Alterations in distribution of surface and intracellular antigens during epididymal maturation of rat spermatozoa

The surface membrane of mammalian spermatozoa is known to undergo considerable conformational and organizational changes during epididymal maturation. However, much less is known about remodelling of intracellular membranes. In this communication we have used specific immunological markers to study the behavior of several antigens both on and within rat spermatozoa as they mature in the epididymis. Four monoclonal antibodies (McAbs) designated 5B1, 1B5, 2D6, and 1B6 were used to probe testicular and caput and cauda epididymal spermatozoa by indirect immunofluorescence and immunogold labeling techniques. None of the McAbs bound to testicular spermatozoa; in all cases, they became reactive only on spermatozoa which had reached the caput epididymis. McAb 5B1 was restricted to the outer acrosomal membrane (OAM) of the acrosomal cap domain. The epitope first appeared on antigen(s) with molecular mass (Mr) of approximately 200 kDa in immature spermatozoa, but later in mature spermatozoa the antigen(s) had Mr of approximately 160 kDa. The antigen(s) recognized by 1B5 McAb on the other hand was initially distributed over the OAM of the entire acrosomal domain (cap + equatorial segment), but during maturation it became progressively more restricted in area until in cauda spermatozoa only the anterior tip of the OAM bound the McAb. McAb 2D6 also bound to the entire OAM and acrosomal contents of caput spermatozoa, but, unlike 5B1 and 1B5 McAbs, reactivity was transient. That is, staining was first detected in caput spermatozoa but then disappeared in corpus and cauda spermatozoa. In contrast to all of the above, 1B6 McAb bound to the surface membrane overlying the entire head domain of caput spermatozoa, but during maturation it became restricted to the postacrosomal domain. These results indicate that, in addition to remodeling of the surface membrane during epididymal maturation, extensive processing of intracellular membrane antigens also takes place and that it is very active within the acrosome. The nature of these intracellular processing events remains to be elucidated, but they may have important consequences for membrane fusion and cell recognition phenomena during fertilization.     

Development of a maturation antigen on the plasma membrane of rat spermatozoa in the epididymis and its fate during fertilization

The ontogeny of a surface membrane antigen on rat spermatozoa has been investigated using the monoclonal antibody, 2D6. Using indirect immunofluorescence microscopy the 2D6 antigen was first detected on spermatozoa from the proximal corpus epididymidis; no reaction was present on testicular cells. The 2D6 antibody also bound to spermatozoa flushed from the uterus of mated rats and to a sperm-derived antigen on the surface of newly fertilized eggs. When frozen sections of epididymal tissues were stained with 2D6 monoclonal antibody immunofluorescence was confined to the epithelium lining the duct in the proximal and distal corpus epididymidis. Fluorescence in the tissue was androgen-dependent. Immunoblots of proteins in luminal secretions collected by micropuncture from different sites along the epididymal duct showed that in the proximal corpus epididymidis the 2D6 monoclonal antibody recognized a 32 kD antigen, but in secretions from the distal corpus and cauda epididymidis the monoclonal antibody also recognized antigens with molecular weights of 28, 23 and 20 kD. Immunoblots of proteins from spermatozoa collected from the corpus epididymidis revealed a reaction over a 32 kD antigen, while on spermatozoa from the cauda epididymidis the 2D6 monoclonal antibody recognized only a 23 kD antigen. Two hypotheses are proposed to account for the varied reactivity of the monoclonal antibody and their relative merits are discussed.     

