Rat heart gap junctions as disulfide-bonded connexon multimers: Their depolymerization and solubilization in deoxycholate

Summary Unproteolyzed gap junctions isolated from rat heart and liver were analyzed for the presence of inter-subunit disulfide bonds by sodium dodecylsulfate polyacrylamide gel electrophoresis. Rat cardiac junctions contained multiple disulfide bonds connecting theM _r 47,000 subunits of the same connexon and of different connexons. Inter-subunit disulfide bonds were absent in liver junctions. Unproteolyzed rat heart gap junctions were resistant to deoxycholate in their oxidized state, but dissolved readily in the detergent when the disulfide bonds were cleaved with -mercaptoethanol. Disulfide bonding in proteolyzed cardiac junctions was limited to pairs ofM _r 29,500 subunits. These junctions were not soluble in deoxycholate even in the presence of -mercaptoethanol. These results show that heart and liver junctions differ in their quarternary organization.     

Immunological characterization of rat cardiac gap junctions: Presence of common antigenic determinants in heart of other vertebrate species and in various organs

Summary Antibodies to the following synthetic peptide, SALGKLLDKVQAY, were purified by affinity chromatography and characterized by ELISA and immunoblotting. These antibodies, shown to be specific to the major protein constitutent of isolated rat heart junctions: connexin 43, cross-reacted with a homologous protein in immunoreplicas of whole heart fractions of trout, frog, chicken, guinea pig, mouse and rat, suggesting a phylogenic conservation of connexin 43 in vertebrates. By immunoblotting of whole organ fractions it was also demonstrated that these antibodies cross-reacted with major proteins ofM _r 32 and 22 kD in rat and mouse liver, ofM _r 41 kD in rat cerebellum, ofM _r 43 kD in uterus, stomach and kidney of rat, ofM _r 46 and 70 kD in rat lens, suggesting that these proteins share common or related epitopes with the synthetic peptide and connexin 43.     

Tissue and species conservation of the vertebrate and arthropod forms of the low molecular weight (16–18000) proteins of gap junctions

Summary Gap junctions have been isolated from four murine tissues, from rat and Xenopus laevis liver, and from Nephrops norvegicus (Norway lobster) hepatopancreas. The preparations of gap junctions from each vertebrate tissue contain a single major protein, M_r 16000, and those from Nephrops hepatopancreas a protein, M_r 18000. Immunocytochemical studies using affinity-purified antibodies raised against gap junctions from Nephrops show the junctional origin of the 18k protein. Immunological studies using Western blotting and biochemical studies using tryptic peptide mapping show no significant differences between the 16k junctional proteins of mouse and hence provide no evidence of tissue variation. These studies also suggest that the mouse, rat, and Xenopus 16 k proteins and the Nephrops 18 k protein share some common structural features.     

Purification and characterization of a protein inhibitor of calcium-dependent proteases from rat liver

Soluble extracts of rat liver contain a protein inhibitor of calcium-dependent proteases. The inhibitor has an apparent M_r = 250,000 and is separated from the calcium-dependent proteases by gel-filtration chromatography in the presence of EGTA. The inhibitor has been purified by affinity chromatography using a calcium-dependent protease covalently linked to Affi-Gel 15. The inhibitor specifically binds to this affinity resin in a calcium-dependent manner and elutes in the presence of EDTA or EGTA. The purified inhibitor appears as a single protein with M_r = 125,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Presumably it is a dimer under nondenaturing conditions. The inhibitor inhibits each of two calcium-dependent proteases from rat liver and from other tissues and species. However, it has no effect on any other protease tested.     

