A characteristic property of vitamin K-dependent plasma protein Z

Evidence is presented for rapid, limited proteolysis of protein Z by alpha-thrombin. This alpha-thrombin-catalyzed proteolysis of protein Z occurred at a single peptide linkage, between Arg-365 and Gly-366, located in the COOH-terminal portion. The resulting NH2-terminal large fragment (PZt) and the COOH-terminal peptide (C-peptide) were isolated and chemically characterized. The C-peptide consisted of 31 amino acid residues including one galactosamine-type Thr residue and was assigned to the position from Gly-366 to the COOH-terminal residue of Val-396 in protein Z. The NH2-terminal large fragment, PZt, constituted the remainder of protein Z. The abilities to bind calcium of intact protein Z, PZt, and the derivative of protein Z devoid of the NH2-terminal gamma-carboxyglutamic acid (Gla) domain (Gla-domainless), prepared with the known chymotrypsin treatment, were examined by equilibrium dialysis. The results indicated that intact protein Z and PZt contain four calcium binding sites with dissociation constants of 0.1 mM. Moreover, the Scatchard plot analysis showed positive cooperativity, suggesting the presence of at least two initial sites for calcium binding. In contrast, the Gla-domainless protein Z had no calcium binding site, indicating that the domain of protein Z functional for calcium binding occurs within the NH2-terminal Gla domain. This differed from factor X, factor IX, protein S, and protein C, all of which contain one or two calcium binding site(s) independent on their Gla-domains.     

A kinetic model for the alpha-thrombin-catalyzed conversion of plasma levels of fibrinogen to fibrin in the presence of antithrombin III

In this study we report a kinetic model for the alpha-thrombin-catalyzed production of fibrin I and fibrin II at pH 7.4, 37 degrees C, gamma/2 0.17. The fibrin is produced by the action of human alpha-thrombin on plasma levels of human fibrinogen in the presence of the major inhibitor of alpha-thrombin in plasma, antithrombin III (AT). This model quantitatively accounts for the time dependence of alpha-thrombin-catalyzed release of fibrinopeptides A and B concurrent with the inactivation of alpha-thrombin by AT and delineates the concerted interactions of alpha-thrombin, fibrin(ogen), and AT during the production of a fibrin clot. The model also provides a method for estimating the concentration of alpha-thrombin required to produce a clot of known composition and predicts a direct relationship between the plasma concentration of fibrinogen and the amount of fibrin produced by a bolus of alpha-thrombin. The predicted relationship between the concentration of fibrinogen and the amount of fibrin produced in plasma provides a plausible explanation for the observed linkage between plasma concentrations of fibrinogen and the risk for ischemic heart disease.     

Human prothrombin activation.

Human prothrombin has been purified from American Red Cross Factor IX concentrates. Studies of the activation of the human prothrombin with the use of sodium dodecyl sulfate electrophoretic analysis of activation products indicated that human prothrombin activation is similar to bovine prothrombin activation. Molecular weight analysis of human prothrombin and intermediated by sodium dodecyl sulfate co-electrophoresis with bovine prothrombin and its intermediates resulted in molecular weights of 70,000 for prothrombin, 51,000 for intermediate 1, 41,000 for intermediate 2, 23,000 for intermediate 3, and 13,000 for intermediate 4. Amino acid compositions of human prothrombin and intermediates are similar to those for bovine prothrombin and intermediates. NH2-terminal sequence studies of human prothrombin, intermediates, and alpha-thrombin A and B chains placed the intermediates in the parent human prothrombin molecule as described for the bovine system. Intermediate 3 is the NH2-terminal of prothrombin, and intermediate 1 is the COOH-terminal segment of the zymogen. Intermediate 4 is the NH2-terminal of intermediate 1. Intermediate 2', the immediate precursor of alpha-thrombin, is the COOH-terminal segment of intermediate 1. In general, a high degree of homology in the primary structure of prothrombin and intermediates was observed between the human and bovine system. The NH2-terminal sequences of human intermediate 2' and alpha-thrombin A chain are identical. However, human intermediate 2' isolated in a manner identical with that used for the isolation of bovine intermediate 2 is homologous with bovine intermediate 2, beginning with residue 14.     