Synthesis of polypeptides by the cervix of the baboon (Papio anubis)

Administration of oestradiol to ovariectomized baboons caused the epithelium of the cervix to differentiate into tall columnar cells that were ciliated or secretory. Administration of progesterone in the presence or absence of oestradiol altered the appearance of the lining epithelium, suggesting a decrease in secretory activity. Fluorographs of media from cultures of tissue from steroid-treated animals reflected changes in polypeptide biosynthesis which correlated with the morphological observations: 6 polypeptides (Mr 88,000-37,000; pI 5.5-6.0) were observed in all treatment groups and, except for relative changes in intensity, these polypeptides were electrophoretically similar to those synthesized by the endometrium. A new group of low molecular weight polypeptides (Mr 23,000-20,000, pI greater than 8.0-5.5) and a basic protein (Mr 160,000) were synthesized and released in the oestradiol-dominated animal. These polypeptides were distinct to the cervical mucosa since they were not observed in the endometrium or oviduct. Progesterone suppressed the synthesis of the low molecular weight acidic polypeptides (Mr 23,000-20,000; pI 6.1-5.5) but maintained the synthesis of the basic polypeptides (Mr 23,000-20,000; pI greater than 8). Treatment with progesterone +/- oestradiol did not appear to induce the synthesis of any new major polypeptides in the cervical epithelium. These results suggest that oestradiol induces the synthesis of a group of cervix-specific polypeptides and progesterone antagonizes the action of oestradiol in the baboon cervix.     

Calcium-dependent proteolysis occurs during platelet aggregation

Control and stimulated platelets were analyzed by two-dimensional polyacrylamide gel electrophoresis to determine whether proteins are altered during platelet activation. Platelets were stimulated with thrombin, collagen, or the calcium ionophore A23187, and aggregation was brought about by stirring in the presence of Ca2+. These activated platelets contained at least three polypeptides not found in control platelets: 1) Mr = 200,000, pI between 6.2 and 6.4; 2) Mr = 100,000, pI = 6.3; and 3) Mr = 91,000, pI = 6.1. An additional polypeptide, polypeptide 4, with Mr = 97,000 and pI = 5.9, was present only in platelets activated by thrombin. When aggregation was prevented, either by adding 5 mM ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) to the platelet suspension or by incubating the platelet suspension without stirring, polypeptides 1-3 were not formed. Partial hydrolysis of polypeptides 2 and 4 with Staphylococcus aureus V8 protease yielded distinct sets of peptide hydrolytic fragments. These differed from those produced by the hydrolysis of alpha-actinin, a major platelet protein, which has a molecular weight similar to polypeptides 2 and 4. Polypeptides 1-3 were also produced during incubation of platelet lysates in the presence of Ca2+. Generation of these polypeptides in lysates was prevented either by chelation of Ca2+ with EGTA or by the addition of N-ethylmaleimide, leupeptin, or mersalyl, inhibitors of the calcium-dependent protease. These data show that the calcium-dependent protease is activated during aggregation of platelets by physiological agents and suggest that this protease could have a role in platelet response to stimulation.     

Identification and distribution of actin in spermatogenic cells and spermatozoa of the rabbit

Using a monoclonal antibody as a highly specific probe and a seminal particle-free fraction of rabbit ejaculated spermatozoa, actin has been localized in the postacrosomal region of mature rabbit spermatozoa. The sperm actin has been extracted and identified on two-dimensional PAGE immunoblots as a single spot of pI = 5.45 and Mr = 43,000. Rabbit sperm actin is present in a nonfilamentous form and is not removed by removing the plasma membrane. Unlike mature spermatozoa, however, filamentous actin is present in spermatogenic cells, as determined by rhodamine phalloidin staining. Starting as diffusely distributed in spermatocytes, actin accumulates in the subacrosomal space and appears as a band in conjunction with the developing acrosome. This band lengthens throughout the spermatid stage and becomes continuous with the postacrosomal region staining in testicular spermatozoa. Actin may therefore function during spermatogenesis to both shape the acrosome to the nucleus and to anchor inner acrosomal membrane proteins.     

Characterization of an acrosomal matrix protein in hamster and bovine spermatids and spermatozoa.

