Isoelectric Species Research Articles

76. The neurotransmitter, vasoactive intestinal peptide (VIP), bolsters the anti-leukemic activities of Ikaros and regulates cellular migration in human T cells

One in three children diagnosed with cancer has leukemia. Leukemia patients that have mutations in Ikaros (IK), a gene that encodes an anti-leukemic factor, have a poor prognosis despite modern chemotherapy. The neurotransmitter receptors, called vasoactive intestinal peptide (VIP) receptor −1 (VPAC1) and VPAC2, which our laboratory has shown to be transcriptionally regulated by IK, are reversed in some leukemia patients. We hypothesized that VIP signaling in leukemia cells bolsters the anti-leukemic activity of IK, while differentially controls transendothelial migration, a hallmark of leukemic transformation, due to VPAC1 for VPAC2 expression reversal. To gain support for this hypothesis, we first asked whether VIP/VPAC1 signaling affects protein levels and/or isoelectric pools of IK (phosphorylation) by conducting two dimensional (2D) gel electrophoresis followed by western analyses. These studies showed that VIP treatment induced a decline in some IK isoforms in activated but not naive lymphoblastic Hut-78 T cells compared to control. Also, VIP addition blocked the appearance of several activated isoelectric immunoreactive IK species. The second question we asked was whether VIP signaling differentially controlled leukemia cell movement by Boyden Chamber assays. Intriguingly, we discovered that both receptors mediated a chemotactic influence towards VIP, but through different signaling pathways. Collectively, these data support the notion that the nervous system naturally contributes to normal blood cell function, but after leukemogenesis, VIP signaling may exacerbate the leukemia phenotype.

Proteomic investigation of changes in human vastus lateralis muscle in response to interval‐exercise training

No previous study has used proteomics to investigate the effects of exercise training on human skeletal muscle. Five recreationally active men completed a 6-wk training programme involving three sessions per week, utilising six 1-min bouts at maximum oxygen uptake (V O(2)max) interspersed with 4 min at 50% V O(2)max. Vastus lateralis was biopsied at standardised times before and after the training intervention. Protein expression profiling was performed using differential analysis of 2-DE gels; complemented with quantitative analysis (iTRAQ) of tryptic peptides from 1-DE gel lane-segments using LC-MALDI MS/MS. Interval training increased average V O(2)max (7%; p<0.001) and was associated with greater expression of mitochondrial components, including succinate dehydrogenase, trifunctional protein-alpha and ATP synthase alpha- and beta-chains. 2-DE resolved 256 spots, and paired t-tests identified 20 significant differences in expression (false discovery rate <10%). Each differentially expressed gene product was present as multiple isoelectric species. Therefore, the differences in spot expression represent changes in post-transcriptional or post-translational processing. In particular, modulation of muscle creatine kinase and troponin T were prominent. Pro-Q Diamond staining revealed these changes in expression were associated with phosphorylated protein species, which provides novel information regarding muscle adaptation to interval training.

5] Isolation of isoelectric species of phosphorylated rhodopsin

5] Isolation of isoelectric species of phosphorylated rhodopsin

Properties of buffer systems with charges immobilized on a gel matrix and their potential use in capillary electrophoresis

Some physicochemical properties, such as conductivity and buffering capacity, are evaluated and compared for two types of buffer systems: soluble, amphoteric, isoelectric species and immobilized weak buffering ions and counter-ions grafted onto a polyacrylamide gel (Immobiline technology). Theoretical calculations are performed for a model system comprising two Immobilines in a wide range of concentrations and compared with the properties of model ampholyte solutions. The results obtained show that the conductivity of any system composed of ampholytes dissolved in pure water is mostly connected with the contributions of the hydrogen and hydroxyl ions, and that the intrinsic ampholyte contribution is negligible at any value of concentration, although it increases when the ampholyte isoelectric point (p I) is removed from neutrality. The buffering capacity behaviour is also analysed in the range of small ampholyte (Immobilines) concentrations as a function of Δp K values (i.e., how far the p K values are removed from the p I of the amphotere). Due to the high flexibility in the preparation of isoelectric, Immobiline buffers (which can cover essentially any pH value), their potential use as background electrolytes for separations in capillary electrophoresis is evaluated.