Staphylocoagulase-binding region in human prothrombin

A staphylocoagulase-binding region in human prothrombin was studied by utilizing several fragments prepared from prothrombin by limited proteolysis. Bovine prothrombin, prethrombin 1, prethrombin 2, and human diisopropylphosphorylated alpha-thrombin strongly inhibited formation of the complex ("staphylothrombin") between human prothrombin and staphylocoagulase, but bovine prothrombin fragment 1 and fragment 2 had no effect on the complex formation, indicating that the binding region of human prothrombin for staphylocoagulase is located in the prethrombin 2 molecule. To identify further the staphylocoagulase-binding region, human alpha-thrombin was cleaved into the NH2-terminal large fragment (Mr = 26,000) and the COOH-terminal fragment (Mr = 16,000) by porcine pancreatic elastase. Of these fragments, the COOH-terminal fragment, which includes Asn-200 to the COOH-terminal end of the alpha-thrombin molecule, partially inhibited the complex formation between staphylocoagulase and human prothrombin. In contrast, the NH2-terminal large fragment did not show any inhibitory effect on the staphylothrombin formation. These results suggest that the staphylocoagulase interacts with human prothrombin through the COOH-terminal region of alpha-thrombin B chain. Other plasma proteins, factor X, factor IX, protein C, protein S, protein Z, all of which are structurally similar to prothrombin, did not inhibit the staphylothrombin formation at all, indicating that a specific interaction site with staphylocoagulase is contained only in the prothrombin molecule.     

Characterization of the kinetic pathway for fibrin promotion of alpha-thrombin-catalyzed activation of plasma factor XIII

Kinetic and thermodynamic studies are presented showing that the cofactor activity of fibrin I (polymerized des-A fibrinogen) in the alpha-thrombin-catalyzed proteolysis of activation peptide (AP) from plasma factor XIII can be attributed to formation of a fibrin I-plasma factor XIII complex (Kd = 65 nM), which is processed by alpha-thrombin more efficiently (kcat/Km = 1.2 x 10(7) M-1 s-1) than free, uncomplexed plasma factor XIII (kcat/Km = 1.4 x 10(5) M-1 s-1). The increase in the specificity constant (kcat/Km) is shown to be largely due to an increase in the apparent affinity of alpha-thrombin for the complex of plasma factor XIII and fibrin I, as reflected by the 30-fold decrease in the Michaelis constant observed for fibrin I bound plasma factor XIII relative to that for uncomplexed plasma factor XIII. Analysis of the initial rates of alpha-thrombin-catalyzed hydrolysis of fibrinopeptide B (FPB) from fibrin I polymer in the presence of plasma factor XIII indicated that alpha-thrombin bound to fibrin I in the ternary complex of alpha-thrombin, plasma factor XIII, and fibrin I polymer is competent to catalyze cleavage of both FPB from fibrin I and AP from plasma factor XIII. This observation is consistent with the view that alpha-thrombin within the ternary complex is anchored to fibrin I polymer through a binding site distinct from the active site (an exosite) and that the active site is alternatively complexed with the AP moiety of plasma factor XIII or the FPB moiety of fibrin I. This conclusion is supported by the observation that a 12-residue peptide, which binds to an exosite of alpha-thrombin and blocks the interaction of alpha-thrombin with fibrinogen and fibrin, competitively inhibits alpha-thrombin-catalyzed release of both FPB and AP from the fibrin I-plasma factor XIII complex.     