ADP-ribosylation of brain synaptosomal proteins correlates with adenylate cyclase activation

ADP-ribosylation of the adenylate cyclase $\text{G}\text{F}$ regulatory subunit by cholera toxin is a major tool for the study of this enzyme. Investigation of the brain enzyme has been hampered up to now by the failure to demonstrate cholera toxin-dependent ADP-ribosylation of membrane-bound proteins. Synaptosomes prepared by flotation from fresh brains homogenized in the presence of protease inhibitors yielded membranes of which several proteins could be ADP-ribosylated by the toxin. The same membranes subjected to mild proteolysis could not be ADP-ribosylated. Adenylate cyclase activation and ADP-ribosylation were simultaneous processes. The major labeled species was of 47,000 M_r. It was solubilized by Lubrol-PX, together with other labeled polypeptides. As analyzed on sucrose gradients, the 47,000 M_r protein was found both in the 3S region, and in the adenylate cyclase containing fraction (9.1S).     

Peptide hormone degradation by a rat mast cell chymase-heparin complex

Material released from rat mast cells by compound $\text{48}\text{80}$ gave parallel responses using ACTH and β-endorphin radioimmunoassays. However, incubation of these labeled compounds under conditions of radioimmunoassay with released material and chromatography on Sephadex G-25 provided evidence that neither ACTH nor β-endorphin were present in the material released from mast cells, but represented an artifact produced by the presence of a protease. Analysis of the released enzyme on Sephadex G-75 under non-dissociative conditions yielded an active enzyme complex with a M_r > 150,000. Under dissociative conditions, the M_r of the enzyme was 25,000. The dissociated enzyme reassociated with purified rat mast cell heparin to form the high molecular weight complex. Further investigation of pH, substrate and inhibitor specificity showed that the peptide degradation is due to a chymotrypsin-like protease, the previously described mast cell chymase, which is active in degrading β-endorphin, ACTH, and ACTH^1–24.     

Protein processing as a regulatory mechanism in the synthesis of the photosynthetic antenna in Chloroflexus

Chloroflexus aurantiacus can be induced to shift from respiratory to photosynthetic energy production by introducing light and/or lowering the oxygen concentration of a culture. After induction, cells synthesize bacteriochlorophyll and proteins for the formation of a functional photosynthetic apparatus. Bacteriochlorophyll is detectable within 2 h after induction. Chlorosome polypeptides are detected after 8–12 h. Two proteins, M_r 60,000 and M_r 47,000, are present in both induced and noninduced cells and react specifically with antibodies against chlorosome polypeptides. Immunological data suggest that these proteins (M_r 60,000 and 47,000) are polyproteins which are transcribed and translated in the dark. When cells are exposed to light or low oxygen tension these proteins are processed into functional polypeptides required in the assembly of the chlorosome. The reaction center polypeptide (M_r 26,000) appears to be part of a separate genetic control system.Dedicated to Prof. G. Drews on occasion of his 60th birthday     

Purification and properties of glutamine synthetase from Bacillus polymyxa

Glutamine synthetase (EC 6.3.1.2) was purified to homogeneity from a free-living nitrogen fixing bacteria, Bacillus polymyxa. The holoenzyme, relative molecular mass (M_r) of 600 000 is composed of monomeric sub-units of 60 000 (M_r). The isoelectric point of the sub-units was 5.2. The pH optimum for the biosynthetic and transferase enzyme activity was 8.2 and 7.8, respectively. The apparent K _m values (K _m ^app ) in the biosynthetic reaction for glutamate, NH₄Cl and ATP were 3.2, 0.22 and 1 mM, respectively. In the transferase reaction the K _m values for glutamine, hydroxylamine and ADP were 6.5, 3.5 and 8×10^-4 mM respectively. L-Methionine-D-L-sulfoximine was a very potent inhibitor in both biosynthetic and transferase reactions. Similar to most Gram positive bacteria there was no evidence of in vivo adenylylation and the enzyme seemed to be mainly regulated by feed-back mechanism.Abbreviations PMSF phenylmethylsulfonylfluoride - TCA trichloroacetic acid - GS glutamine synthetase - MSO L-Methionine-D-L-sulfoximine - SDS-PAGE sodium dodecyl sulfatepolyacrylamide gel electrophoresis - SVPDE snake venum phosphodiesterase     

