Glutathione reductase from human erythrocytes. Molecular weight, subunit composition and aggregation properties.

Glutathione reductase from human erythrocytes exists predominatly as an entity of 100 000 molecular weight under various conditions of pH and ionic strength. The S20,W of 5.5 S and D20W of 50 mum2/s correlate with the molecular weight determined by sedimentation equilibrium. The homogeneity of this species is primarily dependent on the presence of thiols and secondarily on high concentrations of salt. The amino-acid composition of the enzyme shows similarities both with glutathione reductases from other sources and with lipoamide dehydrogenase. From the flavin content and dodecylsulphate-polyacrylamide electrophoresis it is inferred that the native enzyme is a dimer composed of similar subunits of 50 000 molecular weight. In the absence of thiols, glutathione reductase shows a tendency to form tetramers and larger aggregates. Although these larger species are also catalytically active, under cellular conditions the presence of its product, reduced glutathione, should maintain the enzyme as the dimeric entity.     

The relationship between reversibility of fibril formation and subunit composition of rat skin collagen

1. Salt-soluble rat skin collagen was precipitated from solution at neutral pH and 37 degrees . On cooling, a portion of the collagen returned into solution. The fractions were separated, the supernatant was concentrated and the precipitate was redissolved in dilute acetic acid. 2. Solutions of supernatant and precipitate were subjected to the same fractionation procedure, giving four fractions. 3. Each fraction was examined by starch-gel electrophoresis and a relationship between subunit composition and the fractionation procedure was noted. The collagen that redissolved on cooling contained less of the more highly cross-linked components than did either the fraction remaining in the precipitate or the starting material.     

Effects of EDTA treatment upon the protein subunit composition and mechanical properties of mammalian single skeletal muscle fibers

Considerable interest has been focused on the role of myosin light chain LC(2) in the contraction of vertebrate striated muscle. A study was undertaken to further our investigations (Moss, R.L., G.G. Giulian, and M.L. Greaser, 1981, J. Biol. Chem., 257:8588-8591) of the effects of LC(2) removal upon contraction in skinned fibers from rabbit psoas muscles. Isometric tension and maximum velocity of shortening, V(max), were measured in fiber segments prior to LC(2) removal. The segments were then bathed at 30 degrees C for up to 240 min in a buffer solution containing 20 mM EDTA in order to extract up to 60 percent of the LC(2). Troponin C (TnC) was also partially removed by this procedure. Mechanical measurements were done following the EDTA extraction and the readditions of first TnC and then LC(2) to the segments. The protein subunit compositions of the same fiber segments were determined following each of these procedures by SDS PAGE of small pieces of the fiber. V(max) was found to decrease as the LC(2) content of the fiber segments was reduced by increasing the duration of extraction. EDTA treatment also resulted in substantial reductions in tension due mainly to the loss of TnC, though smaller reductions due to the extraction of LC(2) were also observed. Reversal of the order of recombination of LC(2) and TnC indicated that the reduction in V(max) following EDTA treatment was a specific effect of LC(2) removal. These results strongly suggest that LC(2) may have roles in determining the kinetics and extent of interaction between myosin and actin.     

The rat ovarian lutropin receptor. Purification, hormone binding properties, and subunit composition

The luteinizing hormone/human choriogonadotropin (hCG) receptor from superovulated rat ovary was purified to homogeneity. A novel scheme based on reverse immunoaffinity chromatography using immobilized antibodies to membrane proteins from receptor down-regulated ovary and subsequent two-step affinity purification on hCG-Sepharose was used to isolate homogeneous receptor. The purification method was also compared to an alternate scheme involving lectin affinity chromatography followed by hCG affinity chromatography. The purified receptor obtained by the latter method was heterogeneous and highly aggregated. The hormone binding properties, molecular size, and subunit composition of the purified receptor obtained by either method were identical. The stability of the receptor during and following solubilization was markedly improved by using 20% glycerol. The pure receptor consists of four nonidentical subunits of molecular weight 79,300 (alpha), 66,400 (beta), 55,300 (gamma), and 46,700 (delta) as indicated by polyacrylamide gel electrophoresis under reducing conditions. All receptor subunits generally, but occasionally excepting the alpha-subunit, were specifically labeled with iodinated hCG in membrane and soluble receptor preparations using bifunctional cross-linking agents. Analysis of the cross-linked hormone-receptor complexes under nonreducing conditions showed the molecular mass of the undissociated receptor to be 268,000 daltons. Hormone binding studies demonstrated that the isolated receptor retained all of the specific binding characteristics expected for the luteinizing hormone/hCG receptor. In combination, these results indicate that the functional and structural properties of the receptor were not altered during purification.     

