Stimulation by interferon of induction of differentiation of human promyelocytic leukemia cells

F₁-ATPase was isolated from yeast $\text{S.}\text{cerevisiae}$. The constituent subunits 1 and 2 were purified by gel permeation chromatography, and their amino acid compositions determined. Both subunits have a similar composition except for $\text{1}\text{2}$ cystine, methionine, leucine, histidine, and tryptophan. When F₁ is treated for three hours with 5′-p-[³H]fluorosulfonylbenzoyl adenosine in dimethylsulfoxide, 90% of the activity is lost. Disc gel electrophoresis of the modified complex showed that over 90% of the label was associated with subunit 2. A labelled peptide from a $\text{S.}\text{aureus}$ digest of subunit 2 was isolated and sequenced. It had the following amino acid sequence: His-Try¹-Asp-Val-Ala-Ser-Lys-Val-Gln-Glu, whereby Tyr¹ is the modified amino acid residue. This sequence shows homology to other sequences obtained from maize, beef heart, and $\text{E.}\text{coli}$ F₁-ATPases.     

Heterogeneity of large subunits of ribulose-1,5-bisphosphate carboxylase from Hydrogenomonas eutropha

Homogeneous ribulose-1,5-bisphosphate carboxylase purified from autotrophically grown $\text{Hydrogenomonas eutropha}$ can be dissociated with sodium dodecylsulfate into small 15,000-dalton subunits and large 56,000- and 52,000-dalton subunits (the latter in a mole ratio of 5:3). The overal mole ratio of small to large subunits is 1.08. Considering the molecular weight of the native enzyme (516,000), the simplest quarternary structure of this enzyme consists of 8 large (mixed) and 8 small subunits. Isolation of the enzyme from cells under conditions that should minimize proteolysis has no effect upon the observed heterogeneity of the large subunits.     

Biogenesis of the cytochrome bc1 complex in isolated rat hepatocytes

Isolated rat hepatocytes were labeled with [³⁵S]methionine in the absence or presence of cycloheximide or chloramphenicol. The cytochrome $\text{bc}$₁ complex was isolated from labeled cells by a micromethod and analyzed by SDS-polyacrylamide gel electrophoresis and fluorography. All subunits except the two smallest, subunits VII and VIII, were labeled in the absence of translational inhibitors. In the presence of cycloheximide only subunit III (molecular weight, 30 000) was labeled. This polypeptide, identified as an apo-cytochrome b, was weakly labeled with [³⁵S]methionine in the presence of cycloheximide, indicating a strict dependence of cytoplasmically synthesized products for its assembly. In the presence of chloramphenicol, labeling was inhibited only in subunit III.     

Binding of glycoproteins of microsomal and Golgi membranes to lectins.

Four subunits of the acetylcholine receptor molecule, obtained from the electric organ of $\text{Torpedo ocellata}$, have been isolated using polyacrylamide gel electrophoresis, and assayed by titration with a fluorescent lanthanide, terbium, and by affinity-labeling with $\text{p}$-($\text{N}$-maleimido)benzyl [trimethyl-³H] ammonium iodide. The site with which the activator-analogue affinity label reacts, as well as the terbium-binding sites, are mainly associated with the smallest of the subunits of an apparent molecular weight of 40,000. Calcium competes with terbium for these binding sites. The affinity for terbium is the same in the intact molecule as in the subunit (K_Tb ? 19 ± 1 μM), but the affinity for calcium decreases by a factor of 4 (K_Ca ? 4 mM) in the subunit. Hydrolysis of the receptor, catalyzed by trypsin and chymotrypsin, to peptides with an apparent molecular weight of 8000 or less, does not affect the terbium-binding sites. These experiments indicate that the binding sites for neural activators and for calcium are associated with the same subunit, and that the terbium- and calcium-binding sites reflect structural properties of the polypeptide chain rather than the three-dimensional structure of the protein.     

Dephosphorylation of 40S ribosomal subunits by phosphoprotein phosphatase activity from rabbit reticulocytes.

