Chick brain actin and myosin. Isolation and characterization

Brain actin extracted from an acetone powder of chick brains was purified by a cycle of polymerization-depolymerization followed by molecular sieve chromatography. The brain actin had a subunit molecular weight of 42,000 daltons as determined by co-electrophoresis with muscle actin. It underwent salt-dependent g to f transformation to form double helical actin filaments which could be "decorated" by muscle myosin subfragment 1. A critical concentration for polymerization of 1.3 microM was determined by measuring either the change in viscosity or absorbance at 232 nm. Brain actin was also capable of stimulating the ATPase activity of muscle myosin. Brain myosin was isolated from whole chick brain by a procedure involving high salt extraction, ammonium sulfate fractionation and molecular sieve chromatography. The purified myosin was composed of a 200,000-dalton heavy chain and three lower molecular weight light chains. In 0.6 M KCl the brain myosin had ATPase activity which was inhibited by Mg++, stimulated by Ca++, and maximally activated by EDTA. When dialyzed against 0.1 M KCl, the brain myosin self-assembled into short bipolar filaments. The bipolar filaments associated with each other to form long concatamers, and this association was enhanced by high concentrations of Mg++ ion. The brain myosin did not interact with chicken skeletal muscle myosin to form hybrid filaments. Furthermore, antibody recognition studies demonstrated that myosins from chicken brain, skeletal muscle, and smooth muscle were unique.     

Isolation and characterization of alpha-macroglobulin from guinea pig plasma

Guinea pig alpha-macroglobulin was purified to apparent homogeneity by sequential chromatography on Sephacryl S-300, DEAE-cellulose, and hydroxyapatite. A molecular weight of 780,000 was obtained by equilibrium sedimentation. The preparation migrated as a single band of Mr = 180,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. Rabbit antiserum raised against the final preparation partially cross-reacted with human and rat alpha-2-macroglobulins but not with rat alpha-1-macroglobulin. Guinea pig alpha-macroglobulin stimulated the amidolytic activity of trypsin towards a small substrate, but inhibited the proteolytic activity of trypsin towards remazol brilliant blue hide powder. When treated with trypsin or methylamine, four thiol groups per molecule were newly generated. The reaction with trypsin proceeded with at least at two different rates: half of the thiol groups were generated in a fast reaction and the remaining half in a slower reaction. On the other hand, such a two-step reaction was not detected in the reaction with methylamine. The methylamine-treated alpha-macroglobulin retained half the capacity to bind trypsin and its mobility in polyacrylamide gel under nondenaturing conditions remained virtually unchanged. These properties are in marked contrast to those reported for human alpha-2-macroglobulin, but resemble those of rat alpha-2- and mouse alpha-macroglobulins. The amidase activity of trypsin bound to guinea pig alpha-macroglobulin was impaired by soybean trypsin inhibitor to a much greater degree than that of trypsin bound to human or rat alpha-2-macroglobulin.     

Isolation and characterization of metallothionein from guinea pig liver

The amino acid composition, and the absorption, circular dichroism (CD) and magnetic circular dichroism spectra of a metalloprotein induced in the livers of guinea pigs by the injection of CdCl₂ are reported. The amino acid composition of this protein closely resembles that of rat liver metallothionein (MT). We show that this protein has spectroscopic properties that closely follow the behaviour previously reported for several other cadmium-containing metallothioneins in its spectral response to changes in pH, and to the addition of cadmium and copper(I). Dramatic changes are observed in the CD spectrum during the addition of copper(I); it is suggested that these changes are the result of the formation of a mixed Cu(I)/Cd(II) cluster that forms in the α domain once the β domain has been saturated with Cu(I). These results are of particular importance in the characterization of this protein as belonging to the metallothionein class of proteins, as spectral changes of this type are directly related to the displacement of Cd²⁺ and Zn²⁺ from the two, thiolatecluster binding sites that are amongst the unique properties of mammalian metallothioneins. It is demonstrated that the CD spectrum provides a sensitive indicator of the presence of these special metal binding sites by indicating changes in the binding geometry and stoichiometry in response to an incoming metal. These results indicate that the guinea pig liver metallothionein induced by injections of CdCl₂ uses the same α and β type of clusters for cadmium binding as rat liver Cd, Zn-MT, even though there are minor differences in the amino acid composition between the guinea pig and rat liver proteins.     