Experiments have been carried out characterizing an Mr 22,000 protein present in the acrosomes of hamster and bull spermatozoa. The Mr 22,000 protein is resistant to solubilization in detergent solutions containing high or low salt and has a pI of -5.2. With various lectins, the protein from hamster sperm was shown to be sparingly glycosylated with N-acetylglucosamine, mannose, and galactose while that from the bull demonstrated a slight reactivity for galactose. Using a specific monoclonal antibody (MAB 4/18), the Mr 22,000 polypeptide has been localized exclusively to the acrosomes of mature testicular and epididymal hamster and bovine sperm. Acrosomal components of differentiating bovine and hamster spermatids in tissue sections did not react with the monoclonal antibody, although the protein was present in immunoblots of round spermatids. In bovine sperm, MAB 4/18-staining at the ultrastructural level with immunogold-labeled second antibody was present as a reticulum throughout the acrosomal cap and as punctate aggregates in the equatorial segment. In hamster sperm, MAB 4/18-reactivity was present along the periphery of the acrosome in conjunction with matrix components (M1 and M2), as well as along the inner acrosomal membrane. These observations indicate that the acrosomes of bovine and hamster sperm possess an immunologically related Mr 22,000 protein and suggest that differences in MAB 4/18-staining of spermatids and spermatozoa is a result of epitope modification and/or a change in accessibility of the epitope to the antibody probe during the course of spermiogenesis. Based on its localization and solubility properties, we suggest that the Mr 22,000 protein, in conjunction with other polypeptides, forms a structural framework to maintain acrosomal shape and/or compartmentalize acrosomal contents.     

Multiple proteases from Streptomyces moderatus. II. Physicochemical and enzymatic properties of the extracellular proteases

The physicochemical and enzymatic properties of five different extracellular proteases of Streptomyces moderatus were studied. The first protease was found to be a metal chelator sensitive protease with a Mr of 21,000 +/- 1000 a and a pI of 4.6. The second enzyme was an anionic trypsin-like protease (Mr 19,000 +/- 1000; pI 3.8) with a Km value of 4.76 X 10(-4) M on N-benzoyl-L-arginine-p-nitroanilide. A Km value of 1.52 X 10(-4) M was obtained when N-benzoyl-L-arginine ethyl ester was used as the substrate. The other three enzymes were found to be serine alkaline proteases with Mr's of 22,000, 29,000, and 23,000 +/- 1000 and with respective pI's of 7.8, 8.4, and 9.2. All the proteases showed optimum activity in the alkaline pH range. One of the three proteases was found to possess chymotrypsin and elastase-like properties. All five proteases were found to be unstable at temperatures above 60 degrees C. Except the trypsin-like protease, which was stable only in acidic pH, all other enzymes were found to be stable over a wide range of pH.     

Topographical rearrangement of a plasma membrane antigen during capacitation of rat spermatozoa in vitro

We have previously described an antigen (termed 2B1) on rat spermatozoa that is present on the plasma membrane overlying the tail domain. The antigen is mobile within the plane of the plasma membrane and a mAb to it blocks fertilization in vitro. In the present study we describe some dynamic properties of this antigen in relation to its topographical distribution. When spermatozoa were incubated in vitro in a capacitation medium and stained with 2B1 mAb/FITC-rabbit anti-mouse F(ab')2, strong fluorescence appeared over the acrosomal domain. Acute exposure of fresh spermatozoa to dissociating reagents (1 M NaCl or 5 mM 2-mercaptoethanol) or inducers of the acrosome reaction (lysolecithin + Ca2+ or A23187 + Ca2+) failed to mimic these effects. Spermatozoa prelabeled with FITC-2B1 IgG and then capacitated in the presence of excess "cold" 2B1 IgG also showed accumulation of fluorescence on the acrosomal domain, suggesting that the antigen had migrated from the tail. Migration was selective and Ca2(+)- and temperature-dependent but was not inhibited by metabolic poisons (NaF or NaN3). Motility was not obligatory for migration. Immunogold-labeling studies at the ultrastructural level showed that 2B1 antigen was restricted to the surface membrane over both the tail and the acrosomal domains and that during migration it did not change the type of membrane into which it was inserted. From a quantitative analysis of fluorescence on spermatozoa prelabeled with FITC-2B1 IgG and then capacitated, the amount of antigen that appeared on the acrosomal domain was approximately equivalent to that lost from the midpiece domain. The Mr of 2B1 antigen extracted from capacitated spermatozoa was 300-500 Da less than that extracted from noncapacitated cells, suggesting that the molecule had undergone processing concomitant with migration. These results are discussed in relation to mechanisms for targeting antigens to sites where they become physiologically active and are correctly positioned to participate in gamete recognition processes.     