Purification properties and subunit composition of pig heart lipoate acetyltransferase.

Lipoate acetyltransferase [acetyl-CoA: dihydrolipoate S-acetyl-transferase, EC 2.3.1.12], the core enzyme of the pyruvate dehydrogenase complex, has been highly purified by gel chromatography on Sepharose 6B and sucrose density gradient centrifugation in the presence of potassium iodide. The native enzyme has a sedimentation coefficient (S020,W) of 26.7S and a diffusion coefficient (D020,W) of 1.25 x 10(-7) cm2.-sec-1. The weight-average molecular weight was estimated to be 1.8 million from the sedimentation equilibrium data. The content of right-handed alpha helix in the enzyme molecule was estimated to be about 25% by optical rotatory dispersion and about 22% from the circular dichroism spectra. The enzyme was found to contain about 23 moles of protein-bound lipoic acid per mole of enzyme; some other properties are also reported. Lipoate acetyltransferase dissociated to yield a single subunit with a molecular weight of 74,000 as estimated by polyacrylamide gel electrophoresis in sodium dodecyl sulfate and by gel filtration on Bio-Gel in 6 M guanidine-HCl. The molecular weight was also estimated to be 74,000 from sedimentation equilibrium data in 6 M guanidine-HCl] containing 0.1 M 2-mercaptoethanol. Evidence is presented that 1 molecule of lipoate acetyltransferase apparently consists of 24 very similar subunits, each of which contains NH2-terminal alanine. Each subunit contains 1 molecule of covalently bound lipoic acid.     

The SC5b-7 complex: formation, isolation, properties, and subunit composition.

Activation of C in C8-depleted serum results in the formation of a soluble complex containing C5, C6, and C7. The complex has an electrophoretic mobility of an alpha-globulin, an s-rate of 18.5S, and a m.w. of 668,000 daltons. This complex was isolated and upon SDS polyacrylamide gel electrophoresis it was found to contain, in addition to C5b, C6 and C7, an 88,000 dalton glycoprotein. The protein was identified as the band V protein of the soluble C5b-9 complex. It is referred to as SIIIs-protein, or S-protein. Since the S-protein does not bind to C5b-6, it is concluded that it is incorporated during the fusion of C5b-6 with C7. The SC5b-7 complex exhibits the same neoantigen as the SC5b-9 complex, but compared to the C5b-6 complex it appears to contain an additionally qualitatively distinct neoantigen.     

Mechanical properties and chemical composition of avian long bones

We have studied the mechanical behaviour of avian long bones as whole structures, by calculating mechanical parameters such as maximum load, stiffness, bending strength and flexural Young's modulus; bones were always tested in three-point bending. Furthermore composition in several chemical elements and amino acids related to collagen content was also analysed. Correlations were established between body mass, mechanical parameters and chemical contents. Both bending strength and Young's modulus were negatively correlated to body mass. Significant correlations were found between nitrogen content and both strength and Young's modulus, with negative slopes in both cases. Magnesium and phosphorus appear to be the most important inorganic elements to the understanding of the mechanical behaviour of avian long bones.     

Role of subunit crosslinking in fibrin solubility.

¹²⁵I-labelled fibrinogen was clotted by thrombin in the presence of activated Factor XIII and the rates of formation of γ dimers and α polymers were measured. These changes in fibrin subunits were correlated with the solubility of fibrin in 1% monochloroacetic acid. In the presence of the factor XIIIa inhibitor, glycine methyl ester, fibrin solubility was found to depend on the level of α polymers formed. A preferential inhibition of α polymer formation rather than γ dimer was observed in the presence of glycine methyl ester.