Protransglutaminase E from guinea pig skin. Isolation and partial characterization

We have isolated protransglutaminase E, the zymogen form of epidermal transglutaminase E, from the skin of the adult guinea pig. This zymogen is the source of the large majority of soluble transglutaminase activity of skin. A molecular weight value for protransglutaminase E of 77,800 +/- 700, estimated by sedimentation equilibrium, is in close agreement with the apparent values determined by exclusion chromatography and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Treatment of the proenzyme with dispase, proteinase K, trypsin, or thrombin produces active enzyme. The enzyme, transglutaminase E, formed by the action of dispase, was observed to exist in the native state as a molecule indistinguishable in size from the zymogen. Under denaturing conditions, however, the enzyme dissociates into two fragments with molecular weights of 50,000 and 27,000. The observation that reducing agents are not needed for this dissociation suggests a noncovalent association of the two peptide chains in the native enzyme. Evidence that the catalytically essential -SH group of the enzyme residues in the Mr 50,000 fragment and that only the Mr 27,000 fragment possesses an unmasked amino terminus provides the basis for a proposed model of zymogen activation. Whether the noncatalytic fragment plays a role in catalysis is not known because separation of the fragments of native enzyme was not achieved.     

Isolation and characterization of corticostatic peptides from guinea pig bone marrow.

Three corticostatic peptides (GP-CS1, GP-CS2 and GP-CS3) were purified from extracts of guinea pig bone marrow. Each was identified on the basis of their ability to inhibit the secretion of corticosterone by isolated rat adrenal cells stimulated by ACTH. GP-CS1 and GP-CS2 were found to be 31 residues in length, rich in arginine and to have six cysteines typical of the corticostatin/defensin family of peptides previously purified from phagocytic cells of the immune system. GP-CS1 was found to be identical to GP-CS2 except for having a leucine at position 21 instead of isoleucine. GP-CS3 was also found to be rich in arginine and cysteine but structurally distinct from the other peptides. A combination of endoprotease mapping, ion-spray mass spectrometry and gas-phase sequencing revealed that GP-CS3 was a novel homo-dimer consisting of two 13 amino acid residue subunits cross-linked through eight cysteines in an anti-parellel configuration.     

Isolation and biochemical characterization of nuclei from immature and mature guinea pig granulocytes

Intact nuclei were isolated in high yield from enriched fractions of immature and mature guinea pig granulocytic leukocytes. These nuclei were used to determine whether any changes in synthesis and content of nuclear proteins accompany the striking increase in chromatin condensation and the nuclear lobation which occur during granulocyte maturation. The results indicate that the synthesis of nuclear proteins and the nuclear RNA content decrease markedly during granulocyte maturation. The incorporation of l-[U-¹⁴C]leucine into the acid-soluble histone-rich fraction of chromatin from immature cells is about 25 times that of mature cells, and the incorporation into the acid-insoluble, nonhistone proteins of chromatin from immature cells is about 6 times that of mature cells. It appears that there is very little quantitative change with respect to the protein components of nuclei from immature and mature granulocytic leukocytes. No significant differences in the amounts of histone, nonhistone protein, or phosphoprotein between nuclei of immature and mature granulocytes could be detected. No major differences in gel electrophoretic patterns of histones or nonhistone proteins could be detected. The fact that the amount of the chromatin proteins remains relatively constant during cell maturation in spite of the pronounced decrease in the rate of synthesis suggests that the rate of turnover of these proteins decreases significantly as the maturation of granulocytic leukocytes proceeds.     

