Identification of protein products encoded by the proto-oncogene int-1.

The proto-oncogene int-1 is activated by adjacent insertions of proviral DNA in mouse mammary tumor virus-induced tumors and has transforming activity in certain mammary epithelial cell lines. The gene is normally expressed in the central nervous system of mid-gestational embryos and in the adult testis. We raised antibodies against synthetic int-1 peptides and used these to identify protein products of the gene in cells transfected or infected with retroviral vectors expressing int-1. Four protein species of 36,000, 38,000, 40,000, and 42,000 Mr were immunoprecipitated by antibodies against two different int-1 peptides and were not present in control cells. Partial degradation with V8 protease showed the four species to be structurally related to each other and to int-1 polypeptide synthesized in vitro. Treatment of the cells with tunicamycin prevented the appearance of all but the 36,000-Mr species, suggesting that the slower-migrating forms are glycosylated derivatives. The unglycosylated 36,000-Mr species migrated faster in polyacrylamide gels than the in vitro translation product of int-1 and has probably undergone cleavage of an amino-terminal signal peptide.     

Elongated cells derived from rat mammary cuboidal epithelial cell lines resemble cultured mesenchymal cells in their pattern of protein synthesis

Two-dimensional gel electrophoresis has been used to identify polypeptide patterns characteristic of rat mammary cuboidal epithelial cells or mesenchyme-derived cells. Elongated cells and cell lines derived from cloned cuboidal epithelial cells in culture possess a polypeptide pattern which resembles that of the cultured mesenchymal cells rather than that of the cuboidal epithelial cells from which they were derived. These elongated converts also resemble cultured mesenchymal cells in possessing a Triton-insoluble matrix in which vimentin and not prekeratin predominates.     

Identification of a cell surface-binding protein for the core protein of the basement membrane proteoglycan

We have identified a protein(s) on the surface of hepatocytes that binds to the core protein of the heparan sulfate proteoglycan of basement membranes. These cells attached and spread on substrates prepared from the basement membrane heparan sulfate proteoglycan (HSPG) and its core protein (HSPG-core). Three proteins (Mr = 38,000, 36,000, and 26,000) were found to bind to a HSPG-core affinity column using extracts of iodinated hepatocytes, whereas proteins extracted from isolated membranes contained primarily the larger protein (Mr = 38,000). Similar results were obtained using a solid phase binding technique using labeled HSPG-core. Binding of HSPG-core to the protein (Mr = 38,000) was not altered by the presence of an excess of heparin, heparan sulfate, fibronectin, laminin, or collagen IV but was reduced by unlabeled HSPG-core. Similar studies showed that the binding protein (Mr = 3,0000) was present in extracts from the membranes of Engelbreth-Holm-Swarm tumor cells, Madin-Darby canine kidney cells, COS cells, melanoma cells, and rat kidney epithelial cells but not in fibroblasts. The protein was found in increased amounts in 3T3 cells treated with retinoic acid. These observations suggest that a variety of cells that contact basement membrane contain the proteoglycan-binding protein.     

Epithelial organoids and mononuclear phagocytes from DMBA-induced mammary tumors of the rat secrete collagenase in vitro

The production of collagenase has been examined in primary cultures of multicellular epithelial organoids and of stromal cells isolated from DMBA-induced mammary tumors of the rat. Plastic culture dishes and dishes coated with collagen fibrils were used to study the effect of such a substrate on collagenase release. Cultures of 51-μm epithelial organoids consisted of cuboidal cells and a myoepithelial-like cell type which formed a continuous layer under the cuboidal cells. A transient low production of collagenase with an apparent molecular weight (MW) of 72 kD was detected on both substrates. Upon separation by trypsin only cuboidal cells released collagenase. Cultures of 27-μm organoids contained only few myoepithelial-like cells. On plastic, they formed dense monolayers of cuboidal cells and released more collagenase than the greater aggregates. On collagen fibrils, these organoids formed cords and ridges and collagenase production was about 4- to 6-fold higher. These results indicate that collagenase release is influenced by the nature of the interaction of cuboidal cells with the substrate on which they grow. Similar organoids prepared from virgin mammary glands failed to secrete collagenase on either substrate. Primary cultures of stromal cells derived from tumor tissues comprised one basic cell type that expressed a series of properties characteristic for monocytes/macrophages. These cultures were capable of producing collagenase with an apparent MW of 56 kD. Collagenase with a similar size was detected in the extracts of 51 from 65 mammary tumors.     