Phosphoprotein phosphatase activities which remove phosphoryl groups from ribosomal protein have been partially purified from rabbit reticulocytes by chromatography on DEAE-cellulose. Two major peaks of phosphoprotein phosphatase activity were observed when 40S ribosomal subunits, phosphorylated $\text{in vitro}$ with cyclic AMP-regulated protein kinases and (γ-³²P)ATP, were used as substrate. The phosphatase activity eluting at 0.14 M KCl was characterized further using ribosomal subunits phosphorylated $\text{in situ}$ by incubation of intact reticulocytes with radioactive inorganic phosphate. Phosphate covalently bound to 40S ribosomal subunits and 80S ribosomes was removed by the phosphatase activity. The enzyme was not active with phosphorylated proteins associated with 60S ribosomal subunits.     

Binding of [14C]tuberactinomycin O, an antibiotic closely related to viomycin, to the bacterial ribosome.

The binding of [¹⁴C]tuberactinomycin O, an antibiotic closely related to viomycin, to $\text{E. coli}$ ribosomes has been examined by equilibrium dialysis method. The antibiotic has been observed to bind to the 70S ribosome, which possesses two binding sites: one on the 30S ribosomal subunit and another on the 50S subunit. The affinity for the large subunit is greater than that for the small subunit. The binding to both ribosomal subunits is reversed by viomycin, indicating that tuberactinomycin O and viomycin have the same binding sites on the ribosome. The results seem to be in accordance with the previous finding that viomycin exhibits dual actions on ribosomal function: the inhibition of fMet-tRNA_F (initiation) and inhibition of translocation of peptidyl-tRNA.     

Action of methanol on the assocation of ribosomal subunits and its effect on the GTPase activity of elongation factor G

Methanol causes association of 30S and 50S ribosomal subunits from $\text{E. coli}$ at MgCl₂ concentrations in which they are normally completely dissociated. The 70S ribosome formed under these conditions shows a lower sedimentation velocity and is functionally active in the EF-G GTPase. Association of ribosomal subunits in the presence as well as absence of methanol is affected by washing the ribosomes with 0.5 M NH₄Cl. Methanol reduces the Mg²⁺ concentration required for subunit association as well as for EF-G GTPase activity. The basic requirement for EF-G GTPase activity both with and without alcohol is shown to be the association of 30S and 50S subunits.     

Dihydrolipoamide transacetylase from Escherichia Coli: evidence for internal gene duplication.

The dihydrolipoamide transacetylase component of the pyruvate dehydrogenase complex from $\text{Escherichia}$ coli consists of identical subunits of 89.000 daltons. During the purification procedure it is partially cleaved into active fragments of 82.000 daltons, 37.000 daltons and 35.000 daltons. Fingerprint analysis shows at least 80% similarity of the fragments compared with the native component. This provides evidence for two large homologous domains within same polypeptide chain.     

Endogenous, cyclic 3'-5' AMP-dependent phosphorylation of human red cell pyruvate kinase.

Ribosomal proteins of $\text{Physarum}\text{polycephalum}$ were labelled $\text{in}\text{vivo}$ with ³²PO₄. Three acid phosphoproteins were observed in the large subunit, while two basic ones were present in the small subunit. Ribosomal phosphoprotein S3 accounted for 70% of the total radioactivity and may be equivalent to S6 from rat liver.     

Identification of the altered subunit in the inactive F1ATPase of an Escherichia coli uncA mutant.

ATPase activity was restored to the inactive coupling factor, F₁ATPase, of $\text{Escherichia coli}$ strain AN120 ($\text{uncA401}$) by reconstitution of the dissociated complex with an excess of wild-type α subunit. Large excesses of α gave the highest levels of activity. The other subunits which are required for the reconstitution of ATPase activity, β and γ, did not complement the mutant enzyme. These results indicate that the α polypeptide of the AN120 ATPase is defective.     

The structure of form I crystals of D-ribulose-1,5-diphosphate carboxylase.