The COOH-terminal domain of hirudin. An exosite-directed competitive inhibitor of the action of alpha-thrombin on fibrinogen

Hirudin, a potent 65-residue polypeptide inhibitor of alpha-thrombin found in the saliva of the leech Hirudo medicinalis, and fragments thereof are potentially useful as antithrombotic agents. Hirugen, the synthetic N-acetylated COOH-terminal dodecapeptide (Ac-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr(SO3)-Leu) of hirudin was shown in the present study to behave as a pure competitive inhibitor (Ki = 0.54 microM) of human alpha-thrombin-catalyzed release of fibrinopeptide A from human fibrinogen. In contrast to this inhibitory activity, hirugen slightly enhanced (increased kcat/Km 1.6-fold) alpha-thrombin-catalyzed hydrolysis of the fluorogenic tripeptide substrate N-p-Tosyl-Gly-Pro-Arg-7-amino-4-methylcoumarin. These observations indicate that hirugen binds to alpha-thrombin at an exosite distinct from the active site, and that interaction with this exosite is a major determinant of the competence of alpha-thrombin to bind fibrinogen. Consistent with this view, hirugen blocked binding of fibrin II to alpha-thrombin. Studies of the effect of hirugen on the rate of inactivation of alpha-thrombin by antithrombin III (AT), the major plasma inhibitor of alpha-thrombin, indicated that binding of hirugen to alpha-thrombin results in less than a 2.5-fold decrease in the rate of inactivation of alpha-thrombin by AT, both in the absence and presence of heparin. This behavior is distinct from that of active site-directed competitive inhibitors of alpha-thrombin which bind to alpha-thrombin and block both conversion of fibrinogen to fibrin and inactivation of alpha-thrombin by AT. Hirugen, an exosite-directed competitive inhibitor, blocks the interaction of alpha-thrombin with fibrinogen while leaving alpha-thrombin competent to react with AT. Thus, unlike active site-directed competitive inhibitors, hirugen should act in concert with AT and heparin to reduce the amount of fibrinogen that is processed during the lifetime of alpha-thrombin in plasma.     

Alpha-thrombin-catalyzed hydrolysis of fibrin I. Alternative binding modes and the accessibility of the active site in fibrin I-bound alpha-thrombin

Steady-state kinetic parameters were determined for the action of human alpha-thrombin on human fibrin I polymer, an intermediate in the alpha-thrombin-catalyzed conversion of fibrinogen to the fibrin matrix of blood clots during the terminal phase of the blood clotting cascade. Values of 49 s-1 and 7.5 microM were determined (at 37 degrees C, pH 7.4, gamma/2 0.17) for kcat and Km, respectively. Studies of the effect of fibrin I on alpha-thrombin-catalyzed hydrolysis of the fluorogenic substrate N-p-Tos-Gly-L-Pro-L-Arg-7-amido-4-methylcoumarin (tos-GPR-amc) and the effect of fibrin I on the reaction of alpha-thrombin with antithrombin III (AT) were presented which indicate that the active site of alpha-thrombin is accessible while it is bound to its substrate fibrin I. Fibrin I inhibited alpha-thrombin-catalyzed hydrolysis of tos-GPR-amc in a manner inconsistent with the pure competitive inhibition expected for an alternative substrate, whereas fibrinogen, an alpha-thrombin substrate, behaved as a pure competitive inhibitor of the alpha-thrombin-catalyzed hydrolysis of tos-GPR-amc. The effect of fibrin I on alpha-thrombin-catalyzed hydrolysis of tos-GPR-amc was shown to be consistent with alpha-thrombin binding to fibrin I in alternative orientations. In one orientation both the active site and a site distinct from the active site (an exosite) of alpha-thrombin are occupied by fibrin I. In the other orientation only the exosite of alpha-thrombin is occupied and the active site is freely accessible to other substrates. The values of both kcat (21 s-1) and Km (less than 0.23 microM) determined for fibrin I-bound alpha-thrombin acting on tos-GPR-amc were decreased relative to the values of kcat (180 s-1) and Km (7.3 microM) observed for the action of uncomplexed alpha-thrombin on tos-GPR-amc. This observation suggests that the active site of alpha-thrombin is altered in fibrin I-bound alpha-thrombin. Studies of the effect of fibrin I on the reaction of AT with alpha-thrombin (at 37 degrees C, pH 7.4, gamma/2 0.17) indicated that when alpha-thrombin is bound to fibrin I in an orientation where the active site of alpha-thrombin is accessible, AT reacts with alpha-thrombin with a rate constant (greater than 4.2 x 10(4) M-1 s-1) that is greater than the rate constant (1.5 x 10(4) M-1 s-1) for reaction of AT with the free enzyme.(ABSTRACT TRUNCATED AT 400 WORDS)     