Nuclear quadrupole coupling constants of BeH, BeH+ and BeH−: ab initio study of their rovibrational dependence

Abstract The 9Be and 2H quadrupole coupling constants of the rovibrational levels of all isotopic variants of BeH(X2Σ+), BeH + ( X 1 Σ + ) and BeH − ( X 1 Σ + ) are calculated from molecular wavefunctions which explicitly describe nuclear displacement.The strength of the 9Be coupling is predicted to be more than an order of magnitude larger than that of the 2H coupling. The vibrational dependence of the nuclear quadrupole coupling constants is found to be quite significant for all the isotopomers. The qualitative features of the 9Be and 2H quadrupole coupling in these species are consistent with those reported for the corresponding series of lithium hydrides LiH ( 1 Σ + ), LiH + ( 2 Σ + ) and LiH − ( X 2 Σ) . A comparison with other isoelectric species BH+ (X2Σ+), BH(X1Σ+) and CH+ (X 1Σ+) is also made

Immunological Identity of Rat Liver Cytosolic Heme-Binding Protein with Purified and Recombinant Liver Fatty Acid Binding Protein by Western Blots of Two-Dimensional Gels

We examined the degree of similarity between rat liver cytosolic heme-binding protein (HBP) and rat liver fatty acid binding protein (L-FABP) purified from rat liver cytosol and recombinant L-FABP (rL-FABP). We compared 1) HBP, 2) L-FABP, and 3) rL-FABP prepared in three different laboratories and probed them with three different antisera also from different laboratories on Western blots. The objective was to determine whether the isoform pattern of the recombinant would resemble those of the purified rat liver proteins and whether heme is bound by the isoforms. To investigate the similarities, we compared the immunoreactivity of purified HBP, L-FABP, and rL-FABP by probing Western blots of 2-D gels with polyclonal antibodies raised against each of these proteins. Ah of the antibodies react with the same isoelectric species for each of the proteins. In addition, [55Fe]-heme binds equally well to the 2 major HBP isoforms.

Molecular cloning and expression of a gamma-interferon-inducible activator of the multicatalytic protease.

The multicatalytic protease (MCP) can be activated by two distinct multisubunit complexes. One is the regulatory component of the 26 S protease, which contains at least 15 distinct subunits. The other is a hexameric activator composed of 31- and 29-kDa subunits. A cDNA for the smaller subunit has been cloned and sequenced. The cDNA encodes a protein of 249 amino acids. Embedded between sequences typical of globular protein domains is a stretch of 28 "alternating" lysine and glutamic acid residues. Similar regions, which we call KEKE motifs, are also found in two MCP subunits, in subunit 12 of the 26 S protease and in a variety of chaperonins including hsp90, hsp70, and calnexin. Expression of the activator cDNA in Escherichia coli produced a functional protein virtually indistinguishable from MCP activator purified directly from red blood cells. The recombinant protein formed three isoelectric species on two-dimensional polyacrylamide gel electrophoresis, and it reacted with antibodies to red blood cell activator. Recombinant activator also bound the multicatalytic protease and stimulated cleavage at the carboxyl terminus of hydrophobic or charged residues. Synthesis of the activator subunit was induced by gamma interferon treatment of HeLa cells. These last two findings have implications for antigen presentation by class I major histocompatibility receptors.

Molecular characterization of human prorenin isoelectric forms.

Mammalian renins and prorenins can be fractionated by isoelectric focusing into multiple species, the individual production and decay rates of which vary with certain physiological and pharmacological stimuli. The relative abundance of renin isoelectric species varies in certain pathological states, and the individual species have been reported to differ in their biological function. The present study was undertaken to clarify further the biochemical basis for isoelectric heterogeneity in human renin. Previous data have suggested that differences in glycosylation contribute to isoelectric heterogeneity in human renin. To determine whether glycosylation was solely responsible for the observed isoelectric heterogeneity, the isoelectric focusing patterns of either native or non-glycosylated human prorenin were compared. The specific activities of the three most abundant isoelectric species of human prorenin were also compared. Tissue culture supernatants from transfected cells expressing either native recombinant human prorenin or human prorenin in which both glycosylation sites were eliminated were fractionated by isoelectric focusing. The expressed prorenins were detected in various fractions by trypsin activation and the angiotensin I generation assay. Specific activities of the various prorenins were estimated by direct comparison of immunoprecipitable radiolabeled prorenin and trypsin-activatable renin activity in the three most abundant fractions. Native human recombinant prorenin was fractionated into at least five isoelectric species, whereas non-glycosylated human recombinant prorenin migrated as a single isoelectric species, the migration of which was unaffected by forskolin treatment. There was a direct correlation between immunoprecipitable and enzymatically determined prorenin in the three major isoelectric species of native prorenin. The results suggest that isoelectric heterogeneity of human renin is due solely to differential glycosylation of the protein. The proposed different biological functions of the various isoelectric forms is not directly correlated with variations in their specific activities.