Single crystals of d-ribulose-1,5-diphosphate carboxylase from tobacco leaves, Nicotiana tabacum (variety Turkish Samsun), have been examined by X-ray diffraction, electron microscopy, and optical diffraction. Twelve molecules are loosely packed into a body-centered cubic unit cell, space group I4¹32 with cell dimension $\text{a = 383}\text{A}\text{̊}$. The asymmetric unit is one quarter of a molecule, and the minimum molecular symmetry is 222. This symmetry when combined with estimates of the two subunit masses and stoichiometry is compatible with a molecular structure of the composition L₈S₈ (L is large subunit, S is small). If all bonds between large and small subunits are equivalent, the true molecular symmetry is 422; this symmetry is consistent with molecular images in micrographs.     

Further proof for the catalytic role of the larger subunit in the spinach leaf ribulose-1,5-diphosphate carboxylase

The catalytically active oligomeric form of the larger subunit, A_m, obtained from spinach leaf ribulose-1,5-diphosphate carboxylase by pretreatment with p-mercuribenzoate at pH 7.5 followed by incubation at pH 9.0, was free of the smaller subunit based on C-terminal amino acid analyses. Valine was the predominant C-terminus of the A_m preparations, the release of tyrosine being negligibly small [cf. Sugiyama and Akazawa, $\text{Biochemistry}$ 9 (1970) 4499]. The pH optimum of the ribulose-1,5-diphosphate carboxylase reaction by A_m was about 8.5, in comparison to the native enzyme which showed an alkaline pH optimum only in the absence of Mg²⁺. The substrate saturation curve of the catalytic subunit with respect to bicarbonate followed the Michaelis-Menten equation, as contrasted to the anomalous reaction kinetics of the native ribulose-1,5-diphosphate carboxylase molecule reported previously. These overall results indicate that the allosteric properties of spinach ribulose-1,5-diphosphate carboxylase are possibly conveyed by a unique structural conformation that requires the presence of the smaller subunit in association with the larger catalytic subunit component of the enzyme molecule.     

Interaction of kanamycin and related antibiotics with the large subunit of ribosomes and the inhibition of translocation.

The binding of [${}^3\text{H}$]kanamycin to $\text{E. coli}$ ribosomes and ribosomal subunits was studied by equilibrium dialysis and Millipore filter methods. The 70S ribosome bound ca. two molecules up to the antibiotic concentration of 10 uM, and more at higher concentrations. Each ribosomal subunit was observed to possess one major binding site, and the affinity of the small ribosomal subunit was greater than that of the large subunit. The binding of [${}^3\text{H}$]kanamycin to ribosomes and ribosomal subunits was reversed by neomycin or gentamicin, but not by streptomycin and chloramphenicol. Kanamycin, neomycin and gentamicin interfered with the binding of [${}^{14}\text{C}$] tuberactinomycin O. Translocation of N-Ac-Phe-tRNA was markedly inhibited by kanamycin, neomycin or gentamicin, but not by streptomycin.     

The purification of protease IV of E.coli and the demonstration that it is an endoproteolytic enzyme

A strong proteolytic activity is unmasked and solubilized when $\text{E. coli}$ outer membrane fragments are preincubated with 0.083% sodium dodecyl sulfate. This proteolytic activity cleaves αS1 casein into the same degradation products as protease IV, a recently described protease of $\text{E.coli}$ located in the outer membrane (Ph. Régnier, preceding paper), it is concluded that sodium dodecyl sulfate solubilizes the same protease. Protease IV has been purified 11,200 fold, probably to homogenetiy, by sodium dodecyl sulfate polyacrylamide gel electrophoresis followed by elution of the protein from gel slices. The purified enzyme is fully active, its molecular weight, determined from its migration in denaturating gels is 23,500. αS1 casein is cleaved by protease IV into two large polypeptides which are not further degraded and some small peptides of about 5,000 daltons. The production of discrete polypeptide species suggests that protease IV is an endoproteolytic enzyme.     

