Purification and amino-terminal sequence of an insulin-like growth factor-binding protein secreted by rat liver BRL-3A cells

A protein preparation that specifically binds insulin-like growth factors (IGFs) I and II was purified from medium conditioned by rat liver BRL-3A cells using molecular sieve chromatography in 1 M acetic acid followed by affinity chromatography on IGF-II-agarose. The affinity-purified IGF-binding protein exhibits a single major band with apparent Mr = 36,300 under reducing conditions on sodium dodecyl sulfate-polyacrylamide gels. The IGF-binding protein is efficiently and specifically cross-linked to either 125I-IGF-I (human) or 125I-IGF-II (rat) using disuccinimidyl suberate. An IGF-binding protein of similar apparent molecular weight was also affinity purified from rat hepatoma H-35 cell conditioned medium and found to differ from the BRL-3A protein such that potent polyclonal antisera prepared in rabbits against the purified BRL-3A IGF-binding protein exhibited a much lower titer for the H-35 protein in an enzyme-linked immunosorbent assay and upon immunoblotting. In order to determine whether a single BRL-3A IGF-binding protein is present in the affinity-purified preparation, the protein was prepared for sequencing on a Sephacryl S-300 column in 6 M guanidine HCl after reduction and alkylation. The amino acid composition (expressed in percentages) of this IGF-binding protein was determined to be: Cys = 5.5, Lys = 4.8, His = 2.8, Arg = 7.8, Asx = 10.2, Thr = 5.1, Ser = 3.9, Glx = 15.7, Gly = 17.4, Ala = 7.3, Val = 4.6, Met = 1.4, Ile = 2.4, Leu = 8.3, Tyr = 1.0, Phe = 1.9. Sequencing of the NH2-terminal portion of this protein led to the identification of 31 amino acids in the following order: Phe-Arg-Cys-Pro-Pro-Cys-Thr-Pro-Glu-Arg-Leu-Ala-Ala-Cys-Gly-Pro-Pro-Pro- Asp-Ala-Pro-Cys-Ala-Glu-Leu-Val-Arg-Glu-Pro-Gly-Cys. We conclude that rat liver BRL-3A cells secrete a single major IGF-binding protein capable of binding both IGF-I and IGF-II.     

A method for culturing chick melanocytes: The effect of BRL-3A cell conditioning and related additives

Summary A method for growing chick embryo melanocytes is described that utilizes medium conditioned by Buffalo Rat liver (BRL-3A) cells. The dissected trunk region of each 72 h (Stages 14 to 19) embryo produces approximately 200,000 melanocytes (purity, 80%) when processed and cultured for 8 d. Thus, a typical experiment involving 20 embryos would produce a total of 4 × 10⁶ melanocytes. Choice of serum, serum concentration, and cell density were determined experimentally. Partially purified multiplication stimulating activity (MSA) from BRL-3A cells and insulin were also tested as medium additives. MSA was not stimulatory, whereas insulin gave a positive response in 2% but not 10 or 0% serum. The final protocol used a modified F12 medium with 10% bovine calf serum conditioned by BRL-3A cells. Cultures were fed every other day. Small colonies of cells became evident by culture Day 3 and increased rapidly to Day 5 when pigmentation became obvious. Colony size continued to increase but more slowly from Days 5 to 8, whereas pigmentation increased rapidly and maximized on Day 8. There is a factor, or factors, present in BRL-3A conditioned medium that stimulates embryonic chick melanocytes to divide preferentially over contaminating cell types. This results in cultures that can provide adequate numbers and purity for biochemical studies. This work was supported by National Institutes of Health grants GM18969 to J. B. and CA17620 to G. S.     