Purification and complete sequence of a small proteolipid associated with the plasma membrane H(+)-ATPase of Saccharomyces cerevisiae.

The purified plasma membrane H(+)-ATPase of Schizosaccharomyces pombe and Saccharomyces cerevisiae display, in addition to the catalytic subunit of 100 kDa, a highly mobile component, soluble in chloroform/methanol. Chloroform/methanol extraction of S. cerevisiae plasma membranes led to isolation of a low molecular weight proteolipid identical to that present in purified H(+)-ATPase. NH2-terminal amino acid sequencing revealed a 38-residue polypeptide with a calculated molecular mass of 4250 Da. The polypeptide lacks the first two NH2-terminal amino acids as compared with the deduced sequence of the PMP1 gene (for plasma membrane proteolipid) isolated by hybridization with an oligonucleotide probe corresponding to an internal amino acid sequence of the proteolipid. The polypeptide is predicted to contain an NH2-terminal transmembrane segment followed by a very basic hydrophilic domain.     

Lipopolysaccharide-sensitive serine-protease zymogen (factor C) of horseshoe crab hemocytes. Identification and alignment of proteolytic fragments produced during the activation show that it is a novel type of serine protease

The horseshoe crab clotting factor, factor C, present in the hemocytes is a serine-protease zymogen activated with lipopolysaccharide. It is a two-chain glycoprotein (Mr = 123,000) composed of a heavy chain (Mr = 80,000) and a light chain (Mr = 43,000) [T. Nakamura et al. (1986) Eur. J. Biochem. 154, 511-521]. In our continued study of this zymogen, we have now also found a single-chain form of factor C (Mr = 123,000) in the hemocyte lysate. The heavy chain had the NH2-terminal sequence of Ser-Gly-Val-Asp-, consistent with that of the single-chain factor C, indicating that the heavy chain is derived from the NH2-terminal part of the molecule. The light chain had an NH2-terminal sequence of Ser-Ser-Gln-Pro-. Incubation of the two-chain zymogen with lipopolysaccharide resulted in the cleavage of a Phe-Ile bond between residues 72 and 73 of the light chain. Concomitant with this cleavage, the A (72 amino acid residues) and B chains derived from the light chain were formed. The complete amino acid sequence of the A chain was determined by automated Edman degradation. The A chain contained a typical segment which is similar in sequence to a family of repeats in human beta 2-glycoprotein I, complement factors B, protein H, C4b-binding protein, and coagulation factor XIII b subunit. The NH2-terminal sequence of the B chain was Ile-Trp-Asn-Gly-. This chain contained the serine-active site sequence-Asp-Ala-Cys-Ser-Gly-Asp-Ser-Gly-Gly-Pro-. These results indicate that horseshoe crab factor C exists in the hemocytes in a single-chain zymogen form and is converted to an active serine protease by hydrolysis of a specific Phe-Ile peptide bond.     

Pig intestinal microvillar maltase-glucoamylase. Structure and membrane insertion

The NH2-terminal sequence (25 residues) of amphiphilic single polypeptide chain maltase-glucoamylase (EC 3.2.1.20) was determined by gas-phase sequencing. The result indicates that the NH2-terminal segment anchors the enzyme to the microvillar membrane. The single-chain form and the proteolytically processed two-chain form have two distinct active sites differing in heat stability. However, both sites are sensitive to chonduritol B-epoxide and have similar substrate specificity. The amphiphilic single-chain maltase-glucoamylase and the amphiphilic proteolytically processed form were inserted into liposomes and studied by electron microscopy. The results showed that the enzyme is predominantly present as a homodimeric complex in the membrane.     

