Squid neurofilaments. Phosphorylation and Ca2+-dependent proteolysis in situ.

Three major polypeptides co-purify with neurofilaments from squid (Loligo forbesi) axoplasm: P60 (apparent Mr 60,000), P200 (apparent Mr 200,000) and Band 1 (apparent Mr 400,000). Anti-IFA, a monoclonal antibody that recognizes an epitope common to all classes of intermediate filaments, binds to P200 and P60. When axoplasm is incubated with [32P]Pi, the major phosphorylated polypeptides are P200 and Band 1. We have investigated Ca2+-dependent proteolysis of [32P]phosphorylated axoplasm in order to localize the major sites of phosphorylation and to probe the arrangement of the polypeptides in the filament. The proteinase preferentially cleaves P200 and Band 1, liberating the phosphorylated domains. Analysis of proteolysed filaments by electron microscopy and gel electrophoresis shows that most of P200 and Band 1 can be cleaved while still maintaining intact filaments. We suggest that P200 is initially cleaved within a single highly sensitive region, generating two major fragments called P100p (apparent Mr 100,000) and P110s (apparent Mr 110,000). P100p contains the Anti-IFA epitope and co-sediments with filaments, whereas P110s is highly phosphorylated and does not sediment with filaments. Band 1 is cleaved to produce a soluble high-Mr fragment that is phosphorylated and that represents a major portion of the undigested component, whereas P60 is relatively resistant to limited proteolysis. Thus proteolysis appears to define two major filament domains: a conserved core that forms the backbone of the filament, and a highly phosphorylated peripheral region that is not essential for filament integrity.     

A binding study of the interaction of beta-D-fructose 2,6-bisphosphate with phosphofructokinase and fructose-1,6-bisphosphatase

The binding of beta-D-fructose 2,6-bisphosphate to rabbit muscle phosphofructokinase and rabbit liver fructose-1,6-bisphosphatase was studied using the column centrifugation procedure (Penefsky, H. S., (1977) J. Biol. Chem. 252, 2891-2899). Phosphofructokinase binds 1 mol of fructose 2,6-bisphosphate/mol of protomer (Mr = 80,000). The Scatchard plots of the binding of fructose 2,6-bisphosphate to phosphofructokinase are nonlinear in the presence of three different buffer systems and appear to exhibit negative cooperativity. Fructose 1,6-bisphosphate and glucose 1,6-bisphosphate inhibit the binding of fructose-2,6-P2 with Ki values of 15 and 280 microM, respectively. Sedoheptulose 1,7-bisphosphate, ATP, and high concentrations of phosphate also inhibit the binding. Other metabolites including fructose-6-P, AMP, and citrate show little effect. Fructose-1,6-bisphosphatase binds 1 mol of fructose 2,6-bisphosphate/mol of subunit (Mr = 35,000) with an affinity constant of 1.5 X 10(6) M-1. Fructose 1,6-bisphosphate, fructose-6-P, and phosphate are competitive inhibitors with Ki values of 4, 2.7, and 230 microM, respectively. Sedoheptulose 1,7-bisphosphate (1 mM) inhibits approximately 50% of the binding of fructose 1,6-bisphosphate to fructose bisphosphatase, but AMP has no effect. Mn2+, Co2+, and a high concentration of Mg2+ inhibit the binding. Thus, we may conclude that fructose 2,6-bisphosphate binds to phosphofructokinase at the same allosteric site for fructose 1,6-bisphosphate while it binds to the catalytic site of fructose-1,6-bisphosphatase.     

Monoclonal antibody identifies a 200-kDa subunit of the dihydropyridine-sensitive calcium channel

A monoclonal antibody, mAb 1A, that immunoprecipitates the [3H]PN200-110-binding complex from rabbit skeletal muscle has been used to study the subunit structure of the dihydropyridine-sensitive, voltage-activated calcium channel. Digitonin-solubilized [3H]PN200-110-binding component, purified by wheat germ agglutinin chromatography, sediments as a 21 S complex. The sedimentation coefficient of the complex is increased to about 24 S after incubation with mAb 1A IgG. Four polypeptides with apparent molecular weights under nonreducing conditions of 220,000, 200,000, 61,000, and 33,000 co-sediment with the 21 S complex. mAb 1A recognizes the Mr 200,000 polypeptide, as shown by Western blotting analysis. [3H] PN200-110 complex purified by wheat germ agglutinin chromatography followed by immunoaffinity chromatography on an mAb 1A column is comprised primarily of the same four polypeptides. When analyzed by sodium dodecyl sulfate gel electrophoresis under reducing conditions, the Mr 220,000 protein migrates as a polypeptide of Mr 143,000; the mobility of the Mr 200,000 protein recognized by mAb 1A is unaffected by reduction. Thus, the Mr 200,000 polypeptide appears to be a previously undescribed component of the dihydropyridine-binding complex and, in association with the other polypeptides, may comprise the voltage-sensitive calcium channel.     