Pyranose oxidase and pyranosone dehydratase: enzymes responsible for conversion of d-glucose to cortalcerone by the basidiomycete Phanerochaete chrysosporium

Pyranose oxidase (glucose 2-oxidase) and pyranosone dehydratase were purified 27.6- and 43.9-fold respectively from mycelial extracts of the fungus Phanerochaete chrysosporium using hydrophobic interaction, anion exchange and gel filtration chromatography. The enzymes appeared substantially homogeneous on SDS-PAGE and were comprised of identical subuntis with apparent M_r values of 69 000 and 99 000 for pyranose oxidase and pyranosone dehydratase, respectively. The apparent M_r's of the native enzymes, based on equilibrium ultracentrifugation, were 308 000 and 221 000. In coupled reactions, the enzymes catalyzed conversion of d-glucose via d-glucosone (d-arabino-2-hexosulose) to the antibiotic -pyrone, cortalcerone. The latter compound was isolated as a diphenylhydrazone derivative and spectroscopically identified.Abbreviations DMAB 3-dimethylaminobenzoic acid - FPLC fast protein liquid chromatography - MBTH 3-methyl-2-benzothiazolinone hydrazone hydrochloride - PD pyranosone dehydratase - PMSF phenylmethylsulfonyl fluoride - POD pyranose oxidase     

Native phytochrome: immunoblot analysis of relative molecular mass and in-vitro proteolytic degradation for several plant species

The relative molecular mass (M_r) of the native phytochrome monomer from etiolated Cucurbita pepo L., Pisum sativum L., Secale cereale L. and Zea mays L. seedlings has been determined using immunoblotting to visualize the chromoprotein in crude extracts subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A single phytochrome band is observed for each plant species when the molecule is extracted under conditions previously demonstrated to inhibit the proteolysis of native Avena sativa L. phytochrome. A comparison among plant species indicates that the M_r of native phytochrome is variable: Zea mays=127000; Secale=Avena=124000; Pisum=121000; Cucurbita=120000. The in-vitro phototransformation difference spectrum for native phytochrome from each species is similar to that observed in vivo in each case and is indistinguishable from that described for native Avena phytochrome. The difference minima between the red- and far-red-absorbing forms of the pigment (Pr-Pfr) are all at 730 nm and the spectral change ratios (A_r/A_fr) are near unity. When incubated in crude extracts, phytochrome from all four species is susceptible to Pr-specific limited proteolysis in a manner qualitatively similar to that observed for Avena phytochrome, albeit with slower rates and with the production of different M_r degradation products. Further examination of the in-vitro proteolysis of Avena phytochrome by endogeneous proteases has identified several additional phytochrome degradation products and permitted construction of a peptide map of the molecule. The results indicate that both the 6000- and 4000-M_r polypeptide segments cleaved by Pr-specific proteolysis are located at the NH₂-terminus of the chromoprotein and are adjacent to a 64000-M_r polypeptide that contains the chromophore.Abbreviations and symbols A_minimum phototransformation difference spectrum (Pr-Pfr) minimum - Ig immunoglobulin - Mops 3-(N-morpholino)propanesulfonic acid - M_r relative molecular mass - PAGE polyacrylamide gel electrophoresis - PMSF phenylmethylsulfonyl fluoride - SDS sodium dodecyl sulfate     

Characteristics of intracellular proteolytic activities ofBacillus megaterium

Intracellular proteolytic activities ofB. megaterium KM occur soluble in the cytoplasm and periplasm and insoluble in the membrane. Two proteolytic enzymes were found in the cytoplasmic fraction by gel filtration on Sephadex G 150 and by polyacrylamide gel electrophoresis. The first enzyme called C_I was stable, had a relative molecular mass ofM _r=105000 (M=105 kg/mol) and was inhibited by EDTA and PMSF, whereas the second, designated C_II, was labile and had a relative molecular mass ofM _r=46000 (M=46 kg/mol). Because of its lability it could not be characterized in detail. In the “periplasm” only a single proteolytic enzyme P (M _r=28000;M=28 kg/mol) inhibited by EDTA could be demonstrated. The extracellular enzyme exhibited similar properties. The membrane proteolytic activity was sensitive to PMSF and EDTA. The membrane enzymes have not yet been solubilized. In cells of the mutant KM 12 that does not produce the extracellular proteinase, only one type of proteinase, in all its properties identical with the cytoplasmic proteinase C_I, could be demonstrated.     