Antigens on rat spermatozoa with a potential role in fertilization

Eight monoclonal antibodies (McAbs), directed against antigens on rat cauda epididymal spermatozoa, were tested for their capacity to interfere with fertilization in vitro as a means of identifying molecules with a potential role in sperm-egg recognition and fusion. Antigens recognized by the McAbs were visualized on live spermatozoa by indirect immunofluorescence (IIF) and characterized by immunoblotting. Five McAbs (designated 1B5, 2C4, 4B5, 5B1, and 8C4) recognized antigens specifically on the sperm acrosome and three (designated 2B1, 2D6, and 6B2) bound to the flagellum. Of the eight McAbs investigated, three (2B1, 2C4, and 6B2) were effective in blocking fertilization in vitro when added as culture supernatants to mixtures of sperm and eggs. McAb 6B2 was inhibitory due to its ability to agglutinate spermatozoa. McAbs 2B1 and 2C4 did not agglutinate capacitated spermatozoa, had no observable effect on motility, and yet blocked fertilization in a dose-dependent manner. McAb 2C4 did not give a reaction on immunoblots, but the 2B1 antigen was identified as an Mr 40 kD glycoprotein. McAb 2B1 appeared to block fertilization at the level of zona binding, whereas the effects of 2C4 were directed more against zona penetration and/or fusion with the vitellus. When sperm-egg complexes were stained with 2C4 or 2B1 McAbs and viewed by IIF, all spermatozoa that were attached to the zona showed fluorescence on the head. These results suggest that different antigens on the rat sperm head participate in different aspects of the fertilization process and that during capacitation there is either exposure of these antigens or else they migrate to their site of action from the flagellum.     

CD38 molecule: structural and biochemical analysis on human T lymphocytes, thymocytes, and plasma cells.

The structure of the CD38 molecule has been evaluated by one- and two-dimensional gel analysis and by enzymatic digestions. The source of the Ag was mainly membrane preparations obtained from MLC cells, from normal thymocytes, and from the plasmocytoma line LP-1. Membranes were solubilized in NP-40 and the extracts fractionated by immunoaffinity chromatography [using a specific anti-CD38 antibody (A10 mAb) covalently linked to Sepharose protein A]. The purified Ag migrated as a single chain of Mr = 45,000 not associated with beta 2-microglobulin. Two-dimensional IEF gel electrophoresis revealed five spots (isoelectric point (pI) range: 6.5 to 6.9). After neuraminidase treatment, the mobility of the five polypeptides shifted to a more basic pI. Endoglycosidase-H treatment reduced the Mr of CD38 by 20%, revealing a broader band centered at Mr = 36,000. Treatment of CD38 molecule with V8 Staphylococcus aureus protease yielded a single dominant band at Mr = 38,000 which was still reactive with A10 mAb. The CD38 molecular was trypsin-resistant in both denatured or native conditions. These results clearly show the glycoprotein nature of CD38 molecule, which includes 2 to 4 N-linked oligosaccharide chains containing sialic acid residues. Furthermore, the present data indicate that the CD38 molecule does not display an apparent biochemical polymorphism among the different CD38+ cells or lines.     

