Recognition of insulin-like growth factor (IGF) serum binding proteins by an antibody raised against a specific IGF-inhibitor

Evidence suggests that a specific inhibitor of the insulin-like growth factors (IGF) which acts by binding to IGF may be structurally related to the native, MW 150K binding protein (BP) in serum. This has now been examined using a polyclonal antiserum (R8) raised against highly purified inhibitor. Western blotting analysis of inhibitor using R8 gave 4 immunoreactive (ir-) bands (MW 34.5K, 23K, 16K and 12K), the most intense being the MW 16K band, identical to the MW of the inhibitor. Ligand blotting using 125I-IGF-I indicated specific IGF-binding activity at MW 29K, 26.5K, 16K and 12K, indicating that at least 2 of the ir-bands (16K and 12K) were IGF-BPs. Western blotting of a salt-precipitated fraction of serum gave 8 ir-bands of which 3 (MW 42K, 38K and 34K) were identical with BP bands detected previously by Hossenlopp et al (Anal. Biochem. (1986) 154, 138-143). These immunological crossreactivities indicate that the inhibitor is structurally related to the higher MW IGF-BPs in serum.     

Partial purification of a specific inhibitor of the insulin-like growth factors by reversed-phase high-performance liquid chromatography

We report here preliminary data using reversed-phase high-performance liquid chromatography for the purification of a specific inhibitor (a molecular weight 16,000–18,000 protein) of the insulin-like growth factor (IGF) or somatomedin family. Crude inhibitor prepared from Cohn fraction IV-1 of human serum was first partially purified using an IGF/CH-Sepharose 4B affinity column. Following elution of the bound inhibitor and resuspension in 0.1% aqueous trifluroacetic acid (mobile phase A), it was injected (100 μl; 2.0 mg protein) onto a Brownlee Aquapore RP-300 column. Application of a linear gradient from 0% to 100% mobile phase B (45% isopropanol−0.1% trifluoroacetic acid) resulted in elution of two peaks of inhibitor activity between 31% and 34% isopropanol associated with a major homogeneous protein peak and a minor heterogeneous protein peak. No inhibitor was recovered when an acetonitrile gradient was used instead of isopropanol, indicating that the inhibitor is very hydrophobic. These data suggest that high-performance liquid chromatography offers a simple procedure for the potential purification of IGF inhibitor(s) from normal human serum.     

The impact of maternal serum on development of enolase activity in fetal rat brain cell culture

The effect of gestational age on serum-mediated changes in enolase activity was tested in a fetal rat brain cell culture. After 6 days exposure to graded concentrations (10 and 20%) of nonpregnant female rat sera, enolase activity in brain cell cultures increased from 2.83 +/- 0.03 to 3.74 +/- 0.19 mumol/min/mg protein, P less than 0.01. By contrast, similar concentrations of 20-day maternal serum progressively decreased enzyme activity from 1.52 +/- 0.14 to 1.19 +/- 0.08 mumol/min/mg protein. The inhibitory effect was apparent at 14 days gestation and became progressively greater during late gestation to reach a maximum at 20 days. Combining equal concentrations of 20-day pregnant with either nonpregnant or adult male serum neutralized the inhibitory activity. When serum from 20-day pregnant rats was partitioned by a dialysis membrane with a 50,000 MW pore size, inhibitory activity could be similarly neutralized by male or nonpregnant female serum. When 20-day maternal serum was passed successively through filters with a greater than 300,000, 100,000, and 50,000 MW exclusion, the inhibitory activity was apparent in all fractions excluded by a molecular weight of 50,000. No inhibition was apparent in fractions that were not excluded by 50,000 MW pore size. Inhibition of enolase activity was greatest in the fraction with MW greater than 300,000. Binding of IGF II could also be demonstrated in this fraction. Binding of IGF II was evident in the fraction greater than 100,000 MW but could not be demonstrated in fractions with a lower molecular weight. The presence of mRNA for IGF II in 20-day fetal rat brain cell cultures was evident when total cellular RNA was analyzed by an RNAase protection assay. It is proposed that a high-molecular-weight component of maternal serum in late gestation can bind endogenously generated IGF II. Such binding, by depleting the necessary growth factors, could inhibit in vitro growth and development of enolase activity.     

Insulin-like growth factor binding proteins of equine serum.

