Comparison of the effects of lung cancer, benign lung disease, and normal aging on pituitary-gonadal function in men.

We retrospectively determined serum total testosterone (T), fraction of T bound, free T index, LH, and FSH levels in 122 men with malignant lung disease, 32 men with benign lung disease, and 106 normal men. Men with malignant and, to a lesser extent, benign lung disease had decreased serum total T and free T index values at the 5th percentiles, with elevations of LH and FSH levels at the 95th percentiles. Linear regression analysis showed reductions in total T and free T index and increases in FSH, but not LH, levels with age in each group. Using multivariate analysis, we found stronger independent effects of disease than age on serum total T and fraction of T bound, but a greater influence of age on free T index. Serum LH values differed by diagnosis, whereas FSH differed by age. Relative to values in the normal men, mean serum total T levels were reduced in men with lung cancer; the fraction of T bound was decreased in the men with lung cancer and increased in the men with benign lung disease, the free T index was decreased in the men with both malignant and benign lung disease, and LH was increased in the men with lung cancer. The hormone and hormone binding results were similar in men with different types of lung cancer. Biochemical evidence of primary and secondary (or combined primary and secondary) hypogonadism was present in 50-59% and 28-32%, respectively, of the men with malignant and benign lung disease vs. 10% of the normal men. These data suggest that 1) there is an increased prevalence of both pituitary gonadotropic and testicular dysfunction in men with malignant and, to a lesser extent, benign chronic lung disease, and 2) the effects of illness are independent of, and quantitatively greater than, those due to age.

Myeloproliferative syndrome evolving in a patient with macromolecular lactate dehydrogenase.

A myeloproliferative syndrome, masked by severe iron deficiency, evolved in a woman with lactate dehydrogenase (LDH) complexed to IgA. Macromolecular LDH is an uncommon cause of increased serum LDH activity. By observing the patient's course for ten years, we were able to understand the initially puzzling clinical findings.

Differences in the carbohydrate moieties of the common alpha-subunits of human chorionic gonadotropin, luteinizing hormone, follicle-stimulating hormone, and thyrotropin: preliminary structural inferences from direct methylation analysis.

The carbohydrate components of combined alpha-subunits of urinary hCG and human pituitary LH (hLH), FSH (hFSH), and TSH (hTSH), each derived from the intact hormone, were studied by direct sugar analysis and methylation analysis. The methods provide a complete survey of the structural elements contained in the complex sugars associated with these glycoproteins, but do not establish the sugar sequences or anomeric configurations of glycosidic bonds. By analogy to N-linked oligosaccharides that occur in many glycoproteins, the data suggest distinct structural features for carbohydrates of alpha-subunits combined with beta-subunits. hCG alpha contains biantennary asparagine-linked chains terminated by either NeuAc alpha 2-3Gal beta 1- or GlcNAc beta 1-2 Man alpha 1- and lacks fucose. hTSH alpha contains biantennary chains with the same termini as hCG alpha plus terminal R-O-4GalNAc and a fucosyl residue linked alpha 1-6 to the inner GlcNAc residue of the N-linked chitobiosyl core. hLH alpha may contain some high mannose chains, but primarily contains biantennary chains terminated by NeuAc alpha 2-3(6)Gal beta 1-, GlcNAc beta 1-, GalNac-1-, R'-O-6GlcNAc-1-, and R"-0-2Man-1-plus a fucosyl residue linked alpha 1-6 to the inner GlcNAc residue of the N-linked chitobiosyl core. hFSH alpha contains more complicated structures that probably include a bisecting GlcNAc residue linked beta 1-4 to a 3,6-di-O-substituted core mannosyl residue, and terminal NeuAc alpha 2-3Gal beta 1-4(+/- Fuc alpha 1-3)GlcNAc-1, Gal beta 1-4(+/- Fuc alpha 1-3)GlcNAc-1-, R"'-O-GalNAc-1-, and GalNAc-1. In addition, the presence of 2,4-di-O-substituted mannose in hFSH alpha indicates that it contains triantennary chains. The identities of the R; R', R", and R"' groups were not determined, but recent studies of glycoprotein hormones suggest that they may be sulfate groups. Our results demonstrate differential glycosylation of virtually identical polypeptide hormone alpha-subunits produced in the same organ or perhaps even in the same cell.

New placental proteins: chemistry, physiology and clinical use.