Effects of ethionine treatment on protein-synthesizing apparatus of rat liver 80 S ribosomes and 40 S ribosomal subunits

The inhibitory effects of ethionine treatment of female rats for 4 h on the protein-synthesizing machineries of 80 S ribosomes and 40 S ribosomal subunits of the liver were investigated. The following results were obtained. (1) The translation of globin mRNA by 80 S ribosomes or 40 S ribosomal subunits, in combination with mouse 60 S subunits, was markedly inhibited by ethionine treatment in a complete cell-free system containing partially purified initiation factors of rabbit reticulocytes and the rat liver pH 5 fraction. (2) The polysome formation of 80 S ribosomes in the complete system described above was inhibited by ethionine treatment. Similar inhibitions by ethionine treatment were observed in the case of incubation of 40 S subunits with reticulocyte lysate, although the polysome formation was rather low even in the case of control 40 S subunits. (3) The pattern of CsCl isopycnic centrifugation of rat liver native 40 S subunits uniformly labeled with [¹⁴C]- or [³H]orotic acid showed that the content of non-ribosomal proteins of native 40 S subunits was decreased by ethionine treatment. The analysis of proteins of native 40 S subunits by SDS-polyacrylamide slab gel electrophoresis revealed that eIF-3 subunits and two unidentified protein fractions of molecular weight of 2.3·10⁴ and 2.1·10⁴ were decreased in ethionine-treated rat liver. (4) 40 S subunits from ethionine-treated or control rat livers were labeled with $\text{N-[}{}^3\text{H]ethylmaleimide}$ or $\text{N-[}{}^{14}\text{C]ethylmaleimide}$, and the ³H to ¹⁴C ratios of individual 40 S proteins on two-dimensional polyacrylamide gel electrophoresis were measured. The results suggested that the conformation of rat liver 40 S subunits was changed by ethionine treatment. (5) These results may indicate that ethionine treatment decreases the activity of rat liver 40 S subunits for the interaction with initiation factors, especially eIF-3, as the results of conformational changes of 40 S subunits.     

Gel electrophoretic studies on ribosomal proteins L7L12 and the Escherichia coli 50 s subunit

Three forms of the 50 S ribosomal subunit of Escherichia coli have been separated by agarose/acrylamide gel electrophoresis. The slowest migrating form, S-50 S, corresponded to native 50 S subunits and contained four copies of proteins $\text{L}\text{7}\text{L}\text{12}$. Removal of the four copies of this protein produced a more rapidly migrating form, M-50 S. The M-50 S form was then converted to the fastest migrating form, F-50 S, by removal of additional proteins, including L10 and L11. A one-step removal of a pentameric complex of four copies of $\text{L}\text{7}\text{L}\text{12}$ plus L10 converted the S-50 S subunit directly to the F-50 S subunit. These proteins recombined specifically with the appropriate protein-deficient 50 S subunit at 3 °C to reform the S-50 S subunit, i.e. the M-50 S subunit was converted back to the S-50 S form by the addition of purified proteins $\text{L}\text{7}\text{L}\text{12}$; and the F-50 S subunit bound the pentameric complex of $\text{L}\text{7}\text{L}\text{12}$ and L10 to form S-50 S. The binding of the pentameric complex, isolated by glycerol gradient centrifugation, supports the model that all four copies of proteins $\text{L}\text{7}\text{L}\text{12}$ are together in one part of the ribosome called the “$\text{L}\text{7}\text{L}\text{12}$ stalk”. Only the four copies of $\text{L}\text{7}\text{L}\text{12}$ were removed from the 50 S subunit in low salt (0.125 m-NH₄Cl) plus 50% ethanol at 0 °C. These ribosomes (in the M-50 S form) had less than 5% of the peptide-synthesizing activity of untreated control ribosomes as measured by a poly(U) translation system in vitro. Peptide-synthesizing activity was restored, upon addition of $\text{L}\text{7}\text{L}\text{12}$, back to the treated ribosomes to give 50 S subunits (S-50 S) with a full complement of four copies of $\text{L}\text{7}\text{L}\text{12}$. Antibody to proteins $\text{L}\text{7}\text{L}\text{12}$ bound only to the S-50 S subunits, producing four new bands separated by gel electrophoresis. The bands represented complexes of one, two, three and four antibodies bound to a 50 S subunit. This result was obtained using either 50 S subunits or 70 S tight couples and indicated that all four copies of $\text{L}\text{7}\text{L}\text{12}$ are either located at a single site in the $\text{L}\text{7}\text{L}\text{12}$ stalk or, much less likely, are divided between two symmetrical sites. Proteins $\text{L}\text{7}\text{L}\text{12}$ were not only accessible to their specific antibody but could also be removed from 70 S ribosomes and polyribosomes without causing their dissociation into subunits. The ribosomes and polyribosomes had an increased gel electrophoretic mobility which was reversed by addition of proteins $\text{L}\text{7}\text{L}\text{12}$.     