Tenascin Mr 220,000 isoform expression correlates with corneal cell migration.

The three isoforms of chicken tenascin, an extracellular matrix glycoprotein, are generated by alternatively spliced fibronectin type III domains. The resulting proteins migrate as bands of Mr 220,000 (ten220), Mr 200,000 (ten200) and Mr 190,000 (ten190) on SDS-PAGE. We describe here two monoclonal antibodies, one specific for ten220 (mAb T17) and another that recognizes all isoforms (mAb T16). These were used to examine the differential expression of isoforms during development. Most impressive is the close correlation between ten220 expression and cell migration in the embryonic cornea. Initially (stage 18), ten190/200 can be detected within the corneal epithelium and along the basement membranes of the lens and sclera. Ten220 appears within the primary stroma immediately prior to the invasion by neural-crest-derived cells. This expression is maintained during the subsequent migration of fibroblasts from the conjunctiva into the primary stroma. With the completion of migration and the marked increase in matrix synthesis by corneal fibroblasts, ten220 disappears. Ten190/200 remains in the region adjoining the endothelium, the Bowman's membrane and the adjacent stroma. The cell-migration-associated isoform is isolated from extracts of embryonic tissues as a homohexamer. Low molecular weight forms appeared absent but a new tenascin band of Mr 210,000 could be detected in brain extracts which may be a new isoform. We conclude that the synthesis of tenascin isoforms is under tight developmental control and speculate that a function of the additional domains is to facilitate cell migration.     

High molecular weight basic and acidic immunosuppressive protein components in uterine secretions of pregnant cows

Immunosuppressive proteinaceous components were determined in bovine uterine milk (UTM) collected during late pregnancy. Crude UTM was separated by ion-exchange (carboxymethylcellulose-CMC) and gel filtration (Sephacryl S-200 and Sepharose CL-6B) chromatography. Basic (CMC+) and acidic (CMC-) protein molecular weight (Mr) components were tested for immunosuppressive activity in an in vitro mitogen (phytohemagglutinin-PHA)-treated lymphocyte blastogenesis assay. For most experiments, cultures containing 1 x 10(5) lymphocytes were incubated with 0.08 micrograms PHA and varying concentrations of test protein in RPMI-1640 with supplements. At 48 +/- 1 h, 0.1 microCi of [3H]thymidine was added to cultures and [3H]DNA was quantified at 60 +/- 1 h of culture. Results were expressed as percentage of control values. Crude UTM, CMC+, and CMC- components exhibited immunosuppressive activity. For immunosuppressive Sephacryl S-200 fractions, activity was greater (p less than 0.05-0.01) for CMC+, S-200 fraction I (greater than or equal to 250,000 Mr, void volume [Vo]) than for CMC-, S-200 fractions I (Vo) and III combined for protein concentrations of 20, 40, and 50 micrograms/ml. For the high Mr Sepharose CL-6B protein components, CMC+, CL-6B fraction I (greater than or equal to 4 x 10(6) Mr, Vo) exhibited greater (p less than 0.05-0.005) activity than CMC-, CL-6B fractions I (greater than or equal to 4 x 10(6) Mr, Vo) and II (2.8 x 10(6) Mr) combined at protein concentrations ranging from 20 to 80 micrograms/ml. In summary, bovine UTM contains basic and acidic immunosuppressive protein components, with the greatest activity being associated with a high Mr, basic component.     