Immunological cross-reactivity of multiplication-stimulating activity polypeptides

The immunoreactivity of the multiple species of multiplication-stimulating activity (MSA) purified from medium conditioned by a rat liver cell line (BRL-3A) has been examined. Antibodies were raised in rabbits following immunization with MSA II polypeptides. Subpopulations of antibodies were purified from one antiserum using DEAE-cellulose chromatography. One antibody subpopulation recognized common antigenic determinants on MSA I and MSA II polypeptides; whereas a second antibody subpopulation recognized common determinants on MSA I, II, and III polypeptides. In a radioimmunoassay utilizing 125I-MSA III-2 and a purified antibody subpopulation, the human somatomedins (somatomedin A, insulin-like growth factor I and II) showed weak, but significant cross-reactivity: insulin-like growth factor II was 10% as potent as MSA II. By contrast, somatomedin partially purified from rat serum, insulin, growth hormone, epidermal growth factor, nerve factor, and fibroblast growth factor, showed no reactivity in the radioimmunoassay.     

The 34 kilodalton insulin-like growth factor binding proteins in human cerebrospinal fluid and the A673 rhabdomyosarcoma cell line are human homologues of the rat BRL-3A binding protein

Three members of a family of insulin-like growth factor binding proteins have been identified by nucleotide sequencing of cDNA clones: the binding subunit of the 150 kDa IGF-binding protein complex in human serum, the 30 kDa IGF binding protein in human amniotic fluid, and a 30 kDa binding protein (BP-3A) isolated from the rat BRL-3A cell line. The present study demonstrates by molecular hybridization and immunoreactivity that the human counterpart of rat BP-3A is a 34 kDa IGF binding protein that is present in human cerebrospinal fluid and is synthesized and secreted by the A673 human rhabdomyosarcoma cell line.     

Nucleotide sequence and expression of a cDNA clone encoding a fetal rat binding protein for insulin-like growth factors

The insulin-like growth factors (IGFs), IGF-I and IGF-II, occur in plasma and tissue fluids complexed to specific binding proteins. Although the role of the binding proteins is not completely defined, they are capable of modulating the biological activity of the IGFs. In order to better understand the function of these proteins, we have isolated a clone from the BRL-3A rat liver cell line that encodes a protein corresponding to the IGF binding protein in fetal rat serum. The cDNA clone encodes a precursor protein of 304 amino acids (32,886 daltons), comprised of a 34-residue hydrophobic prepeptide and a 270-residue mature protein (29,564 daltons). The deduced amino acid sequence agrees with the sequence of 173 amino acid residues determined by Edman degradation. The mature protein contains 18 cysteines and no N-glycosylation sites. It contains an Arg-Gly-Asp (RGD) sequence near the carboxyl terminus. A similar sequence is present on many extracellular matrix proteins and contributes to their recognition by cellular adhesion receptors. The cloned cDNA has been transcribed in vitro and the resulting RNA expressed in Xenopus oocytes. Injected oocytes secrete a 33-kDa protein that is immunoprecipitated by polyclonal antibodies to the BRL-3A binding protein and binds IGF-I and IGF-II with the same affinity and specificity as does purified BRL-3A binding protein. The binding protein cDNA probe hybridizes to an approximately 2-kilobase mRNA in BRL-3A cells and in multiple fetal rat tissues including liver, kidney, intestine, and lung. Levels of this mRNA are greatly reduced in the corresponding adult tissues. The rat IGF binding protein is closely related to the partial amino acid sequences reported for a bovine IGF binding protein and more distantly related to a human IGF binding protein that recently has been cloned. No significant homologies were identified to other proteins. Thus, the rat IGF binding protein that we have cloned appears to be a distinct member of a family of related IGF binding proteins. We postulate that the structurally distinct IGF binding proteins may have different biological functions.     

The binding of rat liver cell multiplication-stimulating activity (MSA) to human placenta and serum proteins.