Structure of the Mouse Mammary Tumor Virus: Polypeptides and Glycoproteins

The polypeptide and glycoprotein compositions of the mouse mammary tumor virus virion from primary monolayer cultures of BALB/cfC3H mouse mammary tumor cells were studied by polyacrylamide gel electrophoresis by using internal and external labeling and Coomassie blue and periodic acid Schiff (PAS) staining. Twelve polypeptides were reproducibly resolved by the combined methods. Five major polypeptides were demonstrable with estimated molecular weights of 52,000, 36,000, 28,000, 14,000, and 10,000. Seven minor polypeptides were also consistently detected and had estimated molecular weights of 70,000, 60,000, 46,000, 38,000, 30,000, 22,000, and 17,000. Carbohydrate was associated with five of these polypeptides as measured by PAS stain or [(3)H] glucosamine labeling, or both. These glycoproteins had estimated molecular weights of 70,000, 60,000, 52,000, 36,000 and 10,000. The majority of the PAS stain and glucosamine was found in the 52,000 and 36,000 dalton peaks.     

Differential response of normal rat mammary epithelial cells to mammogenic hormones and EGF

A simple dissociation procedure and the collagen gel culture system have been utilized to determine the effects of mammogenic hormones and epidermal growth factor (EGF) on the proliferation of normal rat mammary epithelial (RME) cells in serum-free culture. Epithelial fragments, isolated from normal virgin F344 rat mammary glands by enzyme digestion followed by Percoll density gradient centrifugation, were embedded within a rat tail collagen matrix. A three- to four-fold increase in cell number was observed when ovine prolactin (PRL) and progesterone (P) were present in the basal medium during 7 days of culture. Mouse EGF stimulated one cell doubling during the same culture period. Isolated mammary organoids produced a 'stellate' type colony when PRL + P were present in the culture medium. These colonies were composed of small, tightly packed cuboidal cells. The addition of EGF to the basal medium produced a diffuse 'basket' type colony which was composed of large, elongate cells. When the complete hormonal and growth factor combination (PRL + P + EGF) was present, a 'mixed' type colony was observed which contained both the large and small epithelial cell types. Immunocytochemical analysis revealed that both the cuboidal and elongate cells present in the two colony types stained with antibodies to keratin indicating that these cells were epithelial in nature. The small cuboidal cells also expressed thioesterase II and alpha-lactalbumin, both specific for secretory mammary epithelial cells. The large, elongate cell type, however, was positive for actin but did not stain for either secretory epithelial specific marker. The results reported here suggest that normal rat mammary tissue may contain two epithelial populations, one which responds to PRL + P and the other which responds to EGF.     

Characterization of transducin from bovine retinal rod outer segments. II. Evidence for distinct binding sites and conformational changes revealed by limited proteolysis with trypsin

The first stage of amplification in the cyclic GMP cascade in bovine retinal rod is carried out by transducin, a guanine nucleotide regulatory protein consisting of two functional subunits, T alpha (Mr approximately 39,000) and T beta gamma (Mr approximately 36,000 and approximately 10,000). Limited trypsin digestion of the T beta gamma subunit converted the beta polypeptide to two stable fragments (Mr approximately 26,000 and approximately 14,000). The GTPase and Gpp(NH)p binding activities were not significantly affected by the cleavage. Trypsin digestion of the T alpha subunit initially removed a small segment from the polypeptide terminus and resulted in the formation of a single 38,000-Da fragment. When this fragment was recombined with the intact T beta gamma subunit in the presence of membranes containing photolyzed rhodopsin, the reconstituted transducin exhibited greatly reduced GTPase and Gpp(NH)p binding activities. The loss in activities was due to the inability of the cleaved T alpha to bind to the photolyzed rhodopsin. Prolonged digestion converted the 38,000-Da fragment to a transient 32,000-Da fragment and then to two stable 23,000-Da and 12,000-Da fragments. The cleavage of the 32,000-Da fragment, however, can be blocked by bound Gpp(NH)p. The 32,000-Da fragment contains the Gpp(NH)p binding site and retains the ability to activate phosphodiesterase. These results indicate that the guanine nucleotide binding and rhodopsin binding sites are located in topologically distinct regions of the T alpha subunit and proved evidence that a large conformational transition of the molecule occurs upon the conversion of the bound GDP to GTP.     