In the past decade, several new placental proteins have been isolated and studied. The 'pregnancy-specific' beta 1-glycoprotein (SP1) is a major placental product with unusual physicochemical properties that has been extensively investigated, but its biological function remains uncertain. Pregnancy-associated plasma protein A (PAPP-A), a glycoprotein of mol. wt 400,000, has effects in vitro on the coagulation and complement cascades, probably by its properties of protease inhibition. Placental protein 5 (PP5) may be involved in the coagulation and fibrinolytic systems, and in follicle maturation and semen liquefaction. 'Placental protein 12' (PP12) is not a product of the placenta at all; it appears to be produced in the female genital tract under the influence of progesterone and may also be produced by proliferating liver cells. Further study may reveal new roles for these placental proteins beyond their traditional roles as tumour markers.

Oligomerization of pregnancy-specific beta 1-glycoprotein (SP1) at physiologic pH and ionic strength.

Highly purified pregnancy-specific beta 1-glycoprotein (SP1) migrated in gel electrophoresis as a homogeneous species and behaved as a single species in 6 mol/l guanidinium chloride (GdmCl), both in the ultracentrifuge and HPLC. At physiologic pH and ionic strength, in the absence of GdmCl, SP1 existed in the form of oligomers of apparent molecular weights of 40 000 to greater than 300 000. The specific activity of these oligomers varied over a 5-fold range. Electrophoretic mobility also varied among SP1 oligomers, with increasing (alpha-like) mobility shown by oligomers of increasing molecular size. Oligomerization may explain some or all of the reports of SP1 heterogeneity.

A distinctive form of human chorionic gonadotropin beta-subunit-like material produced by cervical carcinoma cells.

The DoT and CaSki human cervical carcinoma cell lines ectopically produce material immunologically similar to the beta-subunit of human chorionic gonadotropin (hCG beta). Culture fluids were analyzed by gel filtration chromatography and radioimmunoassay (RIA) using (a) antiserum directed to conformation-specific (core-directed) determinants not involving the carboxyl-terminal peptide (CTP) in hCG beta purified from urinary hCG (i.e., standard hCG beta) or (b) antiserum directed to the CTP in standard hCG beta. CTP-directed RIA recognized a peak of hCG beta-like immunoreactive material that eluted in the same position as standard hCG beta. However, core-directed RIA recognized additional hCG beta-like material (i.e., ectopic beta-II), most of which eluted before standard hCG beta. CaSki cells were incubated with [3H]mannose, [3H]proline, and [3H] leucine, and the spent medium was immunoprecipitated and analyzed by gel electrophoresis. Several labeled peaks were detected in the lane from the anti-hCG beta X Sepharose immunoprecipitate, one of which corresponded in mobility to standard hCG beta, with two more intense components migrating at higher apparent molecular weights. Carboxypeptidase Y digestion released only 0.2 mol equivalents each of [3H]proline and [3H]leucine from the labeled CaSki material immunoprecipitated with anti-hCG beta X Sepharose, compared to 1 mol equivalent each in similar analysis of standard hCG beta. These findings were consistent with the absence of the 4-carboxy-terminal amino acids from 80% of the hCG beta-like immunoreactive material secreted by CaSki cells. The affinity purified ectopic beta-II failed to combine with standard hCG alpha under conditions in which combination of standard hCG beta with standard hCG alpha was essentially complete. Neither aggregation nor proteolytic degradation was the cause of failure of ectopic beta-II to combine with hCG alpha. We conclude that both the DoT and CaSki cervical carcinoma cell lines secrete a distinctive form of hCG beta-like material, ectopic beta-II. Lack of recognition by CTP-directed antisera and amino acid analysis suggest that ectopic beta-II may lack the CTP, despite its apparent larger size relative to standard hCG beta.

A novel form of ectopic human chorionic gonadotrophin beta-subunit in the serum of a woman with epidermoid cancer.

