The characterization of actin associated with postsynaptic membranes from Torpedocalifornica

SDS-polyacrylamide gel electrophoresis of acetylcholine receptor from $\text{Torpedo}\text{californica}$ electroplax membrane fragments shows, in addition to the four receptor subunits of 40,000, 50,000, 60,000 and 65,000 daltons, other components of apparent molecular weights 43,000, 47,000 and 90,000 daltons. In this study deoxyribonuclease I inhibitory activity has been used to identify actin in $\text{Torpedo}\text{californica}$ receptor-enriched membranes and affinity chromatography on a deoxyribonuclease I agarose column has been used to purify this protein from the membrane preparations. In addition the membrane protein components have been analyzed by electrophoresis on a series of SDS-polyacrylamide gels of varying acrylamide concentrations. Evidence is presented that actin is a component of most preparations of receptor-enriched membrane fragments, having an apparent molecular weight of 47,000 daltons, and is distinct from the 43,000 dalton protein.     

Inhibitory effect of iron on the uptake of lead by erythrocytes.

It is well known that more than 90% of the lead found in blood is associated with the erythrocytes. The present $\text{in vitro}$ experiments show that the uptake of lead-203 by rabbit erythrocytes is inhibited by the presence of non-radioactive lead or iron or by reduction of the incubation temperature. The inhibitory effect of iron on radioactive lead uptake by erythrocytes is also demonstrable $\text{in vivo}$.When lead-203 is incorporated into erythrocytes $\text{in vitro}$, about 10% of the radioactivity is attached to the membrane and the remainder is found in the cytoplasm associated with hemoglobin and an unidentified low molecular weight intracellular component. In the presence of 25 μg/ml of added iron (Fe⁺⁺⁺) the uptake of radioactive lead by erythrocytes is reduced to 21.7±5.1% and membrane binding accounts for approximately 5% of this total. Chromatographic analyses of hemolysates show that the reduction in cytoplasmic labeling is directly related to decreased lead binding to the low molecular weight component, since hemoglobin binding remains unchanged.This work suggests that in addition to the interaction between iron and lead which occurs during the biosynthesis of heme, these metals may directly compete for specific erythrocyte binding sites.     

Absence of ferric enterobactin receptor modification activity in mutants of Escherichia coli K-12 lacking protein a.

The modification activity for the ferric enterobactin receptor in the Triton X-100 solubilized outer membrane of $\text{Escherichia}\text{coli}$ K-12 was adsorbed to a column of $\text{p}$-aminobenzamidine-//-sepharose and eluted with free benzamidine. Recombination of the dialyzed eluate with the filtrate from the column reinstituted conversion of the receptor from 81K to 81K¹, the latter exhibiting an apparent molecular weight of 74,000 daltons in sodium dodecyl sulfate polyacrylamide gel analysis. The eluate from the $\text{p}$-aminobenzamidine column was shown to contain a component, coincident on gels with both protein and modification activity, which by mutational and other analyses appears to be identical with protein $\text{a}$ of the outer membrane.     

Purification of a water-soluble Mg2+-ATPase from human erythrocyte membranes

A water-soluble Mg²⁺-ATPase previously reported (White, M.D. and Ralston, G.B. (1976) Biochim. Biophys. Acta 436, 567–576) has been purified from human erythrocyte membranes. The purified enzyme has a molecular weight of 575 000; the apparent minimum molecular weight was 100 000, corresponding to a soluble protein of the component 3 region. The $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ value for ATP was 1 mM and apparent $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for Mg²⁺ was 3.6 mM. By means of histochemical activity staining in acrylamide gels it was shown that the purified ATPase preparation could be inhibited by Cd²⁺ and Zn²⁺ salts, $\text{p-}\text{chloromercuribenzoate}$ and $\text{N-}\text{ethylmaleimide}$, known inhibitors of membrane endocytosis.     