Sulphated proteins secreted by rat mammary epithelial cells

The main sulphated proteins secreted by rat mammary gland tissue have Mr of approximately 32 000, 27 000 and 25 000 Da. In addition, there are high Mr components which have a diffuse electrophoretic mobility (Mr > 200 000) and most likely corresponded to proteoglycans. The sulphate groups in the proteins with discrete Mr are most likely all linked to carbohydrates. These sulphated molecules were partially purified and identified to isoforms of rat alpha-lactalbumin for the 25-27 kDa bands and to kappa-casein for the 32 kDa band. This pattern of protein sulphation is, as far as we know, quite specific to rat mammary epithelial cells.     

Monoclonal antibodies that coimmunoprecipitate the 1,4-dihydropyridine and phenylalkylamine receptors and reveal the Ca2+ channel structure

Monoclonal hybridoma cell lines secreting antibodies against the (+)-PN 200-110 and the (-)-demethoxyverapamil binding components of the voltage-dependent calcium channel from rabbit transverse-tubule membranes have been isolated. The specificity of these monoclonal antibodies was established by their ability to coimmunoprecipitate (+)-[3H]PN 200-110 and (-)-[3H]demethoxyverapamil receptors. Monoclonal antibodies described in this work cross-reacted with rat, mouse, chicken, and frog skeletal muscle Ca2+ channels but not with crayfish muscle Ca2+ channels. Cross-reactivity was also detected with membranes prepared from rabbit heart, brain, and intestinal smooth muscle. These antibodies were used in immunoprecipitation experiments with 125I-labeled detergent [3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) and digitonin] solubilized membranes. They revealed a single immunoprecipitating component of molecular weight (Mr) 170,000 in nonreducing conditions. After disulfide bridge reduction the CHAPS-solubilized (+)-PN 200-110-(-)-demethoxyverapamil binding component gave rise to a large peptide of Mr 140,000 and to smaller polypeptides of Mr 30,000 and 26,000 whereas the digitonin-solubilized receptor appeared with subunits at Mr 170,000, 140,000, 30,000, and 26,000. All these results taken together are interpreted as showing that both the 1,4-dihydropyridine and the phenylalkylamine receptors are part of a single polypeptide chain of Mr 170,000.     

Identification of a neuronal laminin receptor: an Mr 200K/120K integrin heterodimer that binds laminin in a divalent cation-dependent manner

Neuronal interactions with extracellular matrix (ECM) components are crucial for axon growth and guidance during development and nerve regeneration. Laminin (LN), a prominent ECM glycoprotein, promotes neuronal survival and axon growth. To identify neuronal receptors for LN, we looked for cell surface proteins on the neuronal cell line B50 that bind LN. An integrin alpha/beta 1 dimeric receptor was identified and purified using lectin and LN affinity chromatography. The purified integrin contains two subunits with Mrs of 200 K and 120 K that bind LN specifically in the presence, but not the absence, of divalent cations (Ca2+/Mg2+ or Ca2+/Mn2+). The Mr 120 K protein was identified as the rat integrin beta 1 subunit using two beta 1 subunit-specific antibodies, and was shown to form a noncovalent complex with the Mr 200K putative alpha subunit. Since neurons and neuronal cell lines express similar integrin beta 1-class heterodimers that mediate attachment and process outgrowth on LN, the Mr 200K/120K complex identified here is likely to be an important laminin receptor used by neurons. This integrin may also mediate binding to LN by many nonneuronal cell types.     

The zinc-containing high Km cyclic nucleotide phosphodiesterase of bakers' yeast

The high Km cyclic nucleotide phosphodiesterase of Saccharomyces cerevisiae was purified by an improved procedure. Its amino acid composition is reported. Its pI is 5.85 +/- 0.1. Sedimentation equilibrium analysis of the native enzyme gave Mr = 88,000 +/- 6,000, whilst gel electrophoresis in the presence of dodecyl sulfate gave a molecular weight of 43,000, indicating that the enzyme is a dimer. Preparations of 94 +/- 4% purity contained about 2.4 atoms of zinc/43,000 daltons. Inactivation of the enzyme by 8-hydroxyquinoline was accompanied by removal of about 2 zinc atoms per monomer. Partially inactivated enzyme regained activity during dialysis against zinc, or, with less effect, cobalt salts. 8-Hydroxyquinoline (Ki = 1.1 mM) and 1,10-phenanthroline (Ki = 0.6 mM) were competitive inhibitors. The enzyme was also inhibited by the nonchelating 1,7-and 4,7-phenanthrolines and by thiols and KCN, but not by NaN3. These inhibitors probably act by binding to, but not chelating, enzyme-bound zinc.     