Characterization of a lung-cancer-associated auto-antigen.

An antigen, protein X (Px), was purified from immune complexes isolated from malignant pleural effusions from patients with adenocarcinoma of the lung by EDTA treatment, PEG 8000 precipitation, protein A affinity chromatography, and Sephadex G-200 separation in the presence of 3 M NaCl. The purified antigen had a M(r) 17,000 by SDS-PAGE, and consisted of isoelectric species of pI 6.3 and 6.6. Purified Px recombined with Ig isolated from pleural fluids from patients with lung adenocarcinoma, but not with Ig from patients with breast carcinoma. Using an autologous human and heterologous chicken antibody, Px was found, by immunohistology, in the cytoplasm of some of the well-differentiated lung adenocarcinoma cells, but was not seen in normal lung or a variety of other malignant tissues. A liquid-phase competitive-inhibition RIA was developed. Over 30 ng/ml of Px were found in 9 of 15 pleural fluids from patients with lung carcinoma, none of 20 from patients with breast, ovary, stomach or colon cancer, and in 3 of 15 patients with unknown primary tumor. Our data suggest that Px may be a lung-cancer-associated autoantigen which can elicit a host humoral response in vivo.

Phosphorylation of the budgerigar fledgling disease virus major capsid protein VP1.

The structural proteins of the budgerigar fledgling disease virus, the first known nonmammalian polyomavirus, were analyzed by isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The major capsid protein VP1 was found to be composed of at least five distinct species having isoelectric points ranging from pH 6.45 to 5.85. By analogy with the murine polyomavirus, these species apparently result from different modifications of an initial translation product. Primary chicken embryo cells were infected in the presence of 32Pi to determine whether the virus structural proteins were modified by phosphorylation. SDS-PAGE of the purified virus structural proteins demonstrated that VP1 (along with both minor capsid proteins) was phosphorylated. Two-dimensional analysis of the radiolabeled virus showed phosphorylation of only the two most acidic isoelectric species of VP1, indicating that this posttranslational modification contributes to VP1 species heterogeneity. Phosphoamino acid analysis of 32P-labeled VP1 revealed that phosphoserine is the only phosphoamino acid present in the VP1 protein.

Differing renal effects of activated and nonactivated isoelectric peaks of human prorenin in rats

Isoelectric species of renin are physically heterogeneous. Recent evidence suggests that they may differ functionally, with some species producing natriuresis and diuresis, whereas others have no effect. A physiological function of secreted prorenin has not been documented in any species. The present study was designed to confirm and describe for the first time the renal effects of certain isoelectric species of prorenin. Anesthetized Sprague-Dawley rats were injected (0.1 ml) with trypsin-activated or nonactivated prorenin obtained from human ovarian follicular fluid. The dose chosen was calculated as sufficient to produce 2,300 ng angiotensin I.h-1.100 g rat body wt-1 in the presence of excess sheep substrate. Blood pressure, creatinine clearance, urine flow rate, and urine sodium, potassium, and osmolar excretion were measured. Activated prorenin from isoelectric peaks at isoelectric points (pI) 5.1, 5.2, 5.4, and 5.6 produced marked increases in urine volume (sixfold) and sodium excretion (7- to 10-fold) compared with the group receiving the vehicle (1% albumin in 0.9% saline). Activated prorenin from peaks at pI 4.9 and 5.8 produced no significant increase over the vehicle-only experiments. Captopril pretreatment (1 mg/kg iv) completely blocked the effects of peaks at pI 5.4 and 5.6. Interestingly, injection of nonactivated prorenin from peaks at pI 5.4 and 5.6 produced effects similar to the injection of activated prorenin from these peaks. Similarly, this effect was blocked by pretreatment with captopril. In summary, only certain isoelectric peaks of human prorenin whether activated, to active renin, or nonactivated produced a marked natriuresis and diuresis.(ABSTRACT TRUNCATED AT 250 WORDS)