The stereospecific d-glucose transport protein in cholate extracts of human erythrocyte membranes Molecular sieve chromatography and estimation of molecular weight

Cholate extracts of human erythrocyte membranes (Lundahl, P., Acevedo, F., Fröman, G. and Phutrakul, S. (1981) Biochim. Biophys. Acta 644, 101–107) were fractionated by molecular sieve chromatography on Sepharose 6B, and the size and molecular weight of the active d-glucose transporter were estimated. The eluent contained 10 or 12.5 mM cholate, since higher concentrations inactivated the glucose transporter, and lower concentrations resulted in aggregation. The chromatographic distribution of the transport activity was reproducible, but was broader than one would expect for a homogeneous component. In the presence of 20 mM EDTA and 5 mM dithioerythritol, a combination which affords a highly stable transport activity, a molecular weight of $\text{400 000 ± 20 000}$ (Stokes' radius 5.9 nm) was estimated for the smallest active component. This value represents an upper limit, since the molecular weight of a non-spherical component would have been overestimated, and since bound cholate was calculated to represent about 12% of the molecular weight. The activity was completely recovered upon rechromatography. In 10 mM EDTA and 10 mM 2-mercaptoethanol, the estimated molecular weight of the smallest active component was $\text{210 000 ± 15 000}$, and this component was not stable upon rechromatography in 10 mM EDTA and 10 mM 2-mercaptoethanol. In the absence of chelating and reducing agents, cholate extracts from membranes which had been kept for 5 days at 4°C showed three additional active components smaller than 200 000 in molecular weight. Most of the phospholipids eluted later than the active components of molecular weight 400 000 or 210 000, in all experiments. Electrophoretic analysis in dodecyl sulfate of the chromatographic eluents indicates that at least one of the band 3-polypeptides (nomenclature according to Steck, T.L. (1974) J. Cell Biol. 62, 1–19) is a component of the active transporter. This band 3-polypeptide, which we denote 3.3, has an apparent molecular weight of 88 000. The stable transporter of molecular weight 400 000 might be a tetramer of the 3.3-polypeptide. Alternatively, a dimer of the 3.3-polypeptide in complex with lipids might account for this molecular weight. If the 3.3-polypeptide is the transporter subunit and if it binds cytochalasin B with high affinity ($\text{1.8 · 10}{}^5$ sites/cell) the recovered activity per 3.3-polypeptide is around 40% A degradation product of the 3.3-component (possibly a 4.5-component) might account for the unstable active transporter of molecular weight 210 000.     

Receptors for Dolichos biflorus agglutinin: New cell surface markers on a spontaneous leukemia

$\text{Dolichos biflorus}$ agglutinin (DBA), which is specific for terminal α-N-acetylgalactosamine, bound to a spontaneous leukemia cell of $\text{GRS}\text{A}$ mice, but not to lymphoid cells of the host. The DBA receptors were isolated from the leukemia cell labeled with [³H]-galactose after detergent solubilization and affinity chromatography on DBA-agarose. The major component of the receptors migrated as a glycoprotein of apparent molecular weight 100,000 upon SDS gel electrophoresis. Alkaline treatment degraded the glycoproteins, releasing oligosaccharides of molecular weight around 1,000.     

Regulation of protein synthesis in rabbit reticulocyte lysates: Separation of heme regulated protein kinase into a high and a low molecular weight species

Protein synthesis in rabbit reticulocyte lysates is regulated by heme. In heme deficiency, a heme regulated protein kinase (HRI) is activated that phosphorylates initiation factor eIF-2. Consequently, eIF-2 is inactivated. Results described in this report show that HRI exists in crude and highly purified preparations in two forms; a high molecular weight component which sediments at a sedimentation co-efficient of 14–15S and a previously described 5.8S component (Ranu, R. S. and London, I. M. (1976) $\text{Proc}\text{.}\text{Natl}\text{.}\text{Acad}\text{.}\text{Sci}\text{.}\text{USA}$ 73, 4349–4353). The 14–15S HRI selfphosphorylates poorly and undergoes dissociation into the 5.8S component via an intermediate of 8.5–9S. The 5.8S HRI, on weight basis, is about 5–10 times more active than the 14–15S HRI. In addition, a phosphoprotein phosphatase has been detected in lysates that dephosphorylates selfphosphorylated HRI. This observation suggests that phosphate on HRI turns over. These findings may be relevant ot the mechanism of activation and inactivation of HRI in the absence and presence of heme $\text{in}\text{situ}$.     