Biogenesis, transit, and functional properties of the insulin proreceptor and modified insulin receptors in 3T3-L1 adipocytes. Use of monensin to probe proreceptor cleavage and generate altered receptor subunits

The biogenesis, intracellular transport, and functional properties of the insulin proreceptor and modified insulin receptors were studied in hormone-responsive 3T3-L1 adipocytes. After control cells were labeled with [35S]Met for 7 min, the principal polypeptide that was precipitated by anti-insulin receptor antibodies had a molecular weight (Mr) of 180 000. This initial precursor was rapidly converted (t1/2 = 35 min) to a 200-kilodalton (kDa) polypeptide, designated the insulin proreceptor, by the apparent posttranslational addition of N-linked, high mannose core oligosaccharide units. Mature alpha (Mr 130 000) and beta (Mr 90 000) subunits were derived from sequences within the proreceptor by proteolytic cleavage and late processing steps, and these subunits appeared on the cell surface 2-3 h after synthesis of the 180-kDa precursor. The cation ionophore monensin was used in combination with metabolic labeling, affinity cross-linking, and external proteolysis to probe aspects of proreceptor function, transit, and the development of insulin sensitivity at the target cell surface. At 5 micrograms/mL, monensin potently inhibited the proteolytic cleavage step, and the 200-kDa polypeptide accumulated. Lower concentrations of the ionophore selectively blocked late processing steps in 3T3-L1 adipocytes so that apparently smaller alpha' (Mr 120 000) and beta' (Mr 85 000) subunits were produced. Proreceptor and alpha' and beta' subunits were translocated to the cell surface, indicating that the signal for intracellular transit occurs in the 200-kDa polypeptide and is independent of the posttranslational proteolysis and late processing steps. The alpha' subunit bound insulin both at the surface of intact cells and after solubilization with Triton X-100; the beta' subunit was phosphorylated in an insulin-stimulated manner. The detergent-solubilized 200-kDa proreceptor also exhibited both functional properties. However, the proreceptor that was transported to and exposed on the cell surface was incapable of binding insulin in intact adipocytes. Thus, late processing is not essential for the expression of functions associated with mature alpha and beta subunits. In contrast, it appears that the proteolytic generation of subunits is required for the correct orientation of the hormone binding site in the plasma membrane bilayer and the development of insulin responsiveness in 3T3-L1 adipocytes.     

The calcium channel antagonists receptor from rabbit skeletal muscle. Reconstitution after purification and subunit characterization

The Ca2+ channel antagonists receptor from rabbit skeletal muscle was purified to homogeneity. Following reconstitution into phosphatidylcholine vesicles, binding experiments with (+)[3H]PN 200-110, (-)[3H]D888 and d-cis-[3H]diltiazem demonstrated that receptor sites for the three most common Ca2+ channel markers copurified with binding stoichiometries close to 1:1:1. Sodium dodecyl sulfate gel analysis of the purified receptor showed that it is composed of only one protein of Mr 170,000 under non-reducing conditions and of two polypeptides of Mr 140,000 and 32,000 under disulfide-reducing conditions. Iodination of the protein of Mr 170,000 and immunoblots experiments with antisera directed against the different components demonstrated that the Ca2+ channel antagonists receptor is a complex of Mr 170,000 composed of a polypeptide chain of Mr 140,000 associated to one polypeptide chain of Mr 32,000 by disulfide bridges. One of the problems concerning this subunit structure of the putative Ca2+ channel was the presence of smaller polypeptide chains of Mr 29,000 and 25,000. Peptide mapping of these polypeptide chains and analysis of their cross-reactivity with sera directed against the proteins of Mr 170,000 and 32,000 demonstrated that they were degradative products of the Mr 32,000 component. Both the large (140 kDa) and the small (32 kDa) component of the putative Ca2+ channel are heavily glycosylated. At least 20-22% of their mass were removed by enzymatic deglycosylation. Finally the possibility that both the 140-kDa and 32-kDa components originate from a single polypeptide chain of Mr 170,000 which is cleaved by proteolysis upon purification is discussed.     

Protein analysis of cardiac sarcolemma: effects of membrane-perturbing agents on membrane proteins and calcium transport.

