosphorescence time scale, and are also inconsistent with a mechanism in which quencher encounter with the tryptophan occurs in free solution, as in a protein-opening reaction. The results obtained suggest that the quenching process involves a long-range radiationless transfer.(ABSTRACT TRUNCATED AT 250 WORDS)

Luminescent Measurements↗

PubMed1988

Germ cell-Sertoli cell interactions: analysis of the biosynthesis and secretion of cyclic protein-2.

Cyclic protein-2 (CP-2) is secreted in vitro in substantial amounts by mature rat Sertoli cells in intact Stage VI and Stage VII seminiferous tubules. This stage-dependent secretion has led us to postulate that the biosynthesis of this molecule is stimulated by germ cells at a specific state of development. In order to explore this hypothesis and to examine the steps in CP-2's biosynthesis, we generated a polyclonal antisera against this protein and used it to analyze the biosynthesis and secretion of CP-2. Analysis of the steps in the biosynthesis of CP-2 indicated that its polypeptide core represented most if not all of the translation product of the CP-2 mRNA and that a single aspargine-linked oligosaccharide became attached to this core. Analysis of the rate of biosynthesis of CP-2 at specific stages of the cycle of the seminiferous epithelium was also conducted. Two-millimeter segments of tubules at Stage II, VI, VIIa, b, VIII, and XII were cultured for 1 hr in the presence of [35S]methionine and radiolabeled CP-2 immunoprecipitated from the tubules. Data (35S-CP-2 synthesized per hour) demonstrated that the rate of CP-2's biosynthesis increased 9-fold from Stage II to Stages VI and VIIa, b and then decreased 13-fold by Stage XII. To determine whether these rates of biosynthesis were identical to the rates of secretion, tubules were cultured for 17 hr with [35S]methionine, CP-2 was immunoprecipitated from the culture medium and data were expressed as 35S-CP-2 secreted per hour. This analysis demonstrated that the rate of secretion of CP-2 varied in the same stage-specific manner as its rate of synthesis. However, at each stage, the apparent rate of biosynthesis of the molecule exceeded its apparent rate of secretion. In order to explain this observation, we analyzed the rate of export of newly synthesized CP-2 out of the tubules. This demonstrated that quantitative export of the protein into culture medium required at least 17 hr. This period of time was most likely due to the retention of the protein within the tubular lumen, since primary cultures of Sertoli cells were shown to rapidly secrete newly synthesized CP-2. We, therefore, concluded that CP-2 was biosynthesized in a stage-dependent manner and that all CP-2 was secreted.

Animals↗

PubMed1986

Grating acuity development after early surgery for congenital unilateral cataract.

Longitudinal measures of grating acuity were obtained from 16 children after early surgery, optical correction, and occlusion therapy for congenital unilateral cataract. Compliance with contact lens wear and occlusion therapy was good in this population. Monocular grating acuities were obtained by preferential looking during months 1 through 15 and by an operant procedure during months 16 through 53. Grating acuities of normal eyes did not differ from those obtained in an age-matched normal population and showed no evidence of occlusion amblyopia. During the first year of life, grating acuities of aphakic eyes were typically within the normal range, but lagged behind normal development during years 2 through 4. These results suggest that early surgery is associated with favorable long-term visual results but does not, even with good compliance, completely eliminate deprivation amblyopia.

Cataract↗

PubMed1986

Isolation of cyclic protein-2 from rat seminiferous tubule fluid and Sertoli cell culture medium.

Cyclic Protein-2 (CP-2), a stage-specific secretory product of the rat seminiferous epithelium, has been isolated from seminiferous tubule fluid (STF) and Sertoli cell culture medium. Isolation from STF was accomplished by mixing STF with radiolabeled proteins secreted by Stage VI-VII seminiferous tubules and sequential fractionation of these proteins by hydroxylapatite, DEAE-agarose, and quaternary amine ion-exchange chromatography. Radiolabeled proteins were used to identify the chromatographic fractions that contained CP-2. Through use of these procedures, a highly purified preparation of radioinert CP-2 was obtained from seminiferous tubule fluid. Cyclic Protein-2 was also isolated from Sertoli cell culture medium, indicating that the Sertoli cell is its most likely source. Preliminary characterization of CP-2 was conducted. First, CP-2 appeared to be highly enriched in methionine. Second, the molecular weight of CP-2 was found to be 20,000. Third, analysis by reverse-phase hydrophobic chromatography indicated that CP-2 was relatively hydrophobic. We conclude that CP-2 is a small hydrophobic glycoprotein secreted in vivo and in vitro in a stage-specific manner by Sertoli cells.

Animals↗