Ligand blotting analysis of serum from the horse using radiolabelled IGF-I revealed a protein at 96 kDa which was not present in serum from goat, cow, sheep, deer or donkey. These latter species all displayed five labelled bands in the range 24 to 41 kDa. Conversely, these were only weakly labelled in serum from the horse. Size exclusion chromatography of horse serum pre-incubated with radiolabelled IGF-I revealed reduced binding in the 130-kDa peak compared with goat plasma, and ligand blotting analysis indicated the 96-kDa protein was present in this peak. The 96-kDa protein from horse serum binds IGF-I and IGF-II specifically and appears to be unique to this species. The nature of this protein is at present unknown.     

Utility of electrophoretically derived protein mass estimates as additional constraints in proteome analysis of human serum based on MS/MS analysis

The proteome of a HUPO human serum reference sample was analyzed using multidimensional separation techniques at both the protein and the peptide levels. To eliminate false-positive identifications from the search results, we employed a data filtering method using molecular weight (MW) correlations derived from denaturing 1-DE. First, the six most abundant serum proteins were removed from the sample using immunoaffinity chromatography. 1-DE was then used to fractionate the remaining serum proteins according to the MW. Gel bands were isolated and in-gel digested with trypsin, and the resulting peptides were analyzed by 2-D LC/ESI-MS/MS. A SEQUEST search using the MS/MS results identified 494 proteins. Of these, 202 were excluded formally using protein data filtering as they were single-assignment proteins and their theoretical and electrophoretically-derived MWs did not correlate at high confidence. To evaluate this method, the results were compared with those of 1-D LC/MALDI-TOF/TOF and HUPO Plasma Proteome Project analyses. Our data filtering approach proved valuable in analysis of complex, large-scale proteomes such as human serum.     

Production and glycosylation of sperm constitutive proteins in the lizard Lacerta vivipara. Evolution during the reproductive period

From epididymal fluid samples taken at three different times during the reproductive period (early April, late April, mid-May), the soluble proteins were separated with one dimensional electrophoresis on polyacrylamide gel. Their evolution was studied: firstly quantitatively, after staining with Coomassie blue, or, for one protein (the "L" protein), by immunodetection; secondly, according to their glycosylation after transfer to nitrocellulose and treatment with a set of labelled lectins: from Wheat germ, Ricinus communis, Lens culinaris, Asparagus pea or Canavalia ensiformis, with or without use of their specific inhibitor sugars. At least 15 proteins underwent a quantitative and/or qualitative evolution, mainly during the month of April. Protein "L" (19 kDa), which is androgen dependent and which fixates on to spermatozoa during their epididymal transit, appears to be little or not glycosylated. By contrast its accumulation in the epididymal canal increases considerably during the month of April. Five other proteins proved to be especially interesting because of their evolution during this same period, notably the MW 94, 67, 35, 29 and 25.5 kDa proteins. With the exception of the 67 kDa all the others increased quantitatively. All were decisively enriched in mannose or in methyl-mannoside residues. The proteins of MW 29 and 25.5 kDa were also enriched in galactose or N-acetyl galactosamine residues. These findings are of physiological significance since they are set up concomitantly with the acquisition of maximum motility of spermatozoa in the distal segment of the epididymis, and they coincide with a very great increase in testosteronemia.     

Insulin-like growth factor II overexpression does not affect sorting of lysosomal enzymes in NIH-3T3 cells.

The sorting of newly synthesized mannose 6-phosphate (M6P)-containing proteins and of the major excreted protein (MEP), a lysosomal thiol proteinase, was studied in NIH-3T3 cells transfected with the cDNA of human insulin-like growth factor II (IGF II) or with the vector alone. Extracts from media and cells labelled with [35S] methionine were used for chromatography on a M6P/IGF II receptor affinity matrix or for immunoprecipitation to assess the distribution of newly synthesized M6P-containing proteins and MEP, respectively. The results indicate that the overexpression of IGF II did not affect the synthesis and the sorting of M6P-containing proteins and of MEP. The binding and uptake of the lysosomal enzyme arylsulfatase A were not affected in IGF II overexpressing cells.     

Purification of the type I insulin-like growth factor receptor from human placenta

The type I IGF receptor from human placental membranes was purified to near homogeneity by affinity chromatography on IGF I-Sepharose. SDS-polyacrylamide gel electrophoresis of the affinity purified type I IGF receptor demonstrated a high molecular weight protein with Mr greater than or equal to 300,000 under non-reducing conditions. After reduction with 2-mercaptoethanol two protein bands were found of Mr = 125,000 and 95,000, representing the alpha- and beta-subunits of the receptor molecule, respectively. A co-purification of the insulin receptor through the IGF I-affinity column could be avoided by a preincubation step with insulin.     