Mammary gland Ca2+-binding (-dependent) proteins: Identification as calelectrins and calpactin I/p36

Calcium-binding (-dependent) proteins (CBPs) associated with the spreading of mammary epithelial cell cultures have been identified as various calelectrins and calpactins (p36). In immunoblot analysis, the CBPs of 30–36 kD and 68–70 KD variously react with different calelectrin and calpactin I monomer/p36 antisera. The same immunoreactive proteins were shown to be present in virgin mammary glands and collagen gel mouse mammary epithelial cell cultures. The mammary CBPs show extensive immunochemical relatedness; however, they fail to show cross-reaction with antiserum to calpactin II (lipocortin) antiserum. These immunoreactive CBPs comigrate in electrophoresis with ³⁵S-methionine-labeled CBPs isolated from mammary epithelial cell cultures. Unlike calmodulin, the mammary CBPs that correspond to calelectrins and calpactin I monomer/p36 are not stable to thermal denaturation. The mammary CBPs bind to epithelial cell membranes in a Ca²⁺-dependent manner and are differentially released from ruptured cells, compared with calmodulin, suggesting subcellular localization. Phenothiazineagarose and phenylagarose are equivalent in their ability to bind the mammary CBPs. Thus, mammary gland CBPs of 30–36 kD and 68–70 kD have been shown to be related or equivalent to the calelectrins and to calpactin I monomer/p36. Since these proteins are known to bind Ca²⁺, we conclude that the mammary gland CBPs are also Ca²⁺-binding proteins. The mammary gland CBPs are immunologically related and probably represent members of a larger family of related proteins.     

Increased abundance of a normal cell mRNA sequence accompanies the conversion of rat mammary cuboidal epithelial cells to elongated myoepithelial-like cells in culture.

Rat mammary cuboidal epithelial cell lines in culture convert to elongated myoepithelial-like cells. This conversion is accompanied by the appearance of a 9,000 molecular weight acidic polypeptide (p9ka), abundant in the elongated convertants, but which is hardly detectable in the cuboidal epithelial cells. A cDNA library corresponding to a low-molecular-weight fraction of poly(A)- containing RNA from a myoepithelial-like cell line, has been constructed. Recombinant plasmids containing cDNA complementary to p9ka mRNA have been identified by hybrid-selected translation. The mRNA for p9ka has been identified by Northern blotting and is found to be at least five-times more abundant in cultured myoepithelial-like rat mammary cells when compared to the cuboidal epithelial cells. This cytoplasmic mRNA sequence, which is present in increased abundance in cultured mammary myoepithelial-like cells, is also present, at lower levels, in normal rat tissues, including the mammary glands.     

Glycosylation and secretion of surfactant-associated glycoprotein A

Synthesis of glycoprotein A, the major surfactant-associated protein, was demonstrated in Type II epithelial cells isolated from rat lung. Predominant, secreted forms migrated as glycoproteins with asparagine-linked, complex-type oligosaccharides (32,000-36,000 daltons, pI 4.2-4.8). Primary in vitro translation products of the glycoprotein migrated as five distinct proteins of approximately 26,000 daltons which were processed by pancreatic microsomal membranes in vitro to 30,000-34,000-dalton, endoglycosidase F-sensitive forms. These in vitro processed forms of glycoprotein A co-migrated with intracellular forms immunoprecipitated from [35S]methionine-labeled, Type II cells. Pulse-chase experiments with [35S]methionine-labeled cells demonstrated rapid synthesis of endoglycosidase H-sensitive precursors of 34,000 daltons, pI 4.7-4.8, which were neither secreted from Type II cells nor detected in surfactant from alveolar lavage. These high-mannose forms were slowly processed to more acidic, endoglycosidase H-resistant, neuraminidase-sensitive forms. At between 10 and 180 min, fully sialylated or other endoglycosidase H-resistant forms were a minor fraction of intracellular glycoprotein A. After 16 h, intracellular glycoproteins A were primarily present as endoglycosidase H-resistant forms. Secretion of mature, sialylated, glycoprotein A was first detected 1 h after labeling, and was also readily detected after 16-24 h chase period. Tunicamycin, which blocks N-linked protein glycosylation, resulted in synthesis of three major 26,000-dalton proteins which co-migrated with the nonglycosylated, surfactant-associated proteins A1 present in surfactant from alveolar lavage and with the major in vitro translation products of rat lung poly(A+) mRNA. Tunicamycin inhibited secretion of glycoprotein A. Swainsonine, which inhibits Golgi alpha-mannosidase II, completely inhibited synthesis of the fully sialylated molecule. Swainsonine produced forms of glycoprotein A which were both neuraminidase- and endoglycosidase H-sensitive and were readily secreted. Monensin, an ionophore that alters protein transport, markedly inhibited intracellular sialylation and secretion. These studies demonstrate that pulmonary Type II cells rapidly synthesize and process surfactant-associated glycoprotein A precursors to endoglycosidase H-sensitive forms, which are slowly sialylated prior to secretion.     