Protein composition of cardiac sarcolemmal membranes was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Membranes were observed to contain about 20 polypeptide bands ranging from 18000 to 200 000 dalton mass. Out of these, six bands were prominent and together comprised 57% of the membrane protein. When sarcolemmal membranes, phosphorylated by [gamma-(32)P] ATP in the presence of Ca(2+) or Na+ with and without K+, were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis at pH 2.4, the band III region (Mr 105 000) of gels was found to contain active sites of monomeric Ca-ATPase and (Na,K)ATPase. Bands I (Mr greater than 200 000), II (Mr 150 000), III (Mr 105 000), and VI (Mr 47 000) were accesible to trypsin; the extent of proteolysis was dependent on the time of exposure to, and the concentration of, trypsin (i.e, ratio of sarcolemmal protein/trypsin). Addition of molar sucrose protected sarcolemmal proteins from the tryptic proteolysis. Calcium transport was reduced by the action of trypsin; the degree of reduction was influenced by the time of exposure of membranes to trypsin as well as the concentration of trypsin. (Mg,Ca)ATPase activity, on the other hand, was elevated moderately at lower concentration and reduced at higher concentration of trypsin. Treatment with phospholipase C cium transport and (Mg,Ca)ATPase activity; electrophoretic patterns were unaffected by this treatment. Addition of lecithin to phospholipase C treated membranes produced a moderate increase in calcium transport. Exposure to Triton X-100 (1%) specifically solubilized three protein bands (Mr90 000, 67 000, and 57 000), whereas exposure to deoxycholate (1%) preferentially solubilized high-molecular-weight proteins, including band III (Mr 105 000); Lubrol-PX (1%) caused nonspecific solubilization of proteins, although the extent of solubilization with Lubrol-PX was considerably less than with either Triton or deoxycholate.     

Purification, subunit structure and properties of a high-molecular-mass protein phosphatase capable of dephosphorylating mRNP of the brine shrimp Artemia sp

A phosphoprotein phosphatase active towards casein, phosphorylase a and mRNP proteins has been detected in the cytosol of cryptobiotic gastrulae of Artemia sp. This phosphatase has a relative molecular mass (Mr) of 225,000 as measured by gel filtration on Sephadex G-200 and has been purified to near homogeneity by ion-exchange chromatography on different DEAE-substituted matrices, affinity chromatography on polylysine-agarose, histone-Sepharose 4B and protamine-agarose, hydrophobic chromatography on phenyl-Sepharose 4B and gel filtration on Sephadex G-200. Sodium dodecyl sulphate gel electrophoresis of the final purification step revealed that the enzyme contains two types of subunits, alpha and beta, with Mr of 40,000 and 75,000, respectively. These values, in conjunction with the native Mr and the molar ratios of the subunits estimated by densitometric analysis of the gel, suggested that the subunit composition of the enzyme is alpha 2 beta 2. When treated with 1.7% (v/v) 2-mercaptoethanol at -20 degrees C or with ethanol, the enzyme released the catalytic alpha subunit of Mr 40,000. The protein phosphatase was activated by basic proteins e.g. protamine (A 0.5 = 1 microM), histone H1 (A 0.5 = 1.6 microM) and polylysine (A 0.5 = 0.2 microM) and inhibited by ATP (I 0.5 = 12 microM), NaF (I 0.5 = 3.1 mM) and pyrophosphate (I 0.5 = 0.6 mM). The enzyme is a polycation-stimulated protein phosphatase. Purified mRNP proteins, phosphorylated by the mRNP-associated casein kinase type II, are among the substrates used by the enzyme. The function of reversible phosphorylation-dephosphorylation of mRNP as a regulatory mechanism in mRNP metabolism is discussed.     

Control of glycoprotein synthesis. Purification of UDP-N-acetylglucosamine:alpha-D-mannoside beta 1-2 N-acetylglucosaminyltransferase II from rat liver