Identification and partial purification of a heart mitochondrial membrane proteinase

Membrane-bound proteinase activity was demonstrated by a solid-phase assay system in both beef heart and rat liver mitochondria. The activity was sensitive to SH reagents and assorted proteinase inhibitors. Although stimulated by nonionic detergents, it became labile when solubilized by detergents. The proteinase activity from heart mitochondria copurified with the ADP:ATP translocator protein. Gel electrophoresis of this preparation revealed the translocator polypeptide as well as a number of minor components. In solubilized mitochondria the ADP:ATP translocator polypeptide slowly disappeared upon standing at 0°C as revealed by polyacrylamide gel electrophoresis under denaturing conditions. The loss of this polypeptide was prevented by addition of proteinase inhibitors as well as the translocator affinity ligand, carboxyatractylate. These observations confirm the presence of an integral membrane proteinase in mitochondria and suggest a structural and enzymatic interaction between the proteinase and the ADP:ATP translocator.Abbreviations PMSF phenylmethanesulfonyl fluoride - TPCK l-1-tosylamido-2-phenylethylchloromethyl ketone - TLCK 1-chloro-3-tosylamido-7-amino-l-2-heptanone - NEM N-ethylmaleimide - PCMBS p-chloromercuriphenylsulfonic acid - SDS sodium dodecyl sulfate - MOPS morpholinopropane sulfonate - [I₅₀] concentration of inhibitor required to give 50% inhibition     

Biogenesis of the chloroplast phosphate translocator

Calcium-dependent proteolysis of several polypeptides from rat brain and synaptosomal cytosol was observed including proteolysis of polypeptides of M_r 340 000 and 300 000. These latter polypeptides comigrated with high-M_r microtubule-associated proteins of microtubule preparations from brain or synaptosomal cytosol. Calcium influx into intact synaptosomes due to depolarisation with high potassium or veratridine or treatment with the ionophore A23187 did not result in Ca²⁺-dependent proteolysis of any polypeptides. This may be due to the low calcium sensitivity of the protease since no proteolysis of the M_r 340 000 and 300 000 polypeptides was seen in synaptosomal cytosal at < 10 μM free Ca²⁺.     

The cardiac gap junction protein (Mr 47,000) has a tissue-specific cytoplasmic domain of Mr 17,000 at its carboxy-terminus

The molecular weight of the heart gap junctional protein subunit was, until recently, believed to be about Mr 28,000-30,000, similar to that of other previously characterized gap junctional proteins. A larger polypeptide of about Mr 44,000-47,000, which undergoes proteolysis during isolation, has recently been proposed as the form of the heart junction protein in vivo. We show here that this entity has the same amino-terminal sequence as the previously characterized Mr 29,000-30,000 component. Thus, the cardiac junctional protein has, at its carboxy-terminus, cytoplasmic domain of Mr 17,000; this domain is absent in the liver protein. These observations provide further evidence that gap junction proteins form a highly diversified family.     