Development of mouse mammary gland: identification of stages in differentiation of luminal and myoepithelial cells using monoclonal antibodies and polyvalent antiserum against keratin

The development of the mouse mammary gland was studied immunohistochemically using monoclonal antibodies against cell surface and basement membrane proteins and a polyclonal antibody against keratin. We have identified three basic cell types: basal, myoepithelial, and epithelial cells. The epithelial cells can be subdivided into three immunologically related cell types: luminal type I, luminal type II, and alveolar cells. These five cell types appear at different stages of mammary gland development and have either acquired or lost one of the antibody-defined antigens. The cytoplasmic distribution of several of these antigens varied according to the location of the cells within the mammary gland. Epithelial cells which did not line the lumen expressed antigens throughout the cytoplasm. These antigens were demonstrated on the apical site in situations where the cells lined the lumen. One antigen became increasingly basolateral as the cells became attached to the basement membrane. The basal cells synthesize laminin and deposit it at the cell base. They are present in endbuds and ducts and are probably the stem cells of the mammary gland. Transitional forms have been demonstrated which developmentally link these cells with both myoepithelial and (luminal) epithelial cells.     

Identification of canine pulmonary surfactant-associated glycoprotein A precursors

Surfactant-associated glycoproteins A were identified by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis of crude surfactant from canine alveolar lavage: an unglycosylated form (protein A1), 27,000-28,000 daltons; glycoprotein A2, 32,000-34,000 daltons; and glycoprotein A3, 37,000-38,000 daltons; pH at isoelectric point (pI) 4.5-5.0. Glycoproteins A2 and A3 were electroeluted and used to prepare a monospecific antiserum that identified proteins A1, A2, and A3 in immunoblots of crude surfactant obtained from dog lung lavage. This antiserum precipitated several proteins from in vitro translated canine lung poly(A)+ mRNA; proteins of 27,000 daltons, pI 5.0, and 28,000 daltons, pI 4.8-5.0, which precisely comigrated with proteins A1 from canine surfactant. Cotranslational processing of the primary translation products by canine pancreatic microsomal membranes resulted in larger proteins of 31,000-34,000 daltons, pI 4.8-5.0. Treatment of these processed forms of glycoprotein A with endoglycosidase F, to remove N-linked carbohydrate, resulted in proteins of 27,000-28,000 daltons which precisely comigrated with surfactant protein A1. These observations demonstrate that the polypeptide precursors to the glycoproteins A complex are extensively modified by addition of asparagine N-linked complex carbohydrate and are subsequently secreted as glycoproteins A2 and A3.     

Functional differentiation and alveolar morphogenesis of primary mammary cultures on reconstituted basement membrane

An essential feature of mammary gland differentiation during pregnancy is the formation of alveoli composed of polarized epithelial cells, which, under the influence of lactogenic hormones, secrete vectorially and sequester milk proteins. Previous culture studies have described either organization of cells polarized towards lumina containing little or no demonstrable tissue-specific protein, or establishment of functional secretory cells exhibiting little or no glandular architecture. In this paper, we report that tissue-specific vectorial secretion coincides with the formation of functional alveoli-like structures by primary mammary epithelial cells cultured on a reconstituted basement membrane matrix (derived from Engelbreth-Holm-Swarm murine tumour). Morphogenesis of these unique three-dimensional structures was initiated by cell-directed remodelling of the exogenous matrix leading to reorganization of cells into matrix-ensheathed aggregates by 24 h after plating. The aggregates subsequently cavitated, so that by day 6 the cells were organized into hollow spheres in which apical cell surfaces faced lumina sealed by tight junctions and basal surfaces were surrounded by a distinct basal lamina. The profiles of proteins secreted into the apical (luminal) and basal (medium) compartments indicated that these alveoli-like structures were capable of an appreciable amount of vectorial secretion. Immunoprecipitation with a broad spectrum milk antiserum showed that more than 80% of caseins were secreted into the lumina, whereas iron-binding proteins (both lactoferrin and transferrin) were present in comparable amounts in each compartment. Thus, these mammary cells established protein targeting pathways directing milk-specific proteins to the luminal compartment. A time course monitoring secretory activity demonstrated that establishment of tissue-specific vectorial secretion and increased total and milk protein secretion coincided with functional alveolar-like multicellular architecture. This culture system is unique among models of epithelial cell polarity in that it demonstrates several aspects of epithelial cell polarization: vectorial secretion, apical junctions, a sequestered compartment and formation of a basal lamina. These lumina-containing structures therefore reproduce the dual role of mammary epithelia to secrete vectorially and to sequester milk proteins. Thus, in addition to maintaining tissue-specific cytodifferentiation and function, a basement membrane promotes the expression of tissue-like morphogenesis.     