Preparation and characterization of isolated parenchymal cells from guinea pig liver

1. A method is described for the preparation of isolated cells from guinea pig liver. This involved perfusion in situ, in the non-physiological direction, with collagenase. 2. The cell yield was 20--30%, comparable with those from the livers of other species. 3. The ratio of lactate dehydrogenase to glutamate dehydrogenase in the cells was similar to that in vivo, indicating that there was negligible leakage of cytoplasmic enzymes. 4. The concentrations of K+ and adenine nucleotides were initially lower than in the perfused liver; normal values were obtained on incubation, particularly in the presence of substrate. 5. The L-lactate: pyruvate ratio is 16:1, close to established values. The total beta-hydroxybutyrate: acetoacetate ratio indicates that the mitochondrial redox state is more oxidised than in the perfused liver, but the intracellular ratio is similar to that of the intact liver. 6. Rates of gluconeogenesis and ureogenesis, are within the physiological range. Maximal gluconeogeneis from L-lactate was preceded by a lag period. L-lysine stimulated glucose production from L-lactate but did not abolish the lag phase. 7. The effects of aminooxyacetate and octanoate on L-lactate gluconeogenesis were similar to those in the perfused liver.     

Isolation and characterization of one isoform of actin from cultured soybean cells

Cultured soybean cells (SB-1 cell line) were plasmolyzed and lyophilized. Extraction of the dried powder and fractionation yielded a polypeptide with the following key properties: (a) it has a molecular weight of approximately 45,000 and an isoelectric point of approximately 5.9; (b) it is immunologically cross-reactive with rabbit antibodies affinity purified against the Mr 45,000 polypeptide of calf thymus actin; (c) it is eluted from a DEAE-cellulose column at the same ionic strength as Acanthamoeba actin; (d) it yields peptide maps, after limited proteolysis with V8 protease, similar if not identical to those of rabbit muscle actin; and (e) it binds specifically to deoxyribonuclease I. These molecular and binding properties indicate that we have purified one isoform of actin from soybean cells.     

Isolation and characterization of actin from Entamoeba histolytica

Actin has been identified and purified partially from trophozoites of Entamoeba histolytica HMI-IMSS by a procedure that minimizes proteolysis. In cellular extracts, Entamoeba actin would copolymerize with muscle actin, but would not bind to DNase I or form microfilaments. Fractionation of the extracts by DEAE-cellulose and Sephadex G-150 chromatography yielded a purified actin that would copolymerize with rabbit skeletal muscle actin or polymerize alone into long filaments at 24 degrees C upon addition of 100 mM KC1 and 2 mM MgCl2. These filaments are not cold-stable and will depolymerize at 4 degrees C in 1 or 2 h. Entamoeba actin filaments bind phallotoxin with the same affinity as muscle actin and decorate with rabbit skeletal muscle heavy meromyosin. Entamoeba actin filaments activate the Mg2+ ATPase of heavy meromyosin to the same Vmax as muscle actin, but the Kapp is 2.8 times higher. Entamoeba actin is a single species with a slightly higher molecular weight than muscle actin (45,000) and a more acidic pI (5.4). The purified actin does not bind to DNase I, produce inhibition of the enzymatic activity, or block the binding of muscle actin. Comparison of the peptides obtained by limit digest with protease V8 from Staphylococcus aureus shows sequences with common mobility between alpha-actin and Entamoeba actin, but additional peptides are present which may account for the different properties of the Entamoeba actin. Finally, in vitro translation of mRNA from trophozoites produces a single polypeptide equivalent to the molecule purified from Entamoeba extracts.     

Isolation and partial characterization of intestinal calcium-binding proteins from the cow, pig, horse, guinea pig, and chick.