Purification and properties of subunits of sterol ester hydrolase from rat pancreas.

A procedure has been developed for solubilizing subunits of sterol ester hydrolase (EC 3.1.1.13) from rat pancreas by treatment of a tissue homogenate with 0.5% ($\text{w}\text{v}$) digitonin. The crude, solubilized enzyme subunit was shown to have a molecular weight of approximately 70,000 by Sephadex G-200 gel filtration. This enzyme subunit has been purified 500-fold by a combination of ammonium sulfate fractionation, hydroxylapatite, and gel-filtration chromatography. The 70,000 molecular-weight subunits have been shown to aggregate in the presence of cholic acid or sodium taurocholate to a 400,000 molecular-weight form which is the active enzyme. Studies on binding of cholic acid to the subunit protein suggest that after the binding of one molecule of the bile acid, the subunit undergoes a conformational change(s) which makes additional binding sites for cholic acid available. Three types of differential inactivation studies (thermal, guanidine hydrochloride, and pH) indicated significantly greater stability of the active enzyme when compared to the subunits. These data are consistent with the tentative conclusion that a conformational change(s) accompanies the binding of the bile salt to the enzyme subunits, which results in their aggregation and enzyme activity. The theoretical and physiological significance of this interaction between the subunit protein and bile salt is discussed.     

Studies on (K+ + H+)-ATPase. V. Chemical composition and molecular weight of the catalytic subunit

(1) A $\text{(K}{}^{\mathrm{+}}\text{+ H}{}^{\mathrm{+}}\text{)-ATPase}$ preparation from porcine gastric mucosa is solubilized in sodium dodecyl sulfate, and is subjected to gel filtration. (2) A main subunit fraction is obtained, which is a protein carbohydrate lipid complex, containing 88% protein, 7% carbohydrate and 5% phospholipid. The detailed composition of the protein and carbohydrate moieties are reported. (3) Sedimentation analysis of the subunit preparation, after detergent removal, reveals no heterogeneity, but the subunits readily undergo aggregation. (4) Acylation of the subunit preparation with citraconic anhydride causes a clear shift of the band obtained after SDS gel electrophoresis, but the absence of broadening and splitting of the band pleads against subunit heterogeneity. (5) Treatment of the subunit preparation with dansyl chloride indicates that the NH₂ terminus is blocked, which favors the assumption of homogeneity of the protein. (6) Binding studies with concanavalin A indicate that at least 86% of the subunit preparation is composed of glycoprotein. (7) These findings, taken together, strongly suggest that there is a single subunit which is a glycoprotein and which represents the catalytic subunit of the enzyme. From sedimentation equilibrium analysis a molecular mass value of 119 kDa (S.E. 3, $\text{n = 6}$) is calculated for protein + carbohydrate and of 110 kDa (S.E. 3, $\text{n = 6}$) for protein only. (8) In combination with the molecular mass of 444 kDa (S.E. 10, $\text{n = 4}$) obtained for the intact enzyme by radiation inactivation we conclude that the enzyme appears to be composed of a homo-tetramer of catalytic subunits.     

A novel subunit separation procedure for cryptomonad phycoerythrin 545.

A method to separate phycoerythrin 545, isolated from the cryptomonad alga, $\text{Rhodomonas}\text{lens}$, into two subunits has been developed. The method uses no denaturants (urea, guanidine, detergent) but relies on dissociation of the dimeric protein and subsequent aggregation of the β subunit at pH 3.0. The absorption spectra and amino acid composition of the subunits are presented. The spectra of the α subunit was red-shifted relative to β in both pH 3.0 buffer and in acidic 8.0 M urea.