A novel form of free human chorionic gonadotrophin beta-subunit (hCG beta) was found in serum from ElBre, a woman with epidermoid carcinoma of unknown origin. ElBre hCG beta was larger than standard (pregnancy urine) hCG beta when analysed by gel chromatography (apparent molecular weight 54 000 vs 44 000). This size difference appeared to be due to a larger carboxyterminal extension (CTE) of ElBre hCG beta since thermolysin cleavage of the CTE from standard hCG beta and Elbre hCG beta yielded core products of the same size. Oligosaccharides, O-linked to serine or threonine, were present in ElBre hCG beta, presumably on its CTE as judged by the complete binding of desialylated ElBre hCG beta to immobilized peanut agglutinin (this lectin is specific for terminal galactose linked beta 1----3 to N-acetylgalactosamine, a disaccharide exposed after desialylation of the O-linked oligosaccharides of standard hCG beta). ElBre hCG beta, however, was incompletely recognized by antisera specific for the CTE of standard hCG beta, especially the carbohydrate-sensitive antiserum R141. The O-linked oligosaccharides of standard hCG beta are heterogeneous in size; 13% are of the largest (hexasaccharide) form. In contrast, over 50% of the O-linked oligosaccharides in hCG beta from the JAr choriocarcinoma cell line are hexasaccharides. Like desialylated ElBre hCG beta, desialylated JAr hCG beta bound completely to peanut agglutinin, but was incompletely recognized by antisera to the hCG beta-CTE. Furthermore, JAr hCG beta was intermediate in size between standard hCG beta and ElBre hCG beta when analysed by gel chromatography (apparent molecular weight 49 000).(ABSTRACT TRUNCATED AT 250 WORDS)

Human chorionic gonadotropin and human placental lactogen in extragonadal tumors. An immunoperoxidase study of ten non-germ cell neoplasms.

The immunoperoxidase localization of the alpha and beta subunits of human chorionic gonadotropin (hCG) and of human placental lactogen (hPL) was studied in ten extragonadal nontrophoblastic tumors associated with raised serum levels of one or more of these placental proteins. Three of the tumors were bronchial carcinomas, one was a gastric carcinoma, two were malignant carcinoids (one bronchial and one gastric), two were pancreatic islet cell carcinomas, and two were metastatic carcinomas with an unknown primary site. The maximum alpha subunit serum level was 33,000 ng/ml (gastric carcinoid), the maximum hCG/hCG-beta level was 705,000 ng/ml, and the maximum hPL level was 50 ng/ml (both in the gastric carcinoma). An indirect immunoperoxidase technique and rabbit polyclonal affinity-purified antibodies and peroxidase conjugates were used on formalin-fixed, paraffin-embedded sections. Five blocks (eight cases) or six blocks (two cases) from various sites were obtained from each patient at surgery and/or autopsy. Positive stains for hCG/hCG-beta were seen in six of seven tumors (25/37 blocks) with raised levels, for the alpha subunit in nine of nine tumors (30/47 blocks), and for hPL in two of five tumors (4/26 blocks). Only a relatively minor number of the cells were positive, and within the same case, there was considerable site-to-site variation in the number of positive cells. Large bizarre cells contained hCG/hCG-beta as well as the alpha subunit, if it was demonstrated in the same tumor as the beta subunit. Otherwise, the alpha subunit was found in small unremarkable cells. Giant cells that were smaller than those positive for hCG/hCG-beta contained in hPL. In some serial sections, hCG-alpha, hCG/hCG-beta, and hPL were segregated in different cell populations, supporting the concepts of their separate genetic control.

Eutopic and ectopic macromolecular human placental lactogen.

Macromolecular forms of human placental lactogen have received little attention because it has been thought that such forms either compose only a small fraction of total immunoactive placental lactogen or are merely laboratory artifacts. We examined serum and placental tissue from women with normal pregnancy (first and third trimesters), serum and tissue from women with eutopic tumors (mole and choriocarcinoma), and serum from men with ectopic placental lactogen production. Samples were chromatographed on dextran gel (Sephadex G-100), and placental lactogen was measured in the fractions by radioimmunoassay. In all specimens examined, immunoactive placental lactogen was found at the void volume of the column (molecular weight greater than 150,000 daltons). This macromolecular placental lactogen comprised less than 4% of total placental lactogen in the third trimester, in mole, and in 16 of 18 first-trimester samples but was significantly higher, up to 19%, in the malignant cases. In two first-trimester placental extracts (but not in their matched sera) macromolecular placental lactogen was the dominant (greater than 45% of the total placental lactogen) immunoactive species. Authentic monomeric placental lactogen was not converted to macromolecular placental lactogen by repeated freezing and thawing. Third-trimester placental macromolecular placental lactogen was unstable; only 13% remained at the void on rechromatography. First-trimester placental macromolecular placental lactogen, on the other hand, was stable to rechromatography. The behavior of immunochemical dilutions of macromolecular placental lactogen from first-trimester placenta was similar to that of monomeric placental lactogen in the same sample. Macromolecular placental lactogen is probably not artifact, and it can comprise a large fraction of the total immunoactive placental lactogen in certain conditions.

