Structural studies on equine chorionic gonadotropin

The amino acid sequence of the subunit of equine chorionic gonadotropin (eCG, also pregnant mare serum gonadotropin, PMSG) has been determined. Overlapping peptides from tryptic and chymotrypic digests were isolated by a two-dimensional peptide mapping technique and sequenced by the Edman procedure. The proposed amino acid sequence of eCG is: (**Denotes carbohydrate attachment points.) This sequence differs significantly from that proposed by Rathnamet al. (1978) for equine follitropin subunit; in particular, their sequence lacked the first fourteen residues.For the subunit we have placed in sequence 104 amino acid residues by direct sequence determination and peptide overlap procedures; in addition, 37 residues have been placed provisionally by homology with the human chorionic gonadotropin (hCG) sequence and composition and/or sequence data for the peptides isolated in the present studies. Difficulties in the procurement of the hormone have stalled completion of the -subunit amino acid sequence determination. The data now available indicate that eCG -subunit is highly homologous to hCG subunit and the subunits of luteinizing hormone from the pituitary gland of the several species so far described. The proposed partial sequence of eCG is:     

Segmental flexibility of ribosomal protein S1 bound to ribosomes and Q beta-replicase

The protonization pattern of the endogenous donor component D₁ which feeds electrons directly into chl-a⁺_II has been analyzed in Tris-washed inside-out thylakoids with the aid of appropriate pH-indicators. It was found that under repetitive flash excitation the amount of protons released is proportional to the extent of D₁-oxidation, depending on the time between the flashes. The kinetics of D₁-oxidation (being practically the same as in normal Tris-washed chloroplasts) are faster than the proton release by two orders of magnitude. The results lead to the conclusion that D₁ is protonized in the reduced state with pK(D^ox₁) < 5 and becomes deprotonized in the oxidized state with pK(D^red₁) ? 8. The proton release is kinetically limited by a transport barrier. Implications on the interpretation of the proton release pattern in preparation with intact water oxidation are discussed.     

Purification and phosphorylation of initiation factor eIF-2 from rabbit skeletal muscle

Core particle DNA unfolding and refolding are followed by stopped-flow circular dichroism technique. When core particles are dissociated in the stopped-flow cuvette, the high CD deviation corresponding to the dissociated state is reached in the first millisecond, which means that the dissociation process is completed within the dead time of the apparatus which is ～1 ms. The same conclusion can be drawn when core particles are reassociated, since the low CD value, typical of the associated state, is immediately reached. Similarly histone release from chromatin is a very fast process. We also include some points of discussion about core particle assembly process.     

Characterization of the subunit composition of HGPRTase from human erythrocytes and cultured fibroblasts

Hypoxanthine-guanine phosphoribosyltransferase is a ubiquitous human enzyme, the inherited deficiency of which leads to a specific metabolic-neurological syndrome. Native acrylamide isoelectric focusing revealed that the human enzyme consists of different numbers of isoenzymes depending on the tissue of origin. The erythrocytic enzyme has the most isoenzymes while the enzyme from cultured fibroblasts has only a single isoenzyme. The isoenzyme pattern of the erythrocytic enzyme changes on storage of the crude hemolysate at 4 C. Treatment of the stored crude hemolysate with 4.5 m urea and 0.35 mm -mercaptoethanol results in an isoenzyme pattern similar to that of the fresh crude extract. Thus the additional isoenzymes are generated on storage not by covalent modification of the enzyme but probably by binding of small molecules to the enzyme or to association of the enzyme molecules. Hypoxanthine-guanine phosphoribosyltransferase has been purified to 80% homogeneity in three steps, DEAE Sephadex chromatography, heat treatment at 85 C for 5 min, and hydroxylapatite chromatography. Denaturing two-dimensional gel electrophoresis of the erythrocytic enzyme revealed that the erythrocytic enzyme is composed of three major types of subunits (1–3) with the same molecular weight but different isoelectric points. In contrast, the fibroblast enzyme is composed of only a single type of subunit, which comigrates with subunit 1 of the erythrocytic enzyme. Since there is a single genetic locus in humans for HGPRTase (the enzyme is X linked) (Nyhan et al., 1967), the observed subunit modification of the erythrocyte enzyme appears to be the result of posttranslational modification. These findings provide a simple explanation for the observed electrophoretic properties of human HGPRTase. A patient with 0.5% of HGPRTase activity in his erythrocytes was found to have small amounts (> 0.5% but < 5% of normal) of the erythrocytic HGPRTase subunits.This work was supported by a grant from NIAMDD, National Institutes of Health, United States Public Health Service. L. J. G. was supported by a fellowship from the National Institute of Child Health and Human Development. D. W. M. is an Investigator, Howard Hughes Medical Institute.     

Alteration of the structure of theEscherichia coli ribosomes on treatment with Fab fragment of immunoglobulin raised against the ribosomes

Rabbits were immunised againstEscherichia coli ribosomes and the partially purified immunoglobulin G fraction had maximum ability to precipitate the ribosomes as well as the extracted ribosomal proteins. By digestion of immuno-globulin G with papain, monovalent Fab fragments were produced. The 70 S ribosome and its subunits (50 S and 30 S) were separately treated with Fab and then tested in the kinetic assay of degradation of ribosomes by ribonuclease I at various Mg²⁺ concentrations. Treated ribosomes and their subunits were degraded at faster rates than the nontreated ones; the rates in both the control and the treated cases were dependent on the concentration of Mg²⁺. These results indicate the unfolding of the structure of the ribosome on treatment with antibody fragments, which may be due to the weakening of the interaction between rRNAs and ribosomal proteins.     

Cryo-EM structures reveal multiple stages of bacterial outer membrane protein folding

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TGF-β isoforms inhibit hepatitis C virus propagation in transforming growth factor beta/SMAD protein signalling pathway dependent and independent manners

Transforming growth factor beta (TGF-β) plays an important role in the viral liver disease progression via controlling viral propagation and mediating inflammation-associated responses. However, the antiviral activities and mechanisms of TGF-β isoforms, including TGF-β1, TGF-β2 and TGF-β3, remain unclear. Here, we demonstrated that all of the three TGF-β isoforms were increased in Huh7.5 cells infected by hepatitis C virus (HCV), but in turn, the elevated TGF-β isoforms could inhibit HCV propagation with different potency in infectious HCV cell culture system. TGF-β isoforms suppressed HCV propagation through interrupting several different stages in the whole HCV life cycle, including virus entry and intracellular replication, in TGF-β/SMAD signalling pathway–dependent and TGF-β/SMAD signalling pathway–independent manners. TGF-β isoforms showed additional anti-HCV activities when combined with each other. However, the elevated TGF-β1 and TGF-β2, not TGF-β3, could also induce liver fibrosis with a high expression of type I collagen alpha-1 and α-smooth muscle actin in LX-2 cells. Our results showed a new insight into TGF-β isoforms in the HCV-related liver disease progression.