Multiplication-stimulation activity (MSA) from the medium of BRL-3A rat liver cells in culture binds to cell membrane and cytosol receptors from human placenta and to serum proteins. The binding of MSA to placental cell membranes is dependent on time, temperature, pH and divalent ion concentration. MSA bound to placental cytosol receptor and serum is not displaced by insulin, whereas that bound to placental cell membranes is displaced by insulin and insulin-like peptides. The affinity of the three receptors for MSA is similar [approximately 10(8) M(-1)]. An assay using 125I-MSA and placental membrane receptor detects somatomedin-like receoptor activity (SmLRA) in unextracted sera from man and animals. A binding protein in serum that competes for 125I-MSA with receptor could not be completely separated from SmLRA by heating, acidification, charcoal treatment and gel chromatography of the serum. The relative activities of SmLRA and serum binding protein remained constant in three disorders of human growth (acromegaly, growth hormone deficiency and Laron's dwarfism) in which values of SmLRA varied widely. However, the binding protein is only partly responsible for the apparent SmLRA of unextracted serum. It is concluded that MSA is a suitable radioligand for the investigation of somatomedin disorders in man either by receptor assays or by studies of tissue receptors.     

Isolation of a palmitoyl CoA-protein complex with properties of the ADP/ATP carrier from bovine heart mitochondria

A palmitoyl CoA-protein complex was isolated from bovine heart mitochondria and purified to homogeneity. The elution profile of the [¹⁴C]palmitoyl CoA bound protein from a hydroxyapatite column was identical to that seen when [³H]carboxyatractylate was used as the bound ligand. A sample of the palmitoyl CoA-protein complex from a peak fraction of the column appeared to be homogeneous by sodium dodecyl sulfate gel electrophoresis. The mobility of the protein bound with palmitoyl CoA was identical to the one bound with carboxyatractylate and the molecular weight was estimated to be 30,000 daltons. Compared to the stable palmitoyl CoA-protein complex, purification of the unliganded carrier from mitochondria at 22°C resulted in a disaggregated protein. These physical characteristics of the palmitoyl CoA-protein complex correspond to those identified for the $\text{ADP}\text{ATP}$ carrier. The results further confirm the specificity of the fatty acyl CoA ligand for the adenine nucleotide translocase and support the concept that it may be a physiological modulator of adenine nucleotide translocation.     

Production of insulin-like growth factor-II (MSA) by endoderm-like cells derived from embryonal carcinoma cells: possible mediator of embryonic cell growth

The present study was carried out to determine if an insulin-like growth factor (IGF) type activity might be produced by embryonal carcinoma-derived cells. The cell line used to condition growth medium for the isolation of secreted growth factors was a newly established Dif 5 cell type. Dif 5 cells are a differentiated endoderm-like cell type derived from F9 embryonal carcinoma cells (which possess properties similar to mouse embryonic stem cells) following extensive exposure to retinoic acid. When growth medium conditioned by Dif 5 cells is chromatographed on Sephadex G-75 in 1 M acetic acid two peaks of activity are observed which compete for specific [125I]iodo multiplication stimulating activity (MSA) binding to PYS cells. MSA is the rat homologue of human IGF-II. The high molecular weight fraction (Mr approximately 60K) apparently corresponds to IGF-binding protein as determined by its ability to bind [125I]iodo-MSA. The low molecular weight fraction (Mr approximately 8K) is biologically active as this fraction stimulates [3H]thymidine incorporation into serum-starved chick embryo fibroblasts. Radioimmunoassay data indicate that the IGF-like activity produced by Dif 5 cells is more closely related to IGF-II than to IGF-I. Undifferentiated embryonal carcinoma stem cell lines (F9, Nulli, and PCC4) produced little of this MSA-like activity, while PYS-2 (parietal endoderm-like) cells produced about 16 ng MSA/10(6) cells/24 hr as determined by radioimmunoassay. Dif 5 and PSA-5E (visceral endoderm-like) cells, are found to secrete significant amounts of MSA into the growth medium (30-50 ng MSA/10(6) cells/24 hr). These findings offer further support to a proposal that MSA (IGF-II) produced by endoderm cells, particularly visceral endoderm, may serve as an early embryonic growth factor.     