Prolactin Action on Luteal Protein Expression in the Corpus Luteum*

Although it is well established that PRL is essential for luteal cell steroidogenesis and growth in the rat, its exact mechanism of action remains unknown. Whereas PRL stimulates growth and induces the content of specific proteins in several target tissues, its effect on proteins in the corpus luteum has not been examined. To determine whether PRL affects the synthesis of specific luteal protein(s), corpora lutea were obtained from rats hypophysectomized on day 3 of pregnancy and then treated with or without PRL (125 micrograms x 2/day) for 4 days. In this rat model, corpora lutea are exquisitely responsive to PRL and produce high levels of progesterone in response to PRL stimulation. Analysis of cytosolic proteins on one- and two-dimensional gel electrophoresis revealed a dramatic inhibitory effect of PRL on a 37,000 mol wt (MW) protein. This PRL-regulated protein (PRP) resolved into five separate isoelectric variants (pIs 5.5, 5.6, 6.15, 6.6, and 6.85). Since differences in phosphorylation state can alter the isoelectric point of proteins, we examined the possibility that the 37,000 MW PRP is a substrate for phosphorylation. Luteal homogenates were incubated with [32P]ATP in the presence or absence of Ca2+, Ca2+/calmodulin, Ca2+/phospholipid, or cAMP. Phosphorylation of PRP was not affected by PRL treatment or the addition of specific cofactors. The least abundant isoelectric species (pI 6.85) was identified by Western blot analysis as annexin I, a known regulator of phospholipase A2 and prostaglandin synthesis. To determine whether the other four isoelectric species represent variants of the same protein, we developed a polyclonal antibody to the 6.15 isoelectric species which is remarkably regulated by PRL. In the absence of PRL, the 6.15 isoelectric species (37K) is the major protein of the PRP band. Conversely, PRL treatment resulted in the virtual disappearance of this luteal protein. The antibody recognized the 37K alone, indicating that the other 37,000 MW isoelectric species were distinct proteins. To examine the time course of PRL action on the expression of the 37K, luteal cells from day 3 pregnant rats were cultured with different doses of PRL from 6 h to 5 days. Western blot analysis of luteal cellular proteins indicated that PRL caused a decrease in the expression of the 37K within 6 h of treatment. Although it is well known that estradiol together with PRL is required for optimal growth of the rat corpus luteum, estradiol alone had no inhibitory action on the 37K nor did it affect the inhibitory action of PRL.(ABSTRACT TRUNCATED AT 400 WORDS)

Redistribution of 23 kDa tubulovesicle-associated GTP-binding proteins during parietal cell stimulation.

Small GTP-binding proteins are important regulators of intracellular traffic. The presence of several small GTP-binding proteins was documented in subfractions of rabbit parietal cells. Upon maximal stimulation of the cells with a combination of histamine and forskolin, one 23 kDa GTP-binding band was observed to decrease in a 50,000 g membrane fraction while increasing in 4000 g membranes. The 23 kDa band resolved into one major and two minor species on two-dimensional gels. GTP-binding species of 23 kDa, 24 kDa and 25 kDa were present in purified preparations of tubulovesicles. The three isoelectric species of the 23 kDa proteins observed in parietal cell 50,000 g microsomes were enriched in tubulovesicle preparations. None of the tubulovesicle-associated GTP-binding proteins were substrates for ADP-ribosylation by a preparation of botulinum D toxin. These results indicate that tubulovesicles contain discrete small GTP-binding proteins which redistribute during parietal cell stimulation.

Expression of carbohydrate binding protein 35 in human fibroblasts: Variations in the levels of mRNA, protein, and isoelectric species as a function of replicative competence

