Douglas M. Brown

Some properties of soybean lipoxygenase

Abstract Commercial soybean lipoxygenase (EC 1.99.2.1) was purified by ammonium sulfate fractionation, gel filtration on Sephadex G-150, and chromatography on DEAE-cellulose. The purified protein was essentially homogeneous as judged by acrylamide gel electrophoresis and ultracentrifugation. The molecular weight is 108,000 as determined by the sedimentation-equilibrium method. The amino acid composition was determined, and it was shown that the protein contains four residues of free sulfhydryl groups and four residues of half-cystine per molecule. Treatment of the protein with guanidine hydrochloride or sodium dodecyl sulfate produces dissociation. Present evidence indicates that the protein is composed of two subunits of 54,000 molecular weight.

Human thyroglobulin: Studies on the native and S-carboxymethylated protein

Abstract 1. 1. The properties of human thyroglobulin prepared from both normal thyroid glands and from subjects with non-toxic colloid goiters have been studied. No significant differences in composition were found with respect to the non-iodinated amino acids, the total amount of carbohydrate, and the kinds of sugar residues found nor were differences observed in electrophoretic and centrifugal properties. The amino acid compositions are also compared to those of sheep and of hog thyroglobulin. 2. 2. The amino acid analyses showed the presence of approximately 180 half-cystine residues per molecular weight of 660 000. Within experimental error all the half-cystine residues were found to be in disulfide linkage as shown by amino acid analyses of preprations which had been treated at pH 8.6 in 8 M urea with sodium iodoacetate with and without prior reduction by mercaptoethanol. This conclusion was supported by results of amperometric titrations. 3. 3. Evidence concerning subunit structure has been obtained by physical studies of the S-carboxymethyl derivative of reduced thyroglobulin. In the presence of sodium dodecyl sulfate at pH 9.6 this derivative undergoes dissociation and sediments as a single component having an s° 20, w of 3.2–3.4 S. The molecular weight of the dissociated species was estimated to be in the range of 110 000–125 000, thus indicating at least 5 or 6 physically identical subunits or peptide chains in the native thyroglobulin molecule.

Cerebral protein synthesis. I. Physical properties of cerebral ribosomes and polyribosomes.

Ribonucleoprotein preparations from rat cerebral cortex, isolated in 0·25 M -sucrose, 4 m M -MgCl2, 25 m M -KCl and 50 m M -tris-HCl buffer (pH 7·4), exhibited 3 major sedimenting species in the analytical ultracentrifuge. The S 20,w values and percentage distribution of these cerebral mixed ribosomes averaged 56% of 78 s (monomer), 33% of 114 s (dimer), and 11% of 147 s (trimer). Occasionally, small amounts of tetrameric ribosomes appeared. Little dependence of S 20,w values on ribosomal concentration was noted. Ultraviolet absorption analyses and RNA : protein ratios revealed that this preparation was relatively free of contamination with non-ribosomal protein. When cerebral mixed ribosomes were suspended in media low in Mg 2+ (1 m M or less) and high in K + (100 m M ), a 57 s RNP† component appeared. An additional, smaller RNP particle was noted after dialysis against buffer of low ionic strength (35 s) and after treatment with EDTA (26 s). Inasmuch as the larger and smaller RNP components could be caused to reassociate to RNP particles with normal S 20,w values (79 sand 121 s), they probably represented normal subunits of cerebral ribosomes. When cerebral ribosomes were prepared by alternate methods which produced relatively large amounts of polyribosomes with rat liver, the proportions of trimers and larger aggregates increased. However, unlike hepatic preparations, large polyribosomes predominated in cerebral ribosomal pellets only when the isolation media contained relatively high concentrations of Mg 2+ (10 to 12 m M ) and K + (100 m M ). Resuspension of polyribosomal pellets in buffer containing 1 m M -Mg 2+ and 25 m M -K + markedly increased the proportion of mono ribosomes in cerebral preparations, but had little effect on the physical properties of hepatic polyribosomes. The maximum yield of ribosomal aggregates from cerebral tissue was very low (about 0·1 mg RNA/g tissue). This value was about one-sixth the recovery value for cerebral mixed ribosomes and only about 10% of the maximum recovery value for liver polyribosomes. The results indicate that the ribosomal population of cerebral cortical tissue is very low compared to that of liver, and suggest that cerebral polyribosomes are relatively unstable.

The formation of bacterial flagella: III. Characterization of the subunits of the flagella of Bacillus subtilis and Spirillum serpens*

The flagellins of Bacillus subtilis strain 19 and Spirillum serpens have been characterized by physical, chemical and immunochemical methods. The purified flagellar structures of both organisms consist of a single type of protein subunit of molecular weight approximately 40,000.