Isolated ectopic production of the free beta subunit of chorionic gonadotropin by an epidermoid carcinoma of unknown primary site.

Metastatic choriocarcinoma was suspected in a 39-year-old woman who presented 7 months postpartum with fatigue, pelvic pain, a massive pleural effusion, and a positive urine pregnancy test. Subsequent evaluation resulted in discovery of the isolated production of the free beta-subunit of chorionic gonadotropin (CG-beta) by a widespread, poorly differentiated epidermoid carcinoma. Chemotherapy was ineffective, the woman died, and at autopsy the primary site of the tumor could not be determined. The patient's serum (185 ng/ml) and a tumor metastasis (720 ng/g) contained large amounts of immunoactive material that diluted in parallel to CG-beta standard, but neither chorionic gonadotropin (CG), its alpha subunit, nor other placental proteins were detected. A monoclonal antibody that recognizes free CG-beta, but not intact CG, was instrumental in implicating an ectopic source of the CG-beta before a tissue diagnosis was obtained. When the patient's serum was chromatographed on a dextran gel, the CG-beta immunoactivity eluted in a position of higher apparent molecular weight than either standard CG or CG-beta, suggesting that this neoplasm secreted an altered molecular form of the CG-beta subunit.

Clinical and laboratory investigation of a virilized woman with placental-site trophoblastic tumor.

The novel association of virilization and placental-site trophoblastic tumor, an uncommon form of gestational trophoblastic disease (formerly called "trophoblastic pseudotumor"), is described in a 32-year old woman. Multiple agent chemotherapy lowered serum concentrations of chorionic gonadotropin (hCG) (8.7 to 2.1 ng/mL), pregnancy-specific beta 1-glycoprotein (32 to 3.9 ng/mL), and testosterone (400 to 74 ng/dL). Subsequent hysterectomy revealed a 2-cm tumor nodule within the myometrium, and one week postoperatively serum concentrations of hCG (0.5 ng/mL), pregnancy-specific beta 1-glycoprotein (1.3 ng/mL), and testosterone (20 ng/dL) had all returned to normal. Percutaneous catheterization of the ovarian veins before chemotherapy demonstrated a 50-fold elevation of ovarian vein testosterone concentrations compared with normal women, whereas ovarian vein 17 beta-estradiol concentrations were only twofold higher than normal. Direct sampling of the uterine veins at the time of hysterectomy documented testosterone and estradiol production, presumably by the placental-site trophoblastic tumor. Ovarian vein testosterone concentrations at this time were only fourfold above normal, probably the result of falling serum concentrations of hCG. Wedge biopsy of the ovaries disclosed minimal histologic changes (thecal cell luteinization, focal thecosis) compatible with hCG stimulation. Failure of the placental-site trophoblastic tumor to produce large amounts of estrogen, in contrast to normal pregnancy and hydatidiform mole, resulted in marked androgen/estrogen imbalance, high circulating concentrations of free testosterone, and virilization.(ABSTRACT TRUNCATED AT 250 WORDS)

Recurrent abortions, thromboses, and a circulating anticoagulant.

A syndrome occurring recently in young women and consisting of recurrent abortions, thromboses, and a circulating anticoagulant is described. It is suggested that an antiphospholipid antibody may be responsible for this clinical complex.

Placental proteins as tumor markers in human monoclonal gammopathies: results in 109 patients.

Sera from 51 patients with multiple myeloma, 48 patients with monoclonal gammopathy of undetermined significance (MGUS), and 10 patients with Waldenstrom's macroglobulinemia (78 men and 31 women) were analyzed by radioimmunoassay for four placental proteins: the common alpha-subunit of glycoprotein hormones (alpha), chorionic gonadotropin and/or its free beta subunit (CG-beta), placental lactogen (PL), and "pregnancy-specific" beta 1-glycoprotein (SP1), to see if these would be useful tumor markers to distinguish benign from malignant monoclonal gammopathies. The 95th percentiles for serum alpha concentrations in our male patients and normal men were 7.0 and 2.0 ng/ml, respectively. When male patients with renal failure (known to be associated with elevated serum alpha) were excluded, the 95th percentile for serum alpha for the remaining 73 non-uremic men was 4.0 ng/ml. Of these, 7 with MGUS, 2 with macroglobulinemia, and 17 with myeloma had serum alpha concentrations above the 95th percentile for normal men, and analysis of covariance showed that both age and disease category were significantly related to serum alpha concentration. When the serum alpha concentrations from our 73 non-uremic men and 119 normal men were pooled, the 90th percentile was 2.7 ng/ml, and 16 of the 19 individuals in the top 10th percentile came from our non-uremic men (p less than 0.00002). For serum SP1, analysis of data combining our 109 patients with 93 controls again revealed a disproportionate number of the top 10% in our patient population. The 95th percentiles for serum alpha in our female patients, and for serum CG-beta in both sexes, were not significantly elevated above controls. Serum PL concentrations exceeded the 95th percentile of normal in only 5% of our patients, and were not further analyzed. Serum alpha and SP1 concentrations, but not those of CG-beta or PL, were significantly higher for our patients than for controls. These placental proteins are unlikely to be generally useful as tumor markers for monoclonal gammopathies, however, because of the overlap among "benign" and malignant groups, and because of a lack of correlation with stage of the disease as observed in our myeloma patients.