Molecular weight determination of two genetically linked cell surface murine antigens: ThB and Ly-6

Various murine tumor lines were screened by FACS analysis for the surface antigens ThB and Ly-6.2. Positive cell lines were used for immunoprecipitation studies. A monoclonal ThB-specific antibody immunoprecipitated a unique acidic protein of approximately 16 000 daltons from several positive tumors and from concanavalin A (Con-A) and LPS activated splenic lymphocytes. Monoclonal Ly-6.2-specific antibody was used to immunoprecipitate a 33 500 dalton protein that was shown to exist in four similarly sized forms with different basic charges. In the course of these studies, the apparent molecular weight of the surface antigen T 30, immunoprecipitated with a monoclonal T 30-specific antibody from the cell line EL4, was found to be approximately 25 000 daltons.     

Characterization of intra- and extracellular transforming growth factors from normal and avian sarcoma virus-transformed rat cells

Intra- and extracellular transforming growth factors (TGFs) have been characterized in an avian sarcoma virus-transformed rat cell line, 77N1, and the nontransformed parental cell line, NRK. Serum-free conditioned medium from 77N1 and cell extracts from NRK and 77N1 were subjected to ion exchange column chromatography. Intracellular TGFs in cell extracts of NRK and 77N1 cells showed a major peak of DNA synthesis-stimulating and colony-forming activities which eluted in the 0.10 to 0.15 M salt concentration region. Extracellular TGF in conditioned medium of 77N1 cells showed a major peak of activity which eluted at 0.05 to 0.06 M salt concentration. Furthermore partially purified extracellular TGF had a molecular weight of about 40,000 daltons, whereas that for intracellular TGF was about 12,000 daltons. These intra- and extracellular TGFs, as well as TGF gamma 2 which was purified from 77N1 cell extract, were heat- and acid-labile polypeptides sensitive to treatment with dithiothreitol. Radioimmunoprecipitation analyses with antiserum against TGF gamma 2 demonstrated that intra- and extracellular TGFs in 77N1 and NRK cells were immunologically identical or very closely related to each other and suggested the possibility that extracellular TGF from 77N1 cells was released into serum-free conditioned medium after formation of complex with other cellular components.     

慢病毒介导的大鼠肝BRL-3A 细胞Tmub1 基因沉默及稳定感染细胞 系的建立

目的:构建Tmub1基因慢病毒干扰载体,建立稳定转染细胞系,检测大鼠肝BRL-3A细胞中Tmub1基因表达的干扰效果。方法:设计并构建4对针对大鼠Tmub1基因的特异性shRNA干扰质粒,酶切鉴定、DNA测序所得质粒。将由pRSV-Rev、pMDLg-pRRE、pMD2G和pll3.7干扰质粒组成的包装系统共转染293T细胞,产生慢病毒。所得慢病毒感染大鼠正常肝细胞BRL-3A,Western Blot检测不同靶点RNAi后Tmub1蛋白表达情况,确定有效靶点。针对有效靶点大量包装慢病毒。测定病毒滴度并以最适感染复数(multiplicity of infection,MOI)感染BRL-3A细胞后,G418抗生素筛选稳定感染细胞系BRL-3A/256。RT-PCR和Western Blot检测各组细胞Tmub1 mRNA和蛋白质的表达差异。结果:结果显示Tmub1 RNAi慢病毒载体构建成功,C0020Sh2-Hops-256干扰靶点RNAi效果最强。成功包装Tmub1基因RNAi慢病毒,测定病毒滴度为2.3×108TU/ml,对293T细胞的最适感染复数为60。成功建立Tmub1 RNAi慢病毒载体稳定感染细胞系BRL-3A/256,且在该细胞系中Tmub1 mRNA和蛋白质表达明显降低。结论:成功构建Tmub1 RNAi慢病毒载体,有效干扰BRL-3A细胞中Tmub1 mRNA和蛋白表达;成功筛选出Tmub1RNAi慢病毒稳定感染细胞系BRL-3A/256。     