Outer membrane of Escherichia coli K-12: Tsx mutants (resistant to bacteriophage T6 and colicin K) lack an outer membrane protein

$\text{Tsx}$ mutants of $\text{Escherichia coli}$ are fully resistant to a set of T6-like bacteriophage and are resistant to colicin K. We demonstrate that these mutants are missing an outer membrane protein (the tsx-protein) of molecular weight 32,000 as measured by SDS-polyacrylamide gel electrophoresis. $\text{Tsx}$ mutants are receptor mutants which are unable to absorb either the bacteriophages or the colicin and the loss of receptor function can be demonstrated using outer membrane preparations.We suggest that the tsx-protein is the receptor for both the bacteriophage and colicin.     

Microvillus membrane vesicles from pig small intestine Purity and lipid composition

Microvillus membrane vesicles from pig small intestine were isolated by a method based on hypotonic lysis, Mg²⁺ aggregation of contaminants and differential centrifugation. The purity of the membrane vesicles were established by measuring the activity of marker enzymes and the RNA and DNA content. The membranes were found free of contamination by other subcellular membrane fragments, except for a minor contamination with basolateral plasma membranes. The lipid composition was established and, based on weight percentage, the membrane contained neutral lipids, phospholipids, neutral glycolipids and gangliosides in the weight ratio of 18:50:29:2%. The amount of individual phospholipids and glycolipids were quantitated. Phosphatidylethanolamine, -choline, -serine, -inositol and sphingomyelin made up 17,17,6,5 and 5%, respectively of the total lipid. The major glycolipids were two monohexosylceramides containing glucose and galactose as the carbohydrate component, a dihexosylceramide containing galactose as the only carbohydrate component and two pentahexosylceramides containing fucose, galactose, glucose and hexosamine (either $\text{N-}\text{acetylglucosamine}$ or $\text{N-}\text{acetylgalactosamine}$) in the molar ratio of 1:2:1:1.     

Identification of the outer membrane protein of E.coli that produces transmembrane channels in reconstituted vesicle membranes

Outer membrane of $\text{Escherichia}$$\text{coli}$ allows a rapid diffusion of saccharides of molecular weights less than 550. This permeability property could be restored in vesicle membranes reconstituted from isolated phospholipids, lipopolysaccharide, and an outer membrane protein. The active protein aggregates were isolated from the insoluble material left after solubilization of cell envelope of $\text{Escherichia}$$\text{coli}$ B with sodium dodecyl sulfate at 35°. Analysis by acrylamide gel electrophoresis, isoelectric focusing and amino terminal amino acid determination revealed that only a single species of protein, with a molecular weight of 36,500 forms the oligoprotein aggregates which produces diffusion channels.     

In vitro formation of assimilatory nitrate reductase: presence of the constitutive component in bacteria

Cell-free extracts of a selected group of bacteria which are capable of metabolyzing dinitrogen and/or nitrate contain a soluble form of the constitutive component which is active in the $\text{in}\text{vitro}$ formation of NADPH-nitrate reductase when mixed with extracts of $\text{N.}\text{crassa}\text{,}\text{nit}\text{-}\text{1}$. The constitutive component in these extracts is dialyzable and is insensitive to trypsin and protease. The constitutive component which substitutes for the absence of the $\text{nit}\text{-}\text{1}\text{gene product in the}\text{in}\text{vitro}$ formation of NADPH-nitrate reductase is postulated to be a low molecular weight cofactor or polypeptide and is shown to be present in a number of unrelated bacteria.     

Characterization of the membrane matrix derived from the microsomal fraction of rat hepatocytes

A highly purified membrane preparation derived from the microsomal fraction of rat hepatocytes has been chemically characterized and fractionated by means of gel filtration. The preparation has been freed of ribosomes and intravesicular protein and has a composition on a w/w basis of 52.1% protein, 45.0% phospholipid, 2.9% carbohydrate and no RNA. $\text{97 ± 2\%}$ of the total membrane phosphorus is accounted for as phospholipid phosphorus.Determination of the molecular weight distribution of the constituent polypeptides by sodium dodecyl sulfate-polyacrylamide gel electrophoresis gave values ranging from 171 000 to 16 000 for the major classes of proteins. Although several membrane glycoproteins have been identified, the most prominent species has an apparent molecular weight of 171 000, 40% of the total microsomal protein is present' in the 49 000–60 000 molecular weight region. Examination of the intrinsic polypeptide composition of membranes obtained from smooth and degranulated rough endoplasmic reticulum revealed no detectable qualitative differences.Sodium dodecyl sulfate-solubilized microsomal membrane proteins were separated by gel filtration into much simplified molecular weight classes, some of which showed predominantly a single electrophoretic component. Amino acid analysis of individual fractions showed a noticeable trend toward a decreasing ratio of acidic to basic residues with decreasing molecular weight.Membrane phosphorus was distributed between two chromatographic fractions: one containing the membrane phospholipid (97% of the total) as well as essentially all the cholesterol, the other, at the inclusion volume of the gel filtration system, containing small molecular weight species (3% of the total phosphorus). The absence of a ribonuclease-resistant RNA component eluting near the void volume clearly distinguishes the microsomal membrane from the nuclear envelope.     