Characterization of protein S, a gamma-carboxyglutamic acid containing protein from bovine and human plasma.

Protein S is a vitamin K dependent protein of unknown function, which is present in mammalian plasma. It was isolated from bovine plasma by barium citrate adsorption and elution, ammonium sulfate fractionation, and column chromatography on DEAE-Sephadex, heparin-agarose, and polyhomoarginine-Sepharose. Bovine Protein S (Mr 64,200) is a single-chain glycoprotein with an amino-terminal sequence of Ala-Asn-Thr-Leu-Leu-. It contains 7.0% carbohydrate and 10 residues of gamma-carboxyglutamic acid per mol of protein. Human Protein S (Mr 69,000) is also a single-chain glycoprotein with an amino-terminal sequence of Ala-Asn-Ser-Leu-Leu-. It contains 7.8% carbohydrate and 10 residues of gamma-carboxyglutamic acid per mol of protein. These results indicate that Protein S from bovine or human plasma shows many similarities to the other vitamin K dependent proteins present in plasma.     

Proteolytic processing of human preproapolipoprotein A-I. A proposed defect in the conversion of pro A-I to A-I in Tangier's disease

The primary translation product of human intestinal apolipoprotein A-I mRNA was isolated from wheat germ and ascites cell-free translation systems. Comparison of its NH2-terminal sequence with that of plasma high density lipoprotein-associated A-I showed that it is initially synthesized as a preproprotein. Like rat preproapolipoprotein A-I, it contains an 18-amino acid prepeptide and a 6-amino acid propeptide. The highly unusual COOH-terminal Gln-Gln dipeptide present in the rat pro-segment is also represented at the same position in the human sequence. The functional division of the 24-amino acid NH2-terminal extention into pro- and presegments was verified by finding that the stable intracellular form of A-I in a human hepatoma cell line was the proprotein. Edman degradation of radiolabeled intracellular and extracellular A-I indicated that this apolipoprotein was secreted without proteolytic cleavage of its hexapeptide prosegment. Therefore, it appears that apolipoprotein A-I undergoes an additional proteolytic processing step before it is fully integrated into plasma high density lipoprotein. Two-dimensional gel electrophoresis of purified proapolipoprotein A-I isolated from the hepatocyte cell culture media indicated that it corresponds to isoforms 2 and 3, the basic A-I isoproteins which are the precursors of plasma A-I and the predominant plasma A-I isoforms found in patients with Tangier's disease (Zannis, V. I., Lees, A. M., Lees, R. S., and Breslow, J. L. (1982) J. Biol. Chem., 257, 4978-4986). Therefore this pathologic state probably arises from a defect in the conversion of proapolipoprotein A-I to apolipoprotein A-I.     

Characterization and identification of heparin-induced nonopioid-binding sites for beta-endorphin in human plasma

We have characterized the specific binding of human beta-endorphin (1-31) to novel binding sites which are formed in human plasma or serum in the presence of heparin. The formation of the binding sites is temperature-dependent and does not occur in the presence of other anticoagulants, such as sodium-EDTA, sodium-oxalate, or sodium-citrate. The specific binding of 125I-beta H-endorphin to heparin-induced binding sites in human plasma is saturable and reversible. It is not inhibited by morphine or naloxone or by various opioid peptides which share their NH2-terminal opioid-active sequence with beta H-endorphin. In contrast, binding is inhibited by the COOH-terminal beta H-endorphin fragment Gly-Glu indicating that binding is to nonopioid sites. Electroimmunoprecipitation techniques revealed that these binding sites are identical with S protein/vitronectin or derivatives thereof. S protein is a plasma alpha 1-glycoprotein involved in attachment and spreading of cells and also in blood coagulation and complement activation. It is possible that the interaction of beta-endorphin with S protein is of physiological significance.     