Synthesis of chorionic gonadotropin subunits in human choriocarcinoma clonal cell line JEG-3: carbohydrate differences in glycopeptides from free and combined alpha-subunits.

The glycoprotein hormone hCG and its free alpha-subunit are secreted by the clonal choriocarcinoma cell line JEG-3. Free hCG alpha has a larger apparent mol wt (22,000-24,000) than the combined hCG alpha (18,000-19,000) obtained by dissociation of the hCG secreted by these cells. Techniques developed for the specific isolation and purification of the free and combined hCG alpha forms and for the preparation of glycopeptides from these subunits have permitted detection of the incorporation of D-[3H]glucosamine [( 3H]GlcN) and L-[3H]fucose into both alpha-subunit forms. Relative to their [35S]methionine content, 2.3-fold more [3H]GlcN and 6-fold more L-[3H]fucose were incorporated into free hCG alpha than into combined hCG alpha. Analyses of [3H]GlcN glycopeptides prepared from free and combined hCG alpha indicate that the 22,000- to 24,000-dalton subunit form contained more [3H]GlcN and 27% more of the GlcN metabolite N-acetylneuraminic acid than the 18,000- to 19,000-dalton hCG alpha-subunit, than both hCG alpha forms contained two major N-linked oligosaccharide chains differing primarily in their NeuAc content, and that most of the [3H]GlcN was incorporated as GlcN or metabolites of GlcN other than N-acetylneuraminic acid. These studies provide direct chemical evidence of a higher content of carbohydrate in the larger free alpha-subunit form.

The syndrome of anosmia with hypogonadotropic hypogonadism: a genetic study of 18 new families and a review.

Among 18 NIH probands with anosmia and hypogonadotropic hypogonadism (AHH), seven had affected relatives and three had consanguineous parents. Both sexes were equally affected and parents were phenotypically normal. Parental age was not increased. Cleft lip and palate occurred in both eugonadal and hypogonadal persons, a previously reported association that may represent variable expression of AHH. Diabetes mellitus, usually insulin-dependent, was frequent in probands and their families. Other common traits included obesity, cryptorchidism, and hearing loss. All probands were chromosomally normal. The frequency of some dermatoglyphic traits of probands differed from normal, but no trait was unique to AHH. Segregation analysis of our proband sibships was consistent with a hypothesis of autosomal-recessive inheritance with variable expression. However, genetic heterogeneity was apparent when previous reports of familial AHH were surveyed. An X-linked or male sex-limited autosomal-dominant form with unilateral renal agenesis, mental retardation, and hypotelorism has been observed. The infrequent reports of direct male-to-male transmission limit characterization of an autosomal-dominant form of AHH. Our phenotypic analysis suggests that the traits of mental retardation, renal anomalies, hypotelorism, diabetes, and hearing loss may help to distinguish various forms of AHH, whereas cryptorchidism, clefts, and obesity appear in several types of families. At present, genetic counseling is dependent upon establishing inheritance pattern after examination for the known associated anomalies.

Butyrate regulates glycosylation of the glycoprotein hormone alpha subunit secreted by "glucose-starved" human liver cells.