Differential expression of laminin chains in hepatic lipocytes

The lipocyte is an important source of laminin in the normal liver. We have investigated the expression of the 3 chains of laminin in isolated rat lipocytes. Both B₁ and B₂ chains, but not A, were found in medium from 5-day-old lipocyte primary cultures by immunoblotting and immunoprecipitation of ³⁵S-labeled proteins after reducing SDS-polyacrylamide gel electrophoresis. An additional polypeptide of M_r=380 000 was identified by immunoprecipitation. Under non-reducing conditions only one M_r=900 000 band was revealed. High levels of B₁ and B₂ mRNAs were also demonstrated in 5-day-old cultured lipocytes while at the time of seeding, only B₂ chain mRNAs were clearly detectable. A chain mRNA was constantly absent. These results suggest that lipocytes produce a variant form of laminin in primary culture and that the M_r=380 000 polypeptide could be unrelated to the A chain of laminin.     

Biosynthesis,processing, and extracellular release of α-l-fucosidase in lymphoid cell lines of different genetic origins

In humans, the quantity of α-l-fucosidase in serum is determined by heredity. The mechanism controlling levels of the enzyme in serum is unknown. Lymphoid cell lines derived from individuals with either low, intermediate, or high α-l-fucosidase in serum were established. Steady-state levels of intracellular and extracellular α-l-fucosidase as well as rates of synthesis and secretion of enzyme overlapped among the cell lines. Thus,_vivo} serum phenotypes were not expressed in this system. No appreciable differences in the qualitative processing of newly made α-l-fucosidase were observed among these lymphoid cell lines. Cells pulse-labeled with³⁵S-methionine from 0.25 to 2 hr had an intracellular form of enzyme with aM _r=58,000. Cells pulsed for 1.5 hr and chased for 21 hr with unlabeled methionine had an intracellular form ofM _r=60,000 and an extracellular form ofM _r=62,000. All three enzyme forms were glycoproteins with a common polypeptide chain ofM _r=52,000 but with different carbohydrate moieties. No evidence for a high molecular mass precursor form of α-l-fucosidase was found. Fucosidosis is a rare, inherited disease in which α-l-fucosidase activity in tissues and body fluids is low or absent. The mutations for fucosidosis and the serum polymorphism map separately. Lymphoid cells from two siblings with fucosidosis had 8-fold to 341-fold less intracellular α-l-fucosidase protein with 11-fold to 56-fold lower specific activities than control cells. Residual mutant enzyme was a glycoprotein with a polypeptide chain virtually the same size (M _r=52,000) as control enzyme. However, residual mutant enzyme was hypoglycosylated and hypersecreted as compared to control enzyme. This research was supported by National Institutes of Health Grant DK 32161.     

Characterization and immunocytochemical demonstration of glucocorticoid receptor using antisera specific to transformed receptor

Cytosolic and nuclear forms of the glucocorticoid receptor were characterized using immunochemical techniques. Antibodies were raised in rabbits to an M_r 58,000 fragment of the transformed (DNA-binding) glucocorticoid receptor purified from rat liver cytosol by DNA-cellulose chromatography and polyacrylamide gel electrophoresis. Antibodies reacted with the transformed receptor form in a radioimmunoassay for glucocorticoid receptor. Western blot analysis of antibody reactivity revealed a single M_r 185,000 receptor form in rat liver cytosol but a smaller M_r 85,000 form in nucleosol, indicating the M_r 85,000 form is the transformed receptor. Furthermore, western blot analysis indicates that the M_r 185,000 receptor undergoes proteolysis during receptor purification and in vitro transformation processes by generating immunochemically similar proteins of smaller molecular weights. An identical M_r 185,000 glucocorticoid receptor was detected in cytosols of four rat tissues; liver, brain, adrenal medulla, and thymus. The glucocorticoid receptor was localized to the cytoplasm and nucleus of rat adrenal medulla cells by immunohistochemistry, demonstrating the existence in vivo of the transformed receptor and translocation of the receptor from cytoplasm to nucleus.     