Plasmodium falciparum antigens synthesized by schizonts and stabilized at the merozoite surface when schizonts mature in the presence of protease inhibitors

Schizonts of Plasmodium falciparum were cultured in medium containing a mixture of 10 micrograms/ml each of leupeptin, chymostatin, pepstatin, and antipain. The protease inhibitors did not inhibit macromolecular synthesis but were associated with decreased reinvasion of red cells and the accumulation of well preserved merozoites clustered around pigment granules (PCM, protease inhibitor clusters of merozoites). The parasite pellet from PCM cultures contained increased amounts of merozoite antigens, particularly at Mr 83, 73, 66, 45, and 17 kDa. The increases of the Mr 83, 73, and 45 kDa surface antigens observed in PCM had been observed also in similar merozoite clusters obtained by culturing schizonts in the presence of inhibitory antibodies. These three antigens are processed products of the abundant Mr 195 kDa schizont surface antigen. Liquid-phase double immunofluorescence of PCM demonstrated a residual red cell membrane through which monoclonal antibodies passed and reacted with the Mr 83, 73, and 45 kDa merozoite surface antigens or their precursors. The processes associated with normal reinvasion apparently involve protease(s), which plays a role(s) in the breakdown of the red cell membrane and the shedding of merozoite surface antigens. Interference with these processes by protease inhibitors is useful in increasing recoveries of merozoite antigens, as well as in elucidating mechanisms of reinvasion.     

Changes in the organization of surface antigens during in-vitro capacitation of boar spermatozoa as detected by monoclonal antibodies

Monoclonal antibodies specific for three major plasma membrane (PM) proteins, previously referenced as PM protein 2.0, 4.85 and 5.0, and one specific for an unreferenced PM protein (Mr 80,000) were used with indirect fluorescence microscopy to detect the effects of capacitation on the localization of these PM proteins. In ejaculated or cauda spermatozoa, incubation in the capacitating medium caused the appearance of fluorescence in the flagellum and either a loss of fluorescence on the PM overlying the sperm head (PM proteins of 5.0 and Mr 80,000) or a delocalization of fluorescence on the head PM (PM proteins 2.0 and 4.85). Labelling spermatozoa with divalent antibody and then capacitating them indicated the PM protein 5.0 and that of Mr 80,000 migrated out of the head plasma membrane into the flagellar PM during capacitation. These antigens re-entered the head PM when fresh seminal plasma was added after the capacitation period or when energy metabolism was inhibited by azide. Cytochalasin D, an inhibitor of the polymerization of actin, prevented movement of PM protein 5.0 and that of Mr 80,000 of the head PM into the flagellum during incubation in the capacitation medium and prevented re-entry of these antigens from the flagellum into the head PM after incubation in this medium. Localization changes occurring with capacitation were time-dependent but independent of the method of preparing samples for microscopy. For the major PM proteins 4.85 and 5.0, a much smaller percentage of caput spermatozoa (approximately 20%) showed specific localization changes compared to those of the cauda (approximately 80%). Chelation of Ca2+ inhibited these changes in ejaculated spermatozoa and fresh seminal plasma, added to capacitated spermatozoa, restored the localization pattern characteristic of uncapacitated spermatozoa. These observations suggest that the organization of major proteins in the plasma membrane overlying the sperm head is altered during capacitation. These changes are reversible, are dependent on sperm maturation and also appear to involve actin filament interactions with the plasma membrane.     

Structure of the murine plasma cell alloantigen PC-1: comparison with the receptor for transferrin

The plasma cell alloantigen PC-1 was isolated from C1.18 myeloma cells by immunoprecipitation and was analyzed by polyacrylamide gel electrophoresis. It was found to consist of two similar or identical disulfide-bonded polypeptide chains, each of Mr 115,000. The mobility of PC-1 in nonequilibrium pH gradient electrophoresis was similar to that of bovine serum albumin (pI 4.9). The PC-1 antigen is therefore similar to the transferrin receptor in Mr, charge, subunit composition, disulfide bonding, and developmental regulation. Similarities can also be detected by peptide mapping with subtilisin, but not with staphylococcal V8 protease. It is suggested that the PC-1 protein and the transferrin receptor may have had a common evolutionary origin, and may have similar functions.     