Morphology and lactose synthesis in tissue culture of mammary alveoli isolated from lactating mice

Summary Mammary epithelial cells from lactating mice synthesize and secrete lactose in culture and retain many features of their in vivo morphology if mammary glands are only partially dissociated to alveoli, rather than completely dissociated to single cells. After 5 d in culture lactose synthesis by alveoli cultured on floating collagen gels is 10 to 20 times higher than in cultures of single cells on floating collagen gels. Moreover, mammary alveoli in culture retain sensitivity to lactogenic hormones; the synthesis of lactose by alveoli depends on the continued presence of insulin and either hydrocortisone or prolactin. In addition, within alveoli the original juxtaposition of constituent epithelial cells is retained, and cells are cuboidal and have many microvilli and fat droplets. In contrast, alveoli on attached gels flatten and lose their secretory morphology. These results indicate that the shape of the cells, presence of lactogenic hormones, and maintenance of epithelial:epithelial cell contacts are required for maintenance of mammary epithelial cell differentiation in culture. This research was supported by Grants CA-16392 and AG-02909 from the National Institutes of Health and Institutional Grant IN 119 from the American Cancer Society.     

Aldosterone does not alter apical cell-surface expression of epithelial Na+ channels in the amphibian cell line A6.

The steroid hormone aldosterone regulates reabsorptive Na+ transport across specific high resistance epithelia. The increase in Na+ transport induced by aldosterone is dependent on protein synthesis and is due, in part, to an increase in Na+ conductance of the apical membrane mediated by amiloride-sensitive Na+ channels. To examine whether an increment in the biochemical pool of Na+ channels expressed at the apical cell surface is a mechanism by which aldosterone increases apical membrane Na+ conductance, apical cell-surface proteins from the epithelial cell line A6 were specifically labeled by an enzyme-catalyzed radioiodination procedure following exposure of cells to aldosterone. Labeled Na+ channels were immunoprecipitated to quantify the biochemical pool of Na+ channels at the apical cell surface. The activation of Na+ transport across A6 cells by aldosterone was not accompanied by alterations in the biochemical pool of Na+ channels at the apical plasma membrane, despite a 3.7-4.2-fold increase in transepithelial Na+ transport. Similarly, no change in the distribution of immunoreactive protein was resolved by immunofluorescence microscopy. The oligomeric subunit composition of the channel remained unaltered, with one exception. A 75,000-Da polypeptide and a broad 70,000-Da polypeptide were observed in controls. Following addition of aldosterone, the 75,000-Da polypeptide was not resolved, and the 70,000-Da polypeptide was the major polypeptide found in this molecular mass region. Aldosterone did not alter rates of Na+ channel biosynthesis. These data suggest that neither changes in rates of Na+ channel biosynthesis nor changes in its apical cell-surface expression are required for activation of transepithelial Na+ transport by aldosterone. Post-translational modification of the Na+ channel, possibly the 75,000 or 70,000-Da polypeptide, may be one of the cellular events required for Na+ channel activation by aldosterone.     

The correlation between Na+ channel subunits and scorpion toxin-binding sites. A study in rat brain synaptosomes and in brain neurons developing in vitro