A new affinity chromatography adsorbant, in which UDP-GlcNAc has been linked to thiopropyl-Sepharose at the 5 position of the uracil via a 5-mercuri mercaptide bond, was utilized to purify UDP-GlcNAc:alpha-D-mannoside beta 1-2 N-acetylglucosaminyltransferase II 60,000-fold from rat liver. After extraction of rat liver membranes with Triton X-100, the enzyme was found to exist in two molecular weight forms of markedly differing size, separable on Sephadex G-200. The low Mr form was separated from the high Mr form on columns of CM-Sephadex and hydroxylapatite, and was further purified by sequential elutions with NaCl, UDP-GlcNAc, and EDTA from the 5-mercuri-UDP-GlcNAc affinity adsorbant. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified low Mr form under reducing conditions revealed two protein bands of Mr 48,000 and 43,000. The purified enzyme catalyzes the transfer of N-acetylglucosamine from UDP-GlcNAc to the compound: (Formula: see text) The high Mr form of the enzyme, which eluted in the void volume of Sephadex G-200, was resistant to a number of treatments in attempts to reduce its molecular weight. These results suggest that the high Mr form of the enzyme may represent either a complex which normally exists in Golgi membranes as a result of strong protein-protein interactions or a protein with one or more "anchor" segments.     

Mechanism of activation of inactive renin in human plasma by puff adder venom

Venom of the puff adder (Bitis arietans) contains a potent, basic, Mr 24,000 metalloproteinase activity that can destroy all detectable trypsin and chymotrypsin inhibitory activity, when venom is incubated with human plasma. We have found that during such incubation, concomitant activation of inactive renin occurs. In an examination of the mechanism involved we now report the activation, in addition, of plasma prekallikrein and serine proteinase activity, but not plasminogen, when human plasma is incubated with venom. Furthermore, venom was not able to release active trypsin from its complex with alpha 1-proteinase inhibitor and human renin was not inhibited by alpha 1-proteinase inhibitor. The activities in venom and venom/plasma mixtures were analysed using Sephacryl S-200 gel filtration and the effect of 10 mM EDTA and 5 mM phenylmethanesulphonyl fluoride on activities in column fractions was tested. The inactive-renin-activating, plasma prekallikrein-activating and serine proteinase-activating activities could be accounted for to a large extent by a venom metalloproteinase which was estimated to have a Mr of 24,000 by sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis. This enzyme activity appeared to complex with alpha 2-macroglobulin when venom was mixed with plasma. Since both EDTA and phenylmethanesulphonyl fluoride could inhibit the activation of inactive renin by this metalloproteinase, it is suggested that the enzyme activates serine proteinase(s), which then activate inactive renin. Plasma kallikrein may have a role in this process. Additional peaks of inactive-renin-activating activity eluted from Sephacryl S-200 at Mr 30,000 and 80,000 (minor) and an additional, minor peak of caseinolytic activity eluted at Mr 60,000. The Mr 24,000 metalloproteinase in venom may have considerable utility in activating inactive renin at physiological pH owing to its ability to destroy plasma proteinase inhibitors at the same time.     

Dimeric structure of rat skeletal muscle AMP-deaminase subunit

AMP-deaminase from rat skeletal muscle was purified by affinity chromatography on phosphocellulose and gel-filtration on Sephadex G-200. It was established that disulfide bridges and hydrogen bonds were not essential for stability of enzyme oligomeric structure. The dimeric structure of enzyme subunit with Mr 76 kDa (S1) was detected by means of PAGE in the presence of SDS: besides the S1 there were also exhibited two additional bands with Mr 42 (S2) and 33 (S3) kDa. Repeated SDS-PAGE of S1 has revealed the same three protein bands. These results indicate the possibility of dissociation of S1-subunit into two subunits with close Mr values.     

Molecular structure and immunochemical properties of highly purified hemagglutinin from Clostridium botulinum type A

A procedure for isolation of highly purified hemagglutinin from a toxic complex of culture filtrates of Cl. botulinum type A is described. This procedure includes precipitation with (NH4)2SO4, chromatography on Sephadex G-100, G-200 and DEAE-cellulose, specific adsorption on human erythrocytes and affinity chromatography. Using polyacrylamide gel electrophoresis, it was shown that hemagglutinin is a heteropolymeric protein consisting of a monomer (Mr 53 000) and a trimer (Mr 160 000). The monomer is made up of two subunits with Mr 13 000 and one subunit with Mr 27 000 covalently linked by SS-crosslinks. The number and nature of the SS-crosslinks and SH-groups in the protein molecule were determined and a hypothetical structural model of hemagglutinin was proposed. Using immunochemical analysis, it was shown that some (but not all) serological properties of the highly purified protein from Cl. botulinum type A and of its partially purified counterpart are similar to those of hemagglutinin from Cl. botulinum type B.     