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.     

Medium tumor antigen of polyomavirus transformation-defective mutant NG59 is associated with 73-kilodalton heat shock protein.

Affinity-purified medium T antigen encoded by NG59, a nontransforming mutant of polyomavirus, is specifically associated with a protein of 72,000 daltons (72K protein). Medium T antigens of wild-type polyomavirus and the transformation-competent mutant dl8 are not associated with the 72K protein. Instead, they form a complex with another protein of 61,000 daltons. Several lines of evidence suggest that the medium T antigen-associated 72K protein is equivalent to the abundant and constitutive 73K heat shock protein. First, on two-dimensional polyacrylamide gels the 72K protein migrated with the same pI (5.6) as did the 73K heat shock protein. Second, the 72K protein was immunoprecipitable with antibodies against heat shock proteins. Third, when digested with V8 protease, the 72K protein gave rise to the same pattern of fragments as did the 73K heat shock protein.     

Changes in the protein composition of rat spermatozoa during maturation in the epididymis

Spermatozoa from the testis and cauda epididymidis were solubilized by detergent treatment and electrophoresis on SDS polyacrylamide gels revealed that the relative amounts of 13 detergent-extractable proteins decreased during passage of spermatozoa through the epididymis, 6 increased, whilst the remainder showed little or no change. Lactoperoxidase-catalysed iodination of plasma membrane proteins showed that the components carrying most of the label in testicular spermatozoa had Mr values of 110 000, 94 000, 84 000, 55 000 and 42 000 whereas on cauda epididymal spermatozoa the Mr values were 47 000, 24 000, 17 000, 14 500 and 13 500. Substantial differences were also noted in the protein composition of rete testis fluid and cauda epididymal plasma. The results support the concept that there is a considerable reorganization of the molecular architecture of the plasma membrane of spermatozoa during maturation in the epididymis.     

Analysis of proteins in normal and regenerating rat liver using two-dimensional electrophoresis

The major proteins of homogenate, cytosol, nuclei and nuclear membrane extract from normal and regenerating rat liver were studied by two-dimensional electrophoresis with a view of detecting proteins involved in DNA replication regulation. Essential quantitative differences in three out of 200 polypeptides separated as spots and dyed with Coomassie R-250 on two-dimensional maps were revealed. The content of the p38 nuclear protein (Mr congruent to 38 kD, pI congruent to 4) increases 6-8-fold in the S-phase. The level of another nuclear protein, p50 (Mr congruent to 50 kD, pI congruent to 6.5) decreases 2-3-fold. The cytoplasmic protein p35 (Mr congruent to 35 kD, pI congruent to 8) also decreases 2-3-fold. Moreover, the p40 protein (Mr congruent to 40 kD, pI congruent to 6) whose content in the nuclei sharply rises up to 20 times after sham operation was revealed.     

The orientation of transhydrogenase in the inner mitochondrial membrane of rat liver

The orientation of the transmembranous enzyme, pyridine dinucleotide transhydrogenase, in the inner mitochondrial membrane of rat liver has been determined by evaluating effects of proteases on the integrity of the enzyme in mitoplasts and submitochondrial particles. Following treatment of these membranes with the nonspecific protease, proteinase K, antigenic proteolytic products were detected by immunoblot analysis using polyclonal antibody prepared against purified bovine heart enzyme. Proteinase K treatment of mitoplasts converted the 110,000 transhydrogenase monomer into a single immunoreactive species having Mr 75,000. This proteolytic product is stable to further incubation with the protease. Treatment of submitochondrial particles with proteinase K resulted in the disappearance of the 110,000 monomer and the transient formation of an intermediate product with Mr 52,000. Information from these proteolysis studies was used to construct a model of the orientation of transhydrogenase in the inner mitochondrial membrane. This model indicates that transhydrogenase (Mr 110,000) contains a core of proteolytically inaccessible proteins within the membrane (Mr 23,000) bounded by extramembranous domains on the matrix (Mr 52,000) and cytoplasmic (Mr 35,000) face of the inner mitochondrial membrane.     