1. Intestinal calcium-binding proteins have been isolated in high purity from mucosal tissue of the cow, pig, horse, guinea pig, and chick. The proteins from all species exhibit rapid, although not identical, electrophoretic mobilities and possesses high affinities for calcium. 2. The intestinal calcium-binding proteins of mammalian origin exhibit a molecular size of approx. 11 000 by calibrated gel filtration and 9000 on the basis of amino acid composition. The analogous chick protein was found to be about 27 000-28 000 molecular weight by these methods. 3. The amino acid composition of each intestinal calcium-binding protein has been determined and indicates a considerable degree of similarity, especially among the mammalian species. 4. Immunoassay procedures have failed to show any species cross-reactivity when tested against antiserum prepared in response to either the bovine or chick intestinal calcium-binding protein.     

Outer acrosomal membrane of guinea pig spermatozoa: Isolation and structural characterization

We describe a protocol to isolate a highly enriched fraction of outer acrosomal membrane from guinea pig spermatozoa and present new data on the ultrastructure of this membrane domain. Cauda epididymal spermatozoa were suspended into a low ionic strength buffer and subjected to brief homogenization; this stripped the plasma membrane from the spermatozoa and severed the acrosomal apical segment from the spermatozoon. The crescent-shaped apical segments retained the outer acrosomal membrane and specific components of the acrosomal matrix. Enriched fractions of apical segments were isolated on discontinuous sucrose gradients and the outer acrosomal membrane purified by subsequent centrifugation onto Percoll density gradients. The isolated outer acrosomal membrane did not form vesicles, but instead rolled up into spiral sheets. Both thin section and negatively stained specimens revealed a paracrystalline arrangement of filaments associated with the luminal surface of the membrane. The isolated outer acrosomal membrane revealed a limited number of polypeptides by SDS-PAGE, and the polypeptide pattern was distinct from the plasma membrane fraction. The isolated acrosomal membranes possessed no oubain sensitive Na+, K+-ATPase activity, whereas about 20% of the ATPase activity of the plasma membrane enriched fraction was inhibited by oubain. The potential function of the structural differentiations of the outer acrosomal membrane in the membrane fusion events of the acrosome reaction is discussed.     

Induction of DNA fragmentation in chronic B-lymphocytic leukemia cells

Chronic lymphocytic leukemia of B cell type (B-CLL) is a neoplastic disorder characterized by the accumulation of small resting lymphocytes in the periphery. The phenotype of these cells suggests that they are "frozen" at an early stage of maturation. Glucocorticoid hormones are commonly used to treat patients with B-CLL, resulting in a reduction in the peripheral lymphocyte count by an undefined mechanism. Here we report that glucocorticoids stimulate DNA fragmentation characteristic of a suicide process known as apoptosis or programmed cell death (PCD) in suspensions of cells from patients with B-CLL. The effects can be mimicked by Ca2+ ionophore and involve a sustained increase in the cytosolic Ca2+ concentration. Specific antibodies binding to membrane-associated IgM on the leukemic cells can also induce PCD by a similar mechanism. Phorbol esters block DNA fragmentation and cell killing in response to all of the agents, suggesting that activation of protein kinase C desensitizes the cells to PCD. Targeting the B-CLL cells with antibodies that induce an unbalanced, sustained Ca2+ increase may therefore represent a rational strategy for the destruction of leukemic cells.     

Isolation and characterization of pig enamelins.

Enamel proteins were extracted from pig developing enamel by sequential extraction procedures. Two proteins identified as enamelins by slab-gel electrophoresis (Mr 67,000 and 63,000) were separated from amelogenins by gel sieving and ion-exchange chromatography. Their enamelin characteristic was confirmed by hydroxyapatite-binding studies and amino acid analysis. Degradation of extracted enamel proteins was also studied in vitro. The larger of the two enamelins appeared to be resistant to degradation by endogenous enamel proteinases. Hydroxyapatite showed strong binding with the enamelins, but did not prevent the degradation of the Mr-63,000 enamelin. These results indicate that at least one high-Mr enamelin in pig developing enamel is a source of enamelin breakdown products.