Characterization of a mutant form of ribosomal protein S1 from Escherichia coli.

An altered form of ribosomal protein S1 from a mutant of Escherichia coli has been isolated and characterized. The mutant protein (denoted m1-S1) has a molecular weight of 57,000 as shown by sodium dodecyl sulfate-gel electrophoresis and the same NH2-terminal sequence as wild type S1. Protein m1-S1 binds poly(U) in the same manner as protein S1 and is active in protein synthesis with either synthetic or natural mRNA. Thus, about 75% of the sequence of protein S1 (which includes the NH2-terminal region) contains essentially all the functional domains of this protein involved in protein biosynthesis.     

A 25-kDa NH2-terminal fragment carries all the antibacterial activities of the human neutrophil 60-kDa bactericidal/permeability-increasing protein

We have isolated, after limited proteolysis of the bactericidal/permeability-increasing protein (BPI) of human neutrophils, a 25-kDa fragment that possesses the bactericidal and envelope-altering activities of the 60-kDa parent protein. On a molar basis, the fragment is as potent as holo-human BPI against rough Escherichia coli, is more potent than holo-BPI against more resistant smooth E. coli, and retains the specificity of BPI toward Gram-negative bacteria. NH2-terminal amino acid sequence analysis shows that the fragment is derived from the NH2 terminus of the BPI molecule. These findings suggest that all of the molecular determinants of the antibacterial properties of BPI reside within the NH2-terminal 25-kDa segment, implying a novel structural/functional organization for a cytotoxic protein.     

The actin filament-severing domain of plasma gelsolin

Gelsolin, a multifunctional actin-modulating protein, has two actin-binding sites which may interact cooperatively. Native gelsolin requires micromolar Ca2+ for optimal binding of actin to both sites, and for expression of its actin filament-severing function. Recent work has shown that an NH2-terminal chymotryptic 17-kD fragment of human plasma gelsolin contains one of the actin-binding sites, and that this fragment binds to and severs actin filaments weakly irrespective of whether Ca2+ is present. The other binding site is Ca2+ sensitive, and is found in a chymotryptic peptide derived from the COOH-terminal two-thirds of plasma gelsolin; this fragment does not sever F-actin or accelerate the polymerization of actin. This paper documents that larger thermolysin-derived fragments encompassing the NH2-terminal half of gelsolin sever actin filaments as effectively as native plasma gelsolin, although in a Ca2+-insensitive manner. This result indicates that the NH2-terminal half of gelsolin is the actin-severing domain. The stringent Ca2+ requirement for actin severing found in intact gelsolin is not due to a direct effect of Ca2+ on the severing domain, but indirectly through an effect on domains in the COOH-terminal half of the molecule to allow exposure of both actin-binding sites.     

Catalytic properties of bovine alpha-thrombin: a comparative steady-state and pre-steady-state study

Values of steady-state and pre-steady-state parameters for the bovine alpha-thrombin-catalyzed hydrolysis of ZArgONp and ZLysONp have been determined between pH 2.5 and 8 (I = 0.1 M) at 21 +/- 0.5 degree C. Kinetic properties of bovine alpha-thrombin have been analyzed in parallel with those of porcine pancreatic beta-kallikrein-B and bovine beta-trypsin, all acting on cationic substrates. The different primary specificity and catalytic behaviour of these three serine proteinases reflect subtle structural differences at their S1 subsite, especially at residue positions 190, 221 and 226 as well as in the 217-219 segment.     