The DCCD-binding polypeptide alone is insufficient for proton translocation through F0 in membranes of Escherichia, coli

In contrast to membrane vesicles of wild-type strains which become leaky to protons on removal of the F₁ ATPase, those of the mutant $\text{Escherichia}\text{,}\text{coli}\text{,}\text{N}{}_{\mathrm{I44}}$, which lacks the F₁ ATPase, can maintain a proton gradient. A normal N,N′-dicyclohexylcarbodiimide (DCCD)-binding polypeptide is present in the F₀ portion of the ATPase complex of the mutant. However, the 19000 molecular weight component of F₀ is absent. We conclude that the latter polypeptide, in addition to the DCCD-binding polypeptide, is required for a functional proton channel in F₀.     

Glycoproteins and glycolipids of oxyntic cell microsomes II. Glycopeptides and glycolipids

Glycosylated compounds associated with the carbohydrate-rich tubular membrane system of the oxyntic cell were investigated. Two glycopeptide fractions, designated Peaks A and B, were isolated from pronase digests of bullfrog oxyntic cell microsomes. Molecular sieve chromatography and cellulose acetate electrophoresis revealed that, although somewhat heterogeneous, each peak was composed primarily of glycopeptides with similar molecular weights and net charge densities. Peak B glycopeptides had a mean molecular weight of about 6000 and contained 70% of the recovered carbohydrate in the following molar ratios: hexose, 1.00; $\text{N-}\text{acetylhexosamine}$, 0.71; fucose, 0.61; sialic acids, <0.03. Peak a glycopeptides were considerably larger (molecular weight approx. 100 000) and contained carbohydrates in molar ratios similar to those of Peak B. In both peaks galactose and $\text{N-}\text{acetylglucosamine}$, respectively, were the predominant hexose and amino sugar isomers.The glycolipid content of bullfrog oxyntic cell microsomes was assessed by qualitative and quantitative thin-layer chromatography. The most abundant glycolipids were monoglucosylceramides (0.098 mole/mole phospholipid) and monogalactosylceramides (0.046 mole/mole phospholipid). Small quantities of sulfatides and gangliosides were also present.A compilation of available data regarding the chemical composition of the microsomes revealed that these membranes resemble plasma membranes in having high molar ratios of cholesterol to phospholipid (approx. 1.0) and large quantities of carbohydrate (225 μg/mg protein). The possible significance of these compositional features in protecting the oxyntic cell is discussed.     

In vitro synthesis of photosynthetic membranes: I. Development of photosystem I activity and cyclic photophosphorylation

First successful $\text{in vitro}$ synthesis of functional photosynthetic phosphorylating membrane is reported. Etioplasts, highly enriched in cytoplasmic and plastid proteins, isolated from etiolated Cucumber cotyledons pretreated with kinetin and gibberellic acid, and illuminated in a cofactor fortified medium showed commencement of chlorophyll (Chl) synthesis immediately after illumination from exogenous δ-aminolevulinic acid, while photosystem I (PS I) activity commenced 15 min after the onset of illumination. When cotyledons pretreated with kinetin and gibberellic acid were illuminated directly, there was a lag phase of 30 min before the commencement of Chl synthesis and PS I activity developed after 1 h of illumination. In plastids developed both $\text{in vivo}$ and $\text{in vitro}$, the electron flow from dichlorophenolindophenol to methyl-viologen was coupled to phosphorylation as observed by an increase in the electron transport rate on the addition of uncouplers. Analysis of polypeptide profiles of the greening plastids $\text{in vitro}$ showed the disappearance of many higher molecular weight proteins during greening. Polypeptides of molecular weight 32, 20.5, 19.5 K absent in etioplasts appeared as distinct bands after 4 h of greening $\text{in vitro}$.     