Photoreactive derivatives of alpha- and beta-scorpion toxins have been used to analyze the subunit composition of Na+ channels in rat brain. In synaptosomes, both types of toxins preferentially labeled (greater than 85%) a component of 34,000 Da and, at a lower level, another component of 300,000 Da. Reduction of disulfide bridges shifted this latter band from 300,000 Da to 272,000 Da but did not modify the migration of the 34,000-Da component. Similarly, two bands were labeled in cultured brain neurons, one at 259,000 Da by alpha-scorpion toxins and the other at 34,000 Da by both alpha- and beta-scorpion toxins. Contrary to what was observed in synaptosomes, in cultured brain neurons reduction of disulfide bridges had no effect on the migration of the labeled high molecular weight component. Labeling of the smaller polypeptide was obtained even when cells were solubilized with sodium dodecyl sulfate immediately after cross-linking which proves that the 34,000-Da component is not a product of proteolysis. Binding sites for alpha- and beta-scorpion toxins, respectively, did not develop in parallel during neuronal maturation in culture: the increase in beta-scorpion toxin-binding site density was lower and later than that for alpha-scorpion toxin. When related to morphological development, the increase in alpha-scorpion toxin-binding sites was correlated to neurite growth, whereas the increase in beta-scorpion toxin-binding sites was associated with the development of chemical synapses. Finally, in cultured neurons, but not in synaptosomes, both the binding of beta-scorpion toxin and the labeling of the 34,000-Da component by beta-scorpion toxin were enhanced by depolarization of the cell membrane.     

Phosphorylation of the Mr = 34,000 protein in normal and Rous sarcoma virus-transformed rat fibroblasts

Antiserum was raised against the M_r = 34,000 chick cell protein which may serve as a substrate for the Rous sarcoma virus transforming gene product. The antiserum specifically immunoprecipitated 2 proteins from [³⁵S]methionine labeled Rous sarcoma virus-transformed rat cell extracts (a M_r = 35,000 and a M_r = 38,000 protein). Partial protease treatment revealed these two proteins to be very closely related. The protein of apparent M_r = 38,000 was phosphorylated and the phosphate was present exclusively on tyrosine residues. The effect of epidermal growth factor on phosphorylation of the M_r = 35,000 protein was examined in several normal rat fibroblast cell lines. EGF treatment had no effect on phosphorylation of the M_r = 35,000 protein for any normal cell line and also failed to elevate overall levels of phosphotyrosine.     

Tandem duplication and divergence of a sea urchin protein belonging to the troponin C superfamily

The Spec1 and Spec2 proteins of the sea urchin Strongylocentrotus purpuratus are related to calmodulin, troponin C, and myosin light chains by sequence similarity in their four calcium binding domains. These domains, the EF-hands, are distinct helix-loop-helix structures of about 40 amino acids. The Spec1 and Spec2 genes are expressed specifically in aboral ectoderm cells of the developing embryo; however, the function of the Spec proteins in these cells is unknown. To find conserved regions of the proteins that might be important for structure and function, Spec homologues from Lytechinus pictus, a distantly related sea urchin, were sought. L. pictus embryos do not synthesize detectable amounts of the 14,000-17,000-Da Spec proteins as determined by two-dimensional gel electro-phoresis, but do synthesize three 34,000-Da proteins that cross-react with Spec1 antibodies and display a similar ontogenetic pattern of expression. cDNA clones were isolated by hybridization to a synthetic oligonucleotide corresponding to the EF-hand. One clone, LpS1, encodes an mRNA with developmental properties like those of the S. purpuratus Spec mRNAs. However, LpS1 contains an open reading frame for a protein of 34,000 Da rather than 17,000 Da, and antibodies raised against part of the LpS1 reading frame demonstrate that LpS1 encodes a 34,000-Da protein in L. pictus embryos. The sequence of LpS1 reveals the presence of eight EF-hand domains, which share structural homology with the Spec1 or Spec2 EF-hands; however, little else in the protein sequence is conserved. The results support the hypothesis that the LpS1 gene arose from a duplication of an ancestral Spec gene and that the overall structural features of the Spec family of proteins are more conserved than the amino acid sequences.     

Multiple molecular forms of mouse liver arginase

Hepatic arginase (L-arginine amidinohydrolase, EC 3.5.3.1) is an oligomer composed of three or four subunits. The present studies indicate heterogeneity in the size and charge of arginase subunits in mouse liver. Two types of arginase subunits with molecular weights of approximately 35,000 and 38,000 have been found. These two subunits are detected in liver cytosol or in purified preparations of arginase after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. Two dimensional SDS-PAGE revealed multiple ionic forms of arginase for both the 35,000 and 38,000 subunits; the subunits contain basic proteins (pI range 7.8-9.1) and acidic proteins (pI range 5.8-6.4). Limited proteolysis by trypsin eliminated the molecular weight differences between the subunits without substantially affecting either their isoelectric points or activity. Comparative peptide maps and amino acid analyses of the 35,000- and 38,000-Da subunits showed that they were very similar. The data indicate that a neutral peptide (approx 3000 Da) is responsible for the differences in subunit molecular weight and that the multiple sized and charged forms are variants of the same protein.