We have compared the expression and localization of carbohydrate binding protein 35 (CBP35) in human SL66 fibroblasts of different replicative capacities. When serum-starved, quiescent young (passage 11, corresponding to ~18 cumulative population doublings) SL66 cells were treated with serum, there was a marked stimulation in the expression of CBP35. This was revealed both by an increase in the percentage of cells positively stained with anti-CBP35 under immunofluorescence and by an increase in the amount of the protein in immunoblots, as well as by an increase in the level of accumulated mRNA in Northern blots. The rise in the expression of CBP35 in proliferating cells was manifested most clearly in the nuclear fraction, with elevation in the levels of the nonphosphorylated (p I 8.7) protein, as well as the phosphorylated (p I 8.2) derivative. In contrast, older (passage 27–35, 55–68 cumulative population doublings) cultures of SL66 fibroblasts appear to have lost the normal proliferation-dependent regulation of CBP35 expression. The level of CBP35 was high in quiescent high-passage cells and decreased somewhat after serum stimulation. Furthermore, the unphosphorylated (p I 8.7) form of the lectin could not be detected in either the nucleus or the cytoplasm of high-passage SL66 cells. Finally, the level of the mRNA for CBP35 was high in quiescent cultures of high-passage cells, but undetectable 17 h after serum stimulation. These results establish that the expression of CBP35 becomes altered as human fibroblasts acquire reduced replicative capacity.

Characterisation of 24‐kD proteins from rat testes using polyclonal sera reactive to human sperm antigens

A group of antigens of 24-kD Mr from rat testes were characterised biochemically. These antigens were part of a larger molecule of approximately 200 kD. On treatment with disulfide bond reducing agent, the 200-kD molecule was reduced to subunits. Immunoreactivity was confined to a doublet of approximately 24 kD and a single band of approximately 50 kD Mr after the reduction. Glycoprotein in nature, this antigen shared immunoreactive epitopes with a 40-kD antigen on human spermatozoa. Antiserum raised in rabbits against the 24-kD antigen from rat testes reacted with antigens on the acrosome of human spermatozoa. Agglutination of sperm could be induced by the antiserum. The carbohydrate residue could be removed by mannosidase digestion. Chemical deglycosylation studies showed a slight decrease in molecular weight. Immunoreactivity was however not completely lost after chemical deglycosylation. Isoelectric focusing of the antigen identified nine isoelectric species. Two relatively minor species showed immunoreactivity. Acrosome-reacted spermatozoa showed loss of antigens from acrosome.

Free-solution isoelectric focusing for the purification of Staphylococcus aureus enterotoxin C 1

A free-solution isoelectric focusing protocol was developed for the preparative purification of Staphylococcus aureus enterotoxin C 1 (SEC 1). A toxin consisting of a single isoelectric species, pi 8.8, was purified. Thirty-nine milligrams of SEC, was recovered from 3 liters of culture supernatant. This significantly improved purification scheme utilized ammonium sulfate precipitation and the Bio-Rad Rotofor isoelectric cell to complete isolation in 2 days, thereby avoiding the protein degradation prevalent when published procedures are used. The purification protocol developed here for SEC 1 is used to illustrate the utility of Rotofor fractionation in the general purification of bacterial exotoxins.

Crystallization of immunoglobulin Fab fragments specific for DNA.

The purification and crystallization of Fab fragments of two mouse monoclonal immunoglobulins specific for different DNA structures are described. In each case, papain digestion of the immunoglobulins produced a mixture of Fab species differing in their isoelectric points. Purification of one of these species was required to obtain suitable crystals. One of these antibodies, Jel 72, is specific for right-handed duplex poly(dG).poly(dC). An Fab fragment of Jel 72 with a pI of 8.8 was purified by anion-exchange chromatography and used to obtain crystals from 56% saturated ammonium sulfate and 50 mM sodium acetate, pH 4.2, that diffract to 2.6-A resolution. They belong to the orthorhombic space group P2(1)2(1)2(1), with cell dimensions of a = 94.6, b = 102.6, c = 92.4 A. The other antibody, Jel 318, binds triple-stranded DNA poly[d(Tm5C)].poly[d(GA)].poly[d(m5C + T)]. Jel 318 Fab fragments with isoelectric points of 7.6 and 7.8 were also purified by anion-exchange chromatography, and crystals were obtained from 12% polyethylene glycol 8000, 50 mM NaCl, and 10 mM Tris.HCl, pH 7.8. These crystals diffract to about 2.4-A resolution and also belong to the orthorhombic space group P2(1)2(1)2(1), with cell dimensions of a = 82.4, b = 139.5, and c = 42.0 A. For both Fab fragments, crystal size and quality improved dramatically upon purification of an individual isoelectric species.