Regular fragmentation of hydrogen peroxide-treated fibronectin

In the presence of low concentrations (less than 0.5 mM) of hydrogen peroxide Mr 350,000 and 170,000 fragments were generated from plasma and fibroblast medium fibronectins (Fns). No other major fragments were detected when H2O2 concentration was raised or the incubation time prolonged. A 200-300-fold concentration of H2O2 was needed for a complete degradation of the protein. The degradation was inhibited or completely prevented by deferoxamine, diethylene-triaminepentaacetic acid, and thiourea or by Chelex-pretreatment of the Fn solution suggesting a Fenton-type reaction to produce .OH radicals from H2O2. In immunoblotting the Mr 170,000 fragment reacted with monoclonal antibodies against the NH2 terminus and mid-molecule but not with those against the cell-binding site and the COOH terminus of Fn. Reduction of the Mr 350,000 fragment produced alpha- and beta-monomers of Fn as well as Mr 95,000 and 85,000 fragments which reacted with monoclonal antibodies against the cell-binding site and the COOH terminus of Fn. These results suggest that the Mr 170,000 fragment is derived from the NH2-terminal part of both subunits of Fn. The rest of the subunits, the Mr 95,000 (from alpha-chain) and Mr 85,000 (from beta-chain), thus remain disulfide-bonded to an intact Fn subunit to form the nonreduced Mr 350,000 polypeptide. The results show that oxygen radical action may generate defined and reproducible fragments from Fn. The high susceptibility of Fn to the radical induced degradation makes it plausible to occur also in vivo.     

In vivo mutagenicity of benzo[f]quinoline, benzo[h]quinoline, and 1,7-phenanthroline using the lacZ transgenic mice

Phenanthrene, a simplest angular polycyclic aromatic hydrocarbon with a bay-region in its molecule, is reported to be non-mutagenic, although most angular (non-linear) polycyclic aromatic hydrocarbons, such as benzo[a]pyrene and chrysene, are known to show genotoxicity after metabolic transformation into a bay-region diol epoxide. On the other hand, benzo[f]quinoline (BfQ), benzo[h]quinoline (BhQ), and 1,7-phenanthroline (1,7-Phe), which are all aza-analogs of phenanthrene, are mutagenic in the Ames test using Salmonella typhimurium TA100 in the presence of a rat liver S9 fraction. In this report, we undertook to investigate the in vivo mutagenicity of BfQ, BhQ and 1,7-Phe by an in vivo mutation assay system using the lacZ transgenic mouse (Muta Mouse). BfQ and BhQ only slightly induced mutation in the liver and lung, respectively. BfQ- and BhQ-induced cII mutant spectra showed no characteristics compared with that of the control. These results suggest that the in vivo mutagenicities of BfQ and BhQ were equivocal. On the other hand, 1,7-Phe induced a potent mutation in the liver and a weak mutation in the lung. Furthermore 1,7-Phe depressed the G:C to A:T transition and increased the G:C to C:G transversion in the liver like quinoline, a hepatomutagen possessing the partial structure of 1,7-Phe, compared with the spontaneous mutation spectrum. These results suggest that the in vivo mutagenicity of 1,7-Phe might be caused by the same mechanism as that of quinoline, which induced the same mutational spectrum change (G:C to C:G transversion).     

Identification, immunoaffinity purification and partial characterization of a human decidua-associated protein

A crude extract of pooled early-pregnancy decidual tissue was enriched for soluble decidual proteins by exhaustive affinity absorption with antibodies to human serum proteins immobilized on Eupergit C. The partly purified extract was used to prepare monoclonal antibodies. A monoclonal antibody was obtained recognizing an antigen present in extract of decidual tissue and not in extract of proliferative endometrium. The monoclonal antibody was used for immunoaffinity purification of the decidua-associated protein. By SDS-PAGE analysis, under reducing conditions it yielded 2 bands at apparent molecular weights of 55,000 and 25,000. Under non-reducing conditions a single protein band at apparent molecular weight of 200,000 was observed. The Mr 200,000 protein was named hDP200 and the Mr 55,000 protein was named hDP55. It is suggested that hDP55 is a subunit of the hDP200. The hDP200 did not react with polyclonal antibodies specific for PP12 and PP14. PP14 has been shown to be immunologically indistinguishable from PEP and alpha 2-PEG. Our data therefore suggest that hDP200 is a novel human decidua-associated protein.