Protein polymorphism of a human plasma apolipoprotein D antigenic epitope

Based on our previous observation that monoclonal antibody anti-apoD-4E11 reacted with several HDL proteins we studied them further with three questions in mind: i) is there common protein polymorphism in healthy individuals? ii) how many proteins are present and what are their characteristics? iii) are they all apolipoproteins and do they have the same lipoprotein distribution as apoD? Isolated, delipidated apoD was used as a standard for radioimmunometric assay of plasma with antibody 4E11. The antigen varied from 3 to 11 mumol-equivalents of apoD per liter of plasma (equivalent to 5-20 mg apoD/dl plasma) with means of 6.1 and 6.8 mumol/l in men and women, respectively. Two-dimensional electrophoresis of plasma found up to eight 4E11-antigenic-proteins of different Mr, each heterogeneous in pI. All plasmas tested contained apoD and an Mr 38,000 antigen, the latter being the most immunoreactive. Six proteins of Mr 70,000-94,000 were found, but the number varied between subjects. Eighty nine percent of the plasma antigen was associated with lipoproteins: 83% with HDL and VHDL, 5% with LDL and VLDL. Lipoproteins of all sizes, separated by polyacrylamide gradient gel electrophoresis, contained the antigen. ApoD was almost the only 4E11-antigen in LDL, and was in two states: the one free, the other an apoD-apoB mixed disulfide complex. The apparent proportions of higher Mr antigens increased with increasing lipoprotein density, and the proportion of apoD decreased reciprocally. None of these 4E11-antigenic-proteins cross-reacted with antiserum to retinol-binding protein.     

Biosynthesis of the common precursor to vasopressin and neurophysin in vitro in transplantable human oat cell carcinoma of the lung with ectopic vasopressin production

Transplantable human oat cell carcinoma cells of the lung with ectopic vasopressin production were incubated with labeled amino acids and immunoreactive neurophysins in cell extracts were analyzed by isoelectric focusing. When the cells were incubated with L-(35S)-cysteine for 20 h, one major peak (isoelectric point; pI=5.3) and several minor peaks (pI=6.1, 5.7, 5.1, 4.9 and 4.7) of labeled proteins were observed. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the relative molecular mass (Mr) of the pI 5.7 protein was estimated to be 20,000 and that of the pI 6.1 species to be 19,000, while the remainder had a Mr of approximately 10,000. The result of the pulse-labeling experiment has clearly shown that the pI 5.7 and 6.1 proteins, which have affinity for concanavalin A, are biosynthetic precursors for the smaller form of neurophysin with a pI 5.3. When subjected to limited proteolysis with trypsin, the pI 5.7 protein generated a Mr 10,000 protein and a smaller peptide. The Mr 10,000 protein thus produced was identified as neurophysin on the basis of its pH-dependent affinity for vasopressin and the migration pattern on isoelectric focusing. The smaller peptide coeluted with synthetic arginine vasopressin and bound to neurophysin suggesting that it possesses a cysteine-tyrosyl sequence at its N-terminus. Similarly, the pI 6.1 protein liberated neurophysin and vasopressin-like peptide after incubation with trypsin. These results suggests that the glycosylated protein with a pI of 5.7 and a Mr of 20,000 is the common precursor to vasopressin and neurophysin in human oat cell carcinoma of the lung with ectopic vasopressin production. The pI 6.1 protein may be an intermediate in the conversion of the precursor to vasopressin and neurophysin.