Changes in Protein Synthesis Induced in Tomato by Chilling

Impaired chloroplast function is responsible for nearly two-thirds of the inhibition of net photosynthesis caused by dark chilling in tomato (Lycopersicon esculentum Mill.). Yet the plant can eventually recover full photosynthetic capacity if it is rewarmed in darkness at high relative humidity. As a means of identifying potential sites of chilling injury in tomato, we monitored leaf protein synthesis in chilled plants during this rewarming recovery phase, since changes in the synthesis of certain proteins might be indicative of damaged processes in need of repair. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of proteins pulse labeled with [(35)S]methionine revealed discrete changes in the pattern of protein synthesis as a result of chilling. A protein of M(r) = 27 kilodaltons (kD), abundantly synthesized by unchilled plants, declined to undetectable levels in chilled plants. Reillumination restored the synthesis of this protein in plants rewarmed for 8 hours. Peptide mapping analysis showed the 27 kD protein to be the major chlorophyll a/b binding protein of the photosystem II light-harvesting complex (LHCP-II). The identity of this protein was confirmed by its immunoprecipitation from leaf extracts by a monoclonal antibody specific for the major LHCP-II species. While chilling abolished the synthesis of the major LHCP-II species, it also induced the synthesis of an entirely new protein of M(r) = 35 kD. The protein was synthesized on cytoplasmic ribosomes, and two-dimensional polyacrylamide gel electrophroesis showed it to exist as a single isoelectric species. This chilling-induced 35 kD protein is structurally distinct from the 27 kD LHCP-II and appears to be synthesized specifically in response to low temperature. While the 35 kD protein was found not to be associated with the chloroplast thylakoid membrane, chilling did cause selective changes in thylakoid membrane protein synthesis. The synthesis of two unidentified proteins, M(r) = 14 and 41 kD, and the beta-subunit of the chloroplast coupling factor were substantially reduced after chilling. These losses may provide clues as to the causes of the overall reduction in net photosynthesis caused by chilling.

Characterization of bovine conceptus proteins produced during the peri- and postattachment periods of early pregnancy.

Bovine conceptuses removed from the uterus during the peri- and postattachment periods of placentation (Days 17-24 and 26-38, respectively) were cultured in a modified minimum essential medium in the presence of L-[3H]leucine to characterize in vitro synthesis of proteins released into the medium. Patterns of protein production were analyzed by two-dimensional polyacrylamide gel electrophoresis followed by fluorography of dried gels. Four groups of low molecular weight acidic proteins (LMWAP) were observed to be synthesized during the peri- and postattachment periods. The number and relative concentrations of these changed with development. One group (A) consisted of three major and two or more minor isoelectric species (pI approximately equal to 5.8-6.8); these were the major synthesized proteins observed from Days 17-22. The major polypeptides of Group A were present at all time points examined through Day 38 and, in several preparations, appeared as doublets (Mr approximately equal to 22,000 and 24,000) through Day 29 but not thereafter. Group A polypeptides from Day 19 and 36 conceptus cultures were demonstrated by immunoblot analysis to cross-react with antiserum produced against ovine trophoblast protein-1 (oTP-1). A second group of proteins (A1) and a single protein (B) in the 20,000-24,000 Mr range were observed between Days 17 and 22. These were acidic relative to Group A and were not detected after Day 22. A fourth group (C) of LMWAP (Mr approximately equal to 14,000-18,000) was first observed around Day 21 and appeared to increase relative to Group A through Day 29. One protein from this group, C3, was the predominant LMWAP at Day 38.(ABSTRACT TRUNCATED AT 250 WORDS)

Partial purification and characterization of chorionic gonadotrophin in plasma and in culture medium of trophoblast cells from the marmoset monkey (Callithrix jacchus).

A biologically active gonadotrophin has been purified from the media of long-term cultures of trophoblast cells of the common marmoset monkey by a combination of precipitation and chromatography. Marmoset chorionic gonadotrophin (CG) is a glycoprotein which binds Concanavalin A and wheat germ agglutinin. The protein purified from culture media exists as several isoelectric species with pI in the range pH 3.5-4.5. On gel filtration it eluted with an apparent molecular weight of 68-72,000 but on PAGE migrated as if it was 58-65,000. A glycoprotein with similar characteristics has been recovered from plasma samples of pregnant marmosets. Biological activity of partly purified CG from media, as determined by a mouse testicular cell bioassay, was 1-3 i.u./mg protein.