Changes in uterine protein secretion during luteal and follicular phases and detection of phosphatases during luteal phase of estrous cycle in buffaloes (Bubalus bubalis)

Changes in uterine proteins during different reproductive states and their functional significance though known in other species have not been established in buffaloes. An attempt has been made to unravel the changes in composition of buffalo uterine secretion with growth and regression of corpora-lutea during early, mid and late luteal and follicular phase of estrous cycle using gel filtration and electrophoresis techniques. Also the phosphatases activities in luteal phase uterine secretions have been studied. Gel filtration chromatography analysis revealed a protein peak in void volume of the column, the intensity of which was more in all the luteal phase samples than follicular phase samples. Alkaline phosphatase was also found eluted in the void volume. The other three uterus-specific peaks (Peaks V-VII) were detected below 13.7 kd molecular weight. There were at least five peaks of acid phosphatases activity in chromatogram. Silver staining of SDS-PAGE gel detected as many as 40 protein bands in the uterine fluid of which nine proteins were glycoproteins. Molecular weight (MW) comparison revealed the major protein band at 66 kd which could be serum albumin. Comparison of uterine proteins with serum protein bands revealed a 93.5 kd glycoprotein in buffalo serum that did not appear in uterine fluid and at least 11 uterus-specific protein bands (506, 470, 241, 114, 49, 38, 33, 26, 19.2, 16, and 14.3 kd). The 38 and 19.2 kd bands were luteal-stage specific. Intense periodic acid Schiff's (PAS) stained bands in uterine proteins compared to serum indicated glycosylation process in endometrial epithelial cells. The study suggested that buffalo uterine secretion contained mainly serum and several uterus-specific proteins of which few were luteal phase specific. Further study on characterizing the unique or most abundant proteins and defining their role in uterine functions would help to address the cause of low reproduction rate in buffaloes.     

Insulin‐like growth factor‐I prevents caspase‐mediated apoptosis in Schwann cells

Both neurons and glia succumb to programmed cell death (PCD) when deprived of growth factors at critical periods in development or following injury. Insulin‐like growth factor‐I (IGF‐I) prevents apoptosis in neurons in vitro. To investigate whether IGF‐I can protect Schwann cells (SC) from apoptosis, SC were harvested from postnatal day 3 rats and maintained in serum‐containing media until confluency. When cells were switched to serum‐free defined media (DM) for 12–72 h, they underwent PCD. Addition of insulin or IGF‐I prevented apoptosis. Bisbenzamide staining revealed nuclear condensation and formation of apoptotic bodies in SC grown in DM alone, but SC grown in DM plus IGF‐I had normal nuclear morphology. The phosphatidylinositol 3‐kinase (PI 3‐K) inhibitor LY294002 blocked IGF‐I–mediated protection. Caspase‐3 activity was rapidly activated upon serum withdrawal in SC, and the caspase inhibitor BAF blocked apoptosis. These results suggest that IGF‐I rescues SC from apoptosis via PI 3‐K signaling which is upstream from caspase activation. © 1999 John Wiley & Sons, Inc. J Neurobiol 41: 540–548, 1999     

Purification of natural human interferon-gamma by antibody affinity chromatography: analysis of constituent protein species in the dimers

A simple procedure for purifying human interferon-gamma from leukocytes was established, based on monoclonal antibody affinity chromatography. The recovery of interferon activity was essentially quantitative, and the specific activity of the product was (4-12) x 10(7) international units/mg protein. SDS-polyacrylamide gel electrophoresis reproducibly revealed four components associated with interferon activity (and no other proteins): two major ones with molecular weights (MW) of 24,000-25,000 (25K) and 19,000-20,000 (20K), a minor one with MW 14,000-15,000 (15K) (these three bands were doublets), and a still less prominent one(s) with MV 40,000-48,000. Gel filtration in neutral solution indicated that all the 25K, 20K, and 15K species exist as oligomers, probably dimers. By means of experiments using a cleavable crosslinking reagent, the dimers were shown to comprise both homo-and heterodimers. Gel filtration in alkali (the condition used during purification) indicated that the molecules are largely in a monomeric state. Thus, the molecules once dissociated in alkali appear to reassociate at random upon neutralization; this process takes place without being accompanied by inactivation.     