Stimulation by insulin-like growth factors is required for cellular transformation by type beta transforming growth factor

Medium conditioned by BRL-3A cells, a known source of insulin-like growth factor II (IGF-II), induced phenotypic transformation (anchorage-independent proliferation) of mouse BALB/c 3T3 fibroblasts but not rat NRK-49F fibroblasts, in the presence of 10% calf serum. A specific radioreceptor assay and a bioassay indicated that BRL-3A conditioned medium contained 0.5-1 ng/ml of type beta transforming growth factor (beta TGF). Purified IGF-II and beta TGF acting together reconstituted the transforming activity of BRL-3A conditioned medium on BALB/c 3T3 cells. Insulin was 5-10% as potent as IGF-II in supporting the transforming action of beta TGF on BALB/c 3T3 cells. NRK-49F cells were phenotypically transformed by beta TGF in the presence of EGF and 10% calf serum as the sole source of IGFs. However, transformation of NRK-49F cells under these conditions was inhibited by addition of purified IGF-binding protein. Addition of an excess of IGF-II prevented the inhibitory action of IGF-binding protein. The different sensitivity of the two cell lines to IGFs was correlated with lower levels of type I IGF receptor and higher levels of type II IGF receptor in NRK-49F cells as compared with BALB/c 3T3 cells. The results suggest that cellular stimulation by IGFs is a prerequisite for transformation of rodent fibroblasts by beta TGF. We propose that transformation of fibroblasts by beta TGF requires concomitant stimulation by the set of growth factors that support normal cell proliferation.     

Characterization of recombinant human lymphotoxin (tumor necrosis factor-beta) produced by a mammalian cell line

Lymphotoxin (LT) was purified from serum-free conditioned media of a recombinant mammalian cell line transfected with human lymphotoxin cDNA. The purification scheme consisted of controlled pore glass chromatography, Q-Sepharose ion-exchange chromatography, and concanavalin A-Sepharose chromatography. The purified protein was found to be homogeneous by reverse-phase high-performance liquid chromatography and had an approximate specific activity of 130 X 10(6) units per milligram protein as determined by the L-929 cytotoxicity assay. Purified LT had an isoelectric point of approximately 6.85 and an apparent molecular weight of 50,000 by gel permeation high-pressure liquid chromatography. However, when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, two distinct bands at approximate molecular sizes of 25 and 20 kDa were observed. Both the bands were immunoreactive by Western blot analysis and found to be associated with biological activity. The two forms of lymphotoxin differed from each other with respect to protein structure. Amino-terminal amino acid sequence analysis revealed that the 25-kDa LT sequence starts with Leu-Pro-Gly-residues whereas that of the 20-kDa LT begins with His-Leu-Ala; thus the latter form is truncated by 20 amino acid residues from the amino terminal. Two species of LT also differed from each other with respect to carbohydrate structure. Enzymatic removal of sialic acid reduced the molecular weight of 25 kDa by approximately 5 kDa whereas that of the 20-kDa LT was unchanged. A reduction in an apparent molecular size by approximately 4 kDa of both species of LT was observed on removal of N-linked oligosaccharides. Treatment with O-Glycanase had minimal effect on either form of LT. The recombinant lymphotoxin described here was found superior in its solubility behavior as compared to bacterial cell derived LT. Overall, mammalian cell line derived recombinant LT appears closer in its properties to natural LT than does bacterial cell derived recombinant LT.     