Unassembled polypeptides of the plastidic ribosomes in heat-treated 70S-ribosome-deficient rye leaves

The polypeptides of the subunits of 70S ribosomes isolated from rye (Secale cereale L.) leaf chloroplasts were analyzed by two-dimensional polyacrylamide gel electrophoresis. The 50S subunit contained approx. 33 polypeptides in the range of relative molecular mass (M_r) 13000–36000, the 30S subunit contained approx. 25 polypeptides in the range of M_r 13000–40500. Antisera raised against the individual isolated ribosomal subunits detected approx. 17 polypeptides of the 50S and 10 polypeptides of the 30S subunit in the immunoblotting assay. By immunoblotting with these antisera the major antigenic ribosomal polypeptides (r-proteins) of the chloroplasts were clearly and specifically visualized also in separations of leaf extracts or soluble chloroplast supernatants. In extracts from rye leaves grown at 32° C, a temperature which is non-permissive for 70S-ribosome formation, or in supernatants from ribosome-deficient isolated plastids, six plastidic r-proteins were visualized by immunoblotting with the anti-50S-serum and two to four plastidic r-proteins were detected by immunoblotting with the anti-30S-serum, while other r-proteins that reacted with our antisera were missing. Those plastidic r-proteins that were present in 70S-ribosome-deficient leaves must represent individual unassembled ribosomal polypeptides that were synthesized on cytoplasmic 80S ribosomes. For the biogenesis of chloroplast ribosomes the mechanism of coordinate regulation appear to be less strict than those known for the biogenesis of bacterial ribosomes, thus allowing a marked accumulation of several unassembled ribosomal polypeptides of cytoplasmic origin.Abbreviations L polypeptide of large ribosomal subunit - M_r relative molecular mass - r-protein ribosomal polypeptide - S polypeptide of small ribosomal subunit - SDS sodium dodecyl sulfate     

Isolation and characterization from potato tubers of two polypeptide inhibitors of serine proteinases

Two polypeptides, isolated to electrophoretic homogeneity from Russet Burbank potato tubers, are powerful inhibitors of pancreatic serine proteinases. One of the inhibitors, called polypeptide trypsin inhibitor, PTI, has a molecular weight of 5100, and inhibits bovine trypsin. The inhibitor is devoid of methionine, histidine, and tryptophan and contains eight half-cystine residues as four disulfide bridges. The second inhibitor, polypeptide chymotrypsin inhibitor II, PCI-II, has a molecular weight of 5700 and powerfully inhibits chymotrypsin. This inhibitor is also devoid of methionine and tryptophan but it contains only six of half-cystines as three disulflde bonds. Both polypeptides strongly inhibit pancreatic elastase. In immunological double diffusion assays, polypeptide trypsin inhibitor and polypeptide chymotrypsin inhibitor II exhibit a high degree of immunological identity (a) with each other, (b) with a polypeptide chymotrypsin inhibitor (PCI-I, M_r 5400) previously isolated from potato tubers, and (c) with inhibitor II, a larger (monomer M_r ~ 12,000) inhibitor of both trypsin and chymotrypsin which has also been previously isolated from potato tubers. The four polypeptide proteinase inhibitors now isolated from Russet Burbank potato tubers cumulatively inhibit all five major intestinal digestive endo- and exoproteinases of animals. The inhibitors are thought to be antinutrients that are present as part of the natural chemical defense mechanisms of potato tubers against attacking pests.     

Cis- and trans-diamminedichloroplatinum(II) binding products different tertiary structural changes on SV40 DNA

The proteases secreted into culture medium by MCF-7 breast cancer cells produced both plasminogen-dependent and -independent proteolysis, as shown by casein-polyacrylamide gel electrophoresis. All of these proteases except the largest (M_r 120,000) were retained on a benzamidine-Sepharose affinity column, a characteristic of trypsinlike proteases. Among the proteases which activated plasminogen, all except a major protease of M_r 59,000 were antigenically similar to urokinase. These urokinaselike proteases (M_r 65,000 to 25,000) were isolated on a antiurokinase-Sepharose affinity column. The findings indicate that in a stable cell line derived from a human breast cancer there are two distinct types of plasminogen activators, opening the possibility that these activator types may be modulated in separate ways.