Bovine, human and porcine thyrotropins: Molecular weights, amino- and carboxyl-terminal studies☆

Abstract The molecular weights and end-group analyses of bovine, human, and porcine thyrotropins (TSH) are reported. Both the hormones and their S-carboxymethyl derivatives behave as physically homogeneous materials during sedimentation equilibrium runs in 6 m guanidine hydrochloride. The weight average molecular weights of all three thyrotropins are approximately 25,000. No evidence was found for separation into peptide chains of smaller molecular size after disulfide bond cleavage. In the absence of a dissociating agent bovine TSH exhibited concentration-dependent aggregation. End-group analyses were in agreement with the above and showed phenylalanine to be the major amino-terminal residue in both bovine and porcine thyrotropins. In human TSH, phenylalanine and valine were present in equal proportions. In addition, 0.1 to 0.2 moles of amino-terminal aspartic acid per mole of hormone was found in each case. The carboxyl-terminal sequences of bovine and human TSH are most probably -His-Tyr-Lys-Ser-Tyr-COOH; that of porcine TSH, -His-Tyr-Lys-Tyr-Ser-COOH. About 0.2 moles of methionine and leucine which could not be accommodated in the above sequences were also found at or near the carboxyl-termini of bovine and human TSH respectively. The amino acid and carbohydrate compositions of human and porcine TSH are compared with those of bovine TSH and purified human and bovine luteinizing hormones.

Isolation and properties of lactate dehydrogenase isozyme X from swiss mice

Abstract Lactate dehydrogenase X, a sixth LDH isozyme in testes and mature sperm, has been purified to homogeneity from adult mouse testes. The isozyme has a molecular weight of 139,000 ± 6000. Dissociation of the molecule with 6 m guanidine produced a homogeneous material with mol wt 37,700 ± 800. Pyruvate at 37 ° was reduced optimally at about pH 7.25 and a concentration of 0.25 m m . Alpha-ketobutyrate has an optimum pH of 7.75 and concentration of 0.5 m m for reduction with a rate about 50% greater than that of pyruvate. Alpha-ketoglutarate has an optimum pH of about 5.5 and is reduced at only about one-fourth the rate of pyruvate. Pyruvate reduction is considerably inhibited by 2.5 m m lactate. Alpha-hydroxybutyrate and α-hydroxyvalerate are oxidized at about the same rate and under similar optimum conditions as lactate. Alpha-hydroxyglutarate is oxidized at only about one-twentieth the rate of lactate.

Characterization of the subunits of swine kidney leucine aminopeptidase

Abstract The swine kidney leucine aminopeptidase obtained in this study was found to have a molecular weight of 255 000 ± 5000, by the Yphantis method. The subunit, obtained in 6 M guanidine, 0.5% β-mercaptoethanol, has a molecular weight of 63 500 as determined by the Yphantis method. The enzyme is probably converted to a lower molecular weight species, s 20, w = 6.0 S from the native enzyme s 20, w = 13.1 S when dialyzed against 4 M urea, 0.05 M Tris solution. At the same time the enzyme is inactivated. When dialyzed against 4 M urea, 0.05 M Tris and 0.05 M MgCl 2 , the enzyme is not converted to the 6.0-S species and retains full enzymic activity. Dialysis of the enzyme against 0.5 mM EDTA (pH 8.0) completely inactivated it and converted it to a s 20, w = 5.5-S protein. It had been demonstrated earlier that 0.05 M barbital and 0.5 mM MgCl 2 protected the enzyme against inactivation by 5 mM EDTA. These results indicate a potential role for Mg 2+ in subunit structure of the leucine aminopeptidase similar to the role suggested by Simpson and Vallee 12 for zinc in alkaline phosphatase.

The structure of human thyroglobulin: Sedimentation equilibrium in 6 M guanidine hydrochloride

Abstract Reduced and alkylated human thyroglobulin has been studied by high speed sedimentation equilibrium in 6 M guanidine-HCl at various pH values and at several rotor speeds. Our results indicate that thyroglobulin is heterodispersed under these conditions. This heterogeneity appears to be due to a combination of the presence of polypeptide moieties of different size derived from the thyroglobulin structure and possible incomplete dissociation. Due to this molecular weight heterogeneity we observe a significant rotor speed dependence of the apparent molecular weight. It is clear, however, that the human thyroglobulin structure contains polypeptide chains of molecular weight significantly below 100 000 and as low as 50 000.