Affinity Labelling and Identification of the High-Affinity Choline Carrier from Synaptic Membranes of Torpedo Electromotor Nerve Terminals with [3H]Choline Mustard

The physiological mechanisms regulating activity of the sodium-dependent, high-affinity choline transporter and the molecular events in the translocation process remain unclear; the protein has not been purified or characterized biochemically. In the present study, [3H]choline mustard aziridinium ion [( 3H]ChM Az), a nitrogen mustard analogue of choline, bound irreversibly to presynaptic plasma membranes from Torpedo electric organ in a hemicholinium-sensitive, and sodium-, time-, and temperature-dependent manner. Specific binding of this ligand was greatest when it was incubated with membranes in the presence of sodium at 30 degrees C. Separation of the 3H-labelled membrane proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that most of the radiolabel was associated with a polypeptide of apparent molecular mass of approximately 42,000 daltons; labelling of this species was abolished in membranes incubated with ligand in the presence of HC-3. Two other 3H-labelled polypeptides were detected, with apparent molecular masses of approximately 58,000 and 90,000 daltons; radiolabelling of the former was also HC-3 sensitive. [3H]ChM Az may be a useful affinity ligand in the purification of the choline carrier from cholinergic neurons.     

Megakaryocyte colony culture using a liver cell conditioned medium

Megakaryocyte colonies may now be grown in semi-solid agar, agarose, and plasma clot systems using a variety of conditioned media. These include mitogen activated mouse spleen cells, L-cells (fibroblasts), and a myelomonocytic cell line. To these is now added a conditioned medium from BRL-3A, a rat liver cell line. Megakaryocytes show full maturation to cytoplasmic fragmentation in this system. It is suggested that colony stimulating factor for megakaryocytes may come from a variety of sources; the relationship of any of these to the physiological regulator or regulators remains quite unknown.     

Study of Glial Fibrillary Acidic Protein in a Human Glioma Cell Line Grown in Culture and as a Solid Tumor

Abstract: A continuous human glioma cell line grown in culture and as a solid tumor was analyzed for glial fibrillary acidic (GFA) protein. This material provided a rich source for GFA protein that could also be manipulated and controlled. Immunoperoxidase staining at the light and electron microscopic levels revealed that the cell culture and tumor specimens were strongly positive for GFA protein. When aqueous soluble fractions of the cell culture and tumor were separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, electroblotted onto nitrocellulose and stained immunochemically, they contained exclusively low molecular weight (41–43 K-dalton) GFA peptides. SDS (0.15%)-soluble fractions contained either low molecular weight only (culture) or a mixture of peptides ranging from 41 to 49K daltons. SDS (1%) extracts of either cell culture or tumor contained only 49K dalton GFA protein. Two-dimensional gel separation revealed that the GFA protein extracted from either the culture or tumor with 1% SDS resolved to two or three spots at pH 5.8. Low molecular weight GFA peptides (<49K daltons) in aqueous and 0.15% SDS-soluble extracts became increasingly more acidic with decreasing molecular weight. The extremely rapid degradation seen suggests that this cell line may be a valuable system for further study of intermediate filament protein turnover.     

Chemotactic response of rat mammary adenocarcinoma cell clones to tumor-derived cytokines

A cytokine with an apparent molecular weight of 53,000 daltons was isolated from serum-free medium conditioned by MTLn3 cells or from homogenates of MTLn3 cells, a highly metastatic variant of the rat 13762NF mammary adenocarcinoma. The chemotactic responses of MTLn3 and the low metastatic variant MTLn2 cells to this cytokine were tested in vitro using modified Boyden chambers. Both the chemotactic and chemokinetic movements of MTLn3 cells were stimulated by the MTLn3-derived cytokine. In addition, the MTLn3-derived cytokine stimulated a relatively small, but significant chemotactic migration of MTLn2 tumor cells, while these cells did not respond to medium conditioned by MTLn2 cells. MTLn3 cells themselves did not respond chemotactically to type I collagen or medium conditioned by MTLn2 cells. These results suggest that the chemotactic response may be a function of metastatic potential of the invading tumor cells. The production of tumor cytokines that enhance tumor cell motility may thus represent a phenotypic difference between 13762NF tumor cell subpopulations of high and low metastatic potential.     