与大鼠Morris肝癌7777相关的染色质蛋白的免疫亲和层析分离与鉴定

 用5mol/L尿素,将大鼠Morris肝癌7777染色质解离为染色质非组蛋白 (UP组分)及染色质沉淀(UC组分)。UP(含90—95％非组蛋白)用免疫亲和层析(与大鼠Morris肝癌7777去组蛋白染色质抗体交联)分级,经2mol/L NaSCN及8mol/L尿素分部洗脱。将UP及UC,来自UP亲和层析的2mol/L NaSCN及8mol/L尿素洗脱组分同时进行SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)。以大鼠Morris肝癌7777去组蛋白染色质抗体作探针,进行免疫显迹(Immunoblot)测定。在UP部分出现二条阳性带,分子量为:200K及116K。UC部分有三条染色不很深的阳性带,分子量为200K,118K及91K。来自UP亲和层析的2mol/L NaSCN及8mol/L尿素洗脱部分分别有一条浓而清晰的阳性带,分子量分别为74K及83K。用酶联免疫吸附法(E1isa)测试从UP凝胶上切割下的阳性区带,其免疫特异性显著。     

Insulin-like growth factor-binding protein from human plasma. Purification and characterization

Insulin-like growth factor (IGF)-binding protein (BP) has been purified from Cohn fraction IV of human plasma by acidification, ion exchange to remove endogenous ligands, and affinity chromatography on agarose-IGF-II. The pure protein appeared as a single peak by high performance reverse-phase and gel permeation chromatography (molecular mass, 45-50 kDa), but on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gave a major band at 53 kDa and a minor band at 47 kDa, unreduced, or 43 and 40 kDa, respectively, reduced. The two bands stained for both protein and carbohydrate. After storage at 2 degrees C for 5 months at pH 3, two additional bands, at 26 and 22 kDa on unreduced gels, were also present. Autoradiography after affinity labeling with IGF-I or IGF-II tracer revealed a single labeled band of 61 kDa. BP, quantitated using a specific radioimmunoassay, was retained by agarose-immobilized IGF-I, IGF-II, concanavalin A, and wheat germ lectin, but not Helix pomatia lectin. Competitive binding curves using pure BP and human IGF-I and IGF-II as both labeled and unlabeled ligands indicated association constants of 2-3 X 10(10) liters/mol for both peptides, with a slightly higher affinity for IGF-II than IGF-I, and 0.9 binding sites for either peptide per 53-kDa protein. The exact relationship of this acid-stable IGF BP to the 150-kDa complex from which it is derived remains to be determined.     

Properties of phospholipase A2 isolated from rat serum

Phospholipase A2 was extensively purified (1300- to 1400-fold) from rat serum using Sephadex G-100 chromatography. It eluted at a position corresponding to a molecular mass of about 15 kDa. This one purification step gave two bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The faster component had a molecular mass of 16 kDa and the slower band likely contained an aggregate of the faster component. Activity was associated with protein bands on nondenaturing gels. Enzyme activity was assessed using phosphatidylcholine or phosphatidylethanolamine labelled at sn position 2 with radioactive arachidonate. Phosphatidylethanolamine gave higher specific activities than phosphatidylcholine. The enzyme has an absolute requirement for Ca2+ and a pH optimum at 7.4. This pH optimum was more prominent for phosphatidylethanolamine. Activity was inhibited by oleate or arachidonate when phosphatidylcholine was used as substrate, but added free fatty acid did not significantly affect the hydrolysis of phosphatidylethanolamine. Addition of bovine serum albumin (fatty acid free) to assays increased the rate of release of arachidonate from phosphatidylcholine, but not from phosphatidylethanolamine. Phospholipase A2 is present in serum likely as a consequence of blood coagulation and may release fatty acids from cellular membranes following hemorrhage.     

Nimbolide inhibits IGF‐I‐mediated PI3K/Akt and MAPK signalling in human breast cancer cell lines (MCF‐7 and MDA‐MB‐231)

The insulin‐like growth factor I (IGF‐I) signalling pathway contributes a major role on various cancer cell proliferation, survival and cell cycle. The present study was aimed to investigate the effect of nimbolide on IGF signalling and cell cycle arrest in MCF‐7 and MDA‐MB‐231 breast cancer cell lines. The protein expression of IGF signalling molecules and cell cycle protein levels was assessed by western blot analysis. In order to study the interaction of nimbolide on IGF‐1 signalling pathway, IGF‐I and phosphoinositide 3‐kinase (PI3K) inhibitor (LY294002) were used to treat MCF‐7 and MDA‐MB‐231 cells. Further, the cell cycle arrest was analysed by flow cytometry. The protein expression of IGF signalling molecules was significantly decreased in nimbolide‐treated breast cancer cells. PI3K inhibitor and IGF‐I with nimbolide treatment notably inhibited phosphorylated Akt. The cell cycle arrest was observed at the G0/G1 phase, and accumulation of apoptotic cells was observed in nimbolide‐treated breast cancer cell lines. Nimbolide also increased the protein expression of p21 and decreased the cyclins in both the cell lines. Nimbolide decreases the proliferation of breast cancer cells by modulating the IGF signalling molecules, which could be very useful for the breast cancer treatment. Copyright © 2014 John Wiley & Sons, Ltd.     