A cobalt porphyrin containing protein reducible by hydrogenase isolated from Desulfovibrio desulfuricans (Norway)

A cobalt-porphyrin containing protein has been isolated from the sulfate-reducer $\text{Desulfovibrio desulfuricans}$ (Norway). This violet-colored protein has a molecular weight of approx. 13,000 daltons and contains 1 cobalt atom/molecule. The apo-protein was estimated to contain 104 amino-acid residues giving a molecular weight of 11,000 daltons. The UV-visible absorption spectrum of the protein exhibiting maxima at 588,418 and 280 nm with a shoulder at 550 nm is characteristic of metalloporphyrin proteins. The molar extinction coefficients of the cobalt-protein at 588, 418 and 280 nm are 31,330 , 64,670 and 17,200 respectively and its absorbance ratio $\text{A}{}_{280}\text{A}{}_{588}$ is 0.54. The protein is reduced by dithionite giving a blue-colored reduced form. Important spectral modifications of the chromophore occurred during the reduction including a shift of the Soret peak from 418 to 381 nm and a shift of the α band in the opposite direction from 588 to 593.5 nm. The Co-protein was slowly reduced by the hydrogenase from $\text{D.desulfuricans}$ under hydrogen in the presence of cytochrome C₃. The reported data suggest that the redox states of the cobalt center of this new electron carrier correspond to the Co(III) and Co(II) states.     

Molecular characterization of sinistirin

The molecular weight distribution of sinistrin (Inutest ®, Laevosan Ges., Linz, Austria), determined by analytical gel-permeation chromatography, using narrow fractions ($\text{M}$_w$\text{M}$_n< 1.07) obtained by preparative gel-permeation chromatography, covered the range 800–16,000 with $\text{M}$_n  2,500 and $\text{M}$_w  3,500. From viscosity measurements on dilute, aqueous solutions, the relation [η]  0.28 X M^0.3 was obtained, indicating a branched molecular structure; the largest molecules can be described by a sphere with r  23 Å. Comparison of the content of glucose and reducing sugars in the fractions with the molecular weight determined by vapour-pressure osmometry indicated that a glucose end-group is present in the majority of the molecules. The percentage of glucose end-groups is higher in the fractions of lower molecular weight. From this finding, speculations on the biosynthesis of sinistrin are made. The specific optical rotation of sinistrin fractions decreases linearly with 1/$\text{M}$_n.     

Purification of the insecticidal toxin from the parasporal crystal of Bacillus thuringiensis subsp. kurstaki

The insecticidal toxin of $\text{Bacillus}\text{thuringiensis}$ subsp. $\text{kurstaki}$ was isolated from parasporal crystals. The toxin, which is stable for several months, is a glycoprotein with an apparent molecular weight of 68,000 that is generated upon solubilization and activation of a higher molecular weight protoxin (MW_app = 1.3 × 10⁵) at alkaline pH. The toxin was purified by gel filtation and anion exchange chromatography and its molecular weight was established by gel filtration chromatography and SDS polyacrylamide gel electrophoresis.     

Compositional characteristics of a chloroform/methanol soluble protein fraction from spinach chloroplast membranes

Extraction of an aqueous suspension of spinach chloroplast lamellae with a chloroform/methanol mixture leads to solubilization of about $\text{1}\text{3}$ of the total membrane protein. Amino acid analysis of the chloroform/methanol-soluble protein shows that this fraction is largely enriched in the hydrophobic residues proline, leucine, alanine and phenylalanine and considerably depleted in polar amino acids, namely lysine and arginine. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the solubilized material reveals the presence of a variety of low molecular weight polypeptides (molecular weight ? 25 000), with more than 50% of the total fraction being contributed by a 25 000 dalton band. This band, which accounts for about 25% of the total chloroplast lamellar protein, has recently been identified as the main component of the light-harvesting chlorophyll-protein complex. The physiological role of most of the chloroform/methanol-soluble protein fraction is not known at present. From its chemical properties and apparent biological inertness, we propose that it plays mainly a structural role in situ, interacting with the lipid moiety of the chloroplast membrane. The material insoluble in the aqueous chloroform/methanol mixture is largely enriched in manganese, iron, cytochrome and water-soluble proteins, such as chloroplast coupling factor and ribulose diphosphate carboxylase.