The alpha-subunit secreted by Chang human liver cells was labeled in vitro with [35S]Met, [3H]GlcN, or [3H]Fuc, and the biosynthetic products were studied by SDS-PAGE. Cells labeled with [35S]Met secreted a homogeneous 23K-24K alpha. In contrast, alpha secreted from cells labeled with [3H]GlcN and [3H]Fuc in the absence of glucose ("glucose-starved") was heterogeneous. This size heterogeneity was altered by glucose and by butyrate, but was little affected by dibutyryl adenosine 3', 5'-monophosphate. In the presence of 0.56 mM (10 mg/dl) or 5.6 mM (100 mg/dl) glucose, or 2 mM butyrate, primarily the larger and presumably more highly glycosylated 24K-25K alpha was secreted. Moreover, the effect of 2 mM butyrate on the alpha secreted by "glucose-starved" cells was qualitatively and quantitatively similar to the effect of 0.56 mM glucose in the absence of butyrate. Likewise, 2 mM butyrate + 0.56 mM glucose was nearly equivalent to 5.6 mM glucose alone. These results demonstrate a novel effect of butyrate on glycoprotein biosynthesis; it is the only agent, reported thus far, which has the same effects as Glc or Man on protein glycosylation in "glucose-starved" cells.

Production of placental protein 5 (PP5) by non-malignant human fibroblasts in culture. Comparison with 'pregnancy-specific' glycoprotein (SP1).

Placental protein 5 (PP5) was detected by radioimmunoassay in the culture media from 11/19 (58%) of cell lines derived from non-malignant human fibroblasts. One line was studied in detail. Its rate of PP5 secretion was polyphasic, with maxima of 3.1 pmol/10(6) cells per 24 h on day 1, 1.7 on day 8 and 2.0 on day 14, and minima of 0.9 on day 4, 1.0 on day 10 and 0.7 on day 15 of culture. A discordance with the secretion of pregnancy-specific glycoprotein (SP1) was demonstrated; the SP1 concentration was constant initially at 2.9 pmol/10(6) cells per 24 h, followed by a steady decrease to 0.5 on day 15. A small amount of PP5 was retained in the cells, but usually less than 5% of that secreted. When the culture medium was changed daily, more PP5 and SP1 were produced than when the cells were grown in conditioned media. A cell line derived from a human fibrosarcoma produced SP1 at rates exceeding that of the normal fibroblasts, but PP5 production was not detected in the fibrosarcoma.

Use of circulating pregnancy-specific beta 1 glycoprotein as a marker in carcinoma of the breast in women.

Pregnancy-specific beta 1 glycoprotein (SP1) was determined by radioimmunoassay in sera from 27 normal women, 33 women with benign breast disease, and 191 women with carcinoma of the breast, staged for extent of the disease. All diagnostic groups exhibited substantial overlap in SP1 values. Those with benign breast diseases tended to have values at least as high as those with cancer. Normal patients tended to have slightly lower values, but this difference may well have been due to the younger ages of the normal patients in our sample, because SP1 values tended to increase with age. Immunochemical dilutions of SP1 in the serum with the highest value (10.2 ng/ml) did not differ significantly from standard placental SP1.

Physicochemical studies of pregnancy-specific beta 1-glycoprotein: unusual ultracentrifugal and circular dichroic properties.

The molecular properties of native and modified pregnancy-specific beta 1-glycoprotein (SP1) from human placenta were evaluated by sedimentation equilibrium, gel electrophoresis, and circular dichroic measurements. Native SP1 contained 6.2% N-acetylneuraminic acid (NANA), 5.8% galactose (Gal), 13% N-acetylglucosamine, 6.5% mannose, and 1.1% fucose but no detectable N-acetylgalactosamine. Treatment with mixed exoglycosidases and alpha-mannosidase removed 79% of the carbohydrate including all of the NANA and Gal. The intensity of the circular dichroic spectrum of SP1 in the far ultraviolet was quite low with a positive maximum at 235 nm and a negative maximum at 215 nm. The 235-nm band was lost upon treatment with reducing agents or with guanidinium chloride (GdmCl), but not by treatment with neuraminidase. Treatment of SP1 with neuraminidase, or with mixed exoglycosidases and alpha-mannosidase, resulted in decreases of the apparent molecular weight obtained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Neither exposure of SP1 to GdmCl nor its reduction and alkylation resulted in the appearance of subunits on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The partial specific volume of SP1 determined experimentally by comparing sedimentation equilibrium profiles in H2O and D2O was 0.695 +/- 0.007 mL/g. The molecular weight of SP1 in 6 M GdmCl (in the presence or absence of reducing agents) by equilibrium sedimentation was 42 300 +/- 400. In the absence of denaturing agents, SP1 existed in the form of aggregates (at least as high as trimeric SP1) that dissociated only slowly upon dilution. The presence of these aggregates may contribute to the reported molecular heterogeneity of SP1.