Insulin-like growth factor inhibition of growth hormone secretion

Somatomedins-insulin-like growth factors (SM/IGF) are growth hormone (GH) dependent serum growth factors. There is some evidence that IGF inhibit GH release (negative feedback) in 3- to 24-h incubations of cultured rat adenohypophysial cells. We have used acutely dispersed noncultured rat adenohypophysial cells to study the dynamics of IGF on GH secretion. In this system both IGF-I and IGF-II (100 ng/mL) slightly, but significantly, decrease the cumulative GH released by human pancreas growth hormone releasing factor 1-40 (GRF) and the phosphodiesterase inhibitor 3-isobutyl-1-methyl xanthine. The inhibition is small (16%) and usually not statistically significant until 2 h of incubation. The inhibition with IGF is additive to that produced with low concentrations of somatostatin. The IGF also significantly decrease the rate of GH release in all time periods tested (0-1, 1-2, 2-3 h). In addition, the IGF decrease the quantity of [14C]leucine protein eluted at the position of labelled rat GH on Sephadex G75, which would include newly synthesized GH extracted from the cells. Thus we conclude that the decreased GH released may be due to an effect of IGF on both rate of release and on GH synthesis.     

Insulin-like growth factor binding protein measurement: sodium dodecyl sulfate-stable complexes with insulin-like growth factor in serum prevent accurate assessment of total binding protein content by ligand blotting.

The possibility that sodium dodecyl sulfate (SDS)-stable complexes of insulin-like growth factor I (IGF-I) and its binding proteins (IGF-BP) exist in rat serum has been examined by using SDS-polyacrylamide gel electrophoresis (PAGE) followed by both [125I]IGF-I ligand blotting and immunoblotting with antisera directed against either IGF-BP3 or IGF-I. While ligand blotting of rat serum only revealed free IGF-BP subunits (Mr approximately 50, 35, and 30 kDa), immunoblotting with either the IGF-BP3 antiserum or IGF-I antiserum revealed major immunoreactive bands with higher molecular weights (greater than 110, approximately 100, and approximately 84 kDa). The IGF-BP3 antiserum also stained the 50-kDa form of the serum IGF-BP. Specifically stained protein bands were identified by comparison with control immunoblots incubated with normal rabbit serum. Treating the serum with 0.1 N HCl prior to electrophoresis reduced the amount of high molecular weight IGF-BP3 immunoreactive species, with a concomitant increase in the amount of the 50-kDa form. A similar result was obtained if the samples were boiled prior to electrophoresis. These data indicate that not all IGF-BP/IGF complexes may dissociate under normal SDS-PAGE conditions. Therefore, data obtained by using ligand blotting alone may underestimate the amount of total IGF-BP present, especially if the mixture being analyzed also contains large amounts of IGF.     

Characterization of Infant Rat Cerebral Cortical Membrane Proteins Phosphorylated In Vivo: Identification of the ACTH-Sensitive Phosphoprotein B-50