Polypeptide inhibitors of DNA synthesis and cellular proliferation produced by cultured BRL-3A rat liver cells

Variants of the NIL8 hamster cell line have been isolated by a procedure that selects against cells that incorporate [³H]thymidine into DNA in response to stimulation with multiplication-stimulating activity (MSA, an insulin-like growth factor produced by cultured BRL-3A rat liver cells). Some of the variant clones proved to be hypersensitive to two inhibitors of [³H]thymidine incorporation that were present in partially purified MSA preparations. The inhibitors have been found to be heat-labile polypeptides. The inhibitors copurify with MSA through ion exchange chromatography on Dowex 50, but are separable from MSA by gel filtration on Sephadex G-75. One of these factors (IN I) has been shown to inhibit DNA synthesis (as determined by autoradiography) and proliferation of the variant cells. Among several other rodent tumor cell lines tested for sensitivity to inhibition by INI, Reuber H-35 rat hepatoma cells and HSV-transformed hamster fibroblasts were not inhibited by the factor, whereas PG19 mouse melanoma cells were slightly inhibited.     

Human fibroblast-conditioned medium contains a 100K dalton glucose-regulated cell surface protein

This report describes the identification and partial characterization of a 100K dalton “glucose-regulated” cell surface protein of human diploid fibroblasts (HDF). This protein is released into and can be recovered virtually intact from the surrounding culture medium. At the present level of analysis, the protein recovered from the culture medium (“conditioned medium”) is indistinguishable from the protein extracted directly from the cell surface by 1 M urea treatment. Both proteins have molecular weights of 100K daltons when analyzed by gel electrophoresis. The protein is readily labeled at the cell surface via lactoperoxidase-catalyzed iodination, and the label can be chased into the released form of this protein in conditioned medium. Antiserum raised against the medium form of the protein reacts with the surface form of the protein but does not react with fibronectin, the major cell surface protein of HDF. Conditioned medium from SV40-transformed human fibroblasts does not contain the 100K protein, but instead contains a component that has a slightly lower molecular weight (97K daltons). The lower molecular weight band does not iodinate at the cell surface and is apparently an underglycosylated form of the 100K protein. Its molecular weight is shifted back to 100K by growing transformed cells in medium containing excess glucose. After the shift, the component becomes accessible to the radioiodine label. We suggest that the 100K protein is a glucose-regulated protein (Shiu, Pouyssegur and Pastan, 1977; Pouyssegur and Yamada, 1978) that is released into the culture medium. An underglycosylated form of the same glycoprotein is released from transformed cells.     

Identification of the bombesin receptor on murine and human cells by cross-linking experiments

The bombesin receptor present on the surface of murine and human cells was identified using 125I-labeled gastrin-releasing peptide as a probe, the cross-linking agent disuccinimidyl suberate, and sodium dodecyl sulfate gels. A clone of NIH-3T3 cells which possesses approximately 80,000 bombesin receptors/cell with a single binding constant of approximately 1.9 X 10(-9) M was used in these studies. In addition, we used Swiss 3T3 cells and a human glioma cell line which possesses approximately 100,000 and approximately 55,000 bombesin receptors/cell, respectively. Under conditions found optimal for binding, it is demonstrated that 125I-labeled gastrin-releasing peptide can be cross-linked specifically to a glycoprotein of apparent molecular mass of 65,000 daltons on the surface of the NIH-3T3 cells. Similar results were obtained when the cross-linked product was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing or non-reducing conditions. Moreover, the cross-linking reaction is specific and saturable and the 65,000-dalton polypeptide is not observed when the cross-linking experiments were performed with a NIH-3T3 cell line which is devoid of bombesin receptors. Interestingly, glycoproteins with apparent molecular weights of 75,000 were labeled specifically by 125I-labeled gastrin-releasing peptide when similar experiments were performed with Swiss 3T3 cells and with human glioma cell line GM-340. These different molecular weights may indicate differential glycosylation as treatment with the enzyme N-glycanase reduced the apparent molecular weight of the cross-linked polypeptide to 45,000. On the basis of these results it is concluded that the cross-linked polypeptides represent the bombesin receptor or the ligand-binding subunit of a putative larger bombesin receptor expressed on the surface of these cells.