An amphiphilic structure of the ninth component of human complement. Evidence from analysis of fragments produced by alpha-thrombin

Purified human C9 was treated separately with three proteolytic enzymes: trypsin, plasmin, and alpha-thrombin, and the digestion products were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Trypsin initially cleaved the Mr = 71,000 C9 to produce a Mr = 47,000 fragment plus numerous smaller fragments and prolonged digestion reduced the molecule to small polypeptides. Plasmin produced a Mr = 37,000 fragment which was stable to further digestion, plus fragments smaller than Mr = 10,000. Human alpha-thrombin cleaved C9 (7.8% carbohydrate) at a single internal site to produce a Mr = 37,000 fragment (11.3% carbohydrate) and a Mr = 34,000 fragment (3.9% carbohydrate). Statistical analysis of the amino acid compositions of the fragments and alkaline polyacrylamide gel electrophoresis showed that C9 is highly amphiphilic; the Mr = 34,000 fragment contains a majority of the acidic amino acids and migrates rapidly on alkaline gels; the Mr = 37,000 fragment is hydrophobic with a slow electrophoretic mobility. The two fragments remain noncovalently associated, but were separated by sodium dodecyl sulfate-hydroxylapatite chromatography. The NH2-terminal sequence analysis of native C9, of alpha-thrombin-cleaved C9, and for the isolated fragments showed that the acidic Mr = 34,000 fragment is the NH2-terminal C9a domain and the more hydrophobic Mr = 37,000 fragment is the carboxyl-terminal C9b domain. Hemolytic activity of C9 was unaffected by alpha-thrombin cleavage.     

The cGMP-gated channel of the rod photoreceptor cell characterization and orientation of the amino terminus.

The molecular properties and orientation of the cGMP-gated cation channel of bovine rod outer segment membranes were studied using biochemical and immunochemical methods. Western blots labeled with anti-channel monoclonal antibodies indicate that the channel has a subunit Mr of 63,000 in bovine rod outer segment membranes prepared in the presence and absence of protease inhibitors and in rod outer segments from other mammalian retinas. The channel has an apparent Mr of 78,000 in both COS-1 cells and Xenopus oocytes expressing the cloned cDNA. NH2-terminal sequence analysis indicates that the lower Mr of the channel in rod outer segments is caused by the absence of the first 92 amino acids predicted by cDNA sequence analysis. Immunofluorescent and immunogold labeling has confirmed that the 63,000 form of the channel is present in rod outer segments. These results indicate that photoreceptor cell-specific co-translational or post-translational cleavage of the NH2-terminal segment of the channel occurs prior or during the incorporation of the channel into the rod outer segment plasma membrane. Immunogold labeling studies using site-directed antibodies also indicate that the NH2-terminal segment of the rod outer segment channel is exposed on the cytoplasmic side of the plasma membrane.     

Studies on human alpha-fetoprotein. Isolation and characterization of monomeric and polymeric forms and amino-terminal sequence analysis.

Human alpha-fetoprotein has been isolated from the serum and ascitic fluid of a patient with hepatoma by a combination of immunoadsorbent column chromatography and Sephadex G-150 gel filtration. Human alpha-fetoprotein is a sialylated glycoprotein with an estimated molecular weight of 67 500, composed of a single-chain polypeptide of approximately 580 amino acid residues and 3.6% carbohydrate. It is a negatively charged protein with an acid isoelectric point (pH 4.57). In addition to the monomeric form of alpha-fetoprotein, we have identified human alpha-fetoprotein polymers, including dimeric and trimeric forms, which dissociate to the monomer only upon exposure to disulfide-reducing reagents, implying that their formation is dependent upon intermolecular disulfide bonds. These polymers are found in human alpha-fetoprotein isolated by isoelectric focusing in both the major (pI 4.57) and minor (pI 5.2) alpha-fetoprotein fractions. The first 17 residues of the NH2-terminal amino acid sequence of the hepatoma-derived human alpha-fetoprotein have been identified. Fetal alpha-fetoprotein is indistinguishable from hepatoma alpha-fetoprotein by several criteria, including immunoelectrophoresis, acryalmide gel electrophoresis, and proclivity for dimerization.