This study on the phosphorylation in vivo of membrane proteins in cerebral cortices of infant rats reports the identification of the adrenocorticotropin (ACTH)-sensitive phosphoprotein B-50 as one of the substrate proteins that are rapidly phosphorylated in vivo following intracisternal administration of 2 mCi [32P]orthophosphate. Rats were sacrificed 30 min after isotope injection. A fraction enriched in membranes, designated neural membranes (NM), was isolated from the cerebral cortices according to the procedure used for preparation of synaptic plasma membranes (SPM) from adult brain. This NM fraction was characterized by electron microscopy. The proteins of NM were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Numerous protein bands of NM in infant rat brain were phosphorylated in vivo. Attention was focussed on the 32P-labeled protein bands in the molecular weight range of 47K-67K. In this region one phosphoprotein band (MW 48K) was more highly labeled than the other bands. The electrophoretic behavior of three of these labeled bands, designated a, c, and e (MW 48K, 55K, and 62K, respectively) was compared with that of protein bands that were phosphorylated in vitro in cerebral membranes isolated from noninjected infant rats. The effects of ACTH1-24 and cyclic AMP in the in vitro system were also studied to probe for the presence of specific membrane proteins known to be sensitive to these modulators. On incubation of NM with [gamma-32P)ATP in the presence and absence of ACTH1-24 in vitro, phosphorylation of a 48K protein band was inhibited in a dose-dependent fashion by the neuropeptide. Two-dimensional electrophoretic separation of NM proteins labeled in vivo indicated that the 48K band had an isoelectric point of 4.5, identical to that of the ACTH-sensitive B-50 protein previously identified. Cyclic AMP stimulated phosphorylation in vitro of two protein bands (MW 55K and 59K) in NM preparations. This result indicates that the in vivo labeled band c may correspond to the cyclic AMP-sensitive 55K protein, whereas phosphoprotein band e, labeled in vivo, appears to be different from the cyclic AMP-sensitive 59K protein band. These observations indicate that neural membranes isolated from infant rat cerebral cortices contain a variety of proteins that can be phosphorylated in vivo. Several of these, for example, the 48K protein band, have the properties of synaptic plasma membrane proteins of adult rat brain that have been characterized by their sensitivity to neuromodulators in endogenous phosphorylating systems in vitro.     

A heparin-binding erythroid cell stimulating factor from fetal bovine serum has the N-terminal sequence of insulin-like growth factor II

An 8 kd heparin-binding peptide which stimulates thymidine incorporation in cultures of fetal calf liver erythroid cells was isolated from fetal bovine serum by affinity chromatography on Heparin-Sepharose, ion exchange chromatography, gel filtration and reversed-phase HPLC. The N-terminal sequence of the isolated peptide was identical to the N-terminal sequence of bovine erythrotropin or insulin-like growth factor II (IGF II). The potential heparin-binding site of IGF II is probably situated in the arginine-rich C-peptide region. The affinities of human recombinant IGF I and II were compared with those of apolipoprotein H (a plasma heparin-binding protein) and bovine insulin in a heparin-affinity column. The retention times were in the order: Apolipoprotein H greater than hrIGF II greater than hrIGF I greater than insulin (no retention). This unusual property of IGF II suggests that it may be captured in the extracellular matrix in a similar way to fibroblast growth factor, interleukin 3 or granulocyte/macrophage colony-stimulating factor.     

The identification and characterization of two separate carboxylesterases in guinea-pig serum.

The esterase activity of guinea-pig serum was investigated. A 3-fold purification was achieved by removing the serum albumin by Blue Sepharose CL-6B affinity chromatography. The partially purified enzyme preparation had carboxylesterase and cholinesterase activities of 1.0 and 0.22 mumol of substrate/min per mg of protein respectively. The esterases were labelled with [3H]di-isopropyl phosphorofluoridate (DiPF) and separated electrophoretically on sodium dodecyl sulphate/polyacrylamide gels. Two main labelled bands were detected: band I had Mr 80 000 and bound 18-19 pmol of [3H]DiPF/mg of protein, and band II had Mr 58 000 and bound 7 pmol of [3H]DiPF/mg of protein. Bis-p-nitrophenyl phosphate (a selective inhibitor of carboxylesterase) inhibited most of the labelling of bands I and II. The residual labelling (8%) of band I but not band II (4%) was removed by preincubation of partially purified enzyme preparation with neostigmine (a selective inhibitor of cholinesterase). Paraoxon totally prevented the [3H]DiPF labelling of the partially purified enzyme preparation. Isoelectrofocusing of [3H]DiPF-labelled and uninhibited partially purified enzyme preparation revealed that there were at least two separate carboxylesterases, which had pI3.9 and pI6.2, a cholinesterase enzyme (pI4.3) and an unidentified protein that reacts with [3H]DiPF and has a pI5.0. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of these enzymes showed that the carboxylesterase enzymes at pI3.9 and pI6.2 corresponded to the 80 000-Mr subunit (band I) and 58 000-Mr subunit (band II). The cholinesterase enzyme was also composed of 80 000-Mr subunits (i.e. the residual labelling in band I after bis-p-nitrophenyl phosphate treatment). The unidentified protein at pI5.0 corresponded to the residual labelling in band II (Mr 58 000), which was insensitive to neostigmine and bis-p-nitrophenyl phosphate. These studies show that the carboxylesterase activity of guinea-pig serum is the result of at least two separate and distinct enzymes.