Daniel Wellner

The hematopoietic growth factor KL is encoded by the SI locus and is the ligand of the c-kit receptor, the gene product of the W locus

Mutations at the steel locus (Sl) of the mouse affect the same cellular targets as mutations at the white spotting locus (W), which is allelic with the c-kit proto-oncogene. We show that KL, a hematopoietic growth factor obtained from conditioned medium of BALB/c 3T3 fibroblasts that stimulates the proliferation of mast cells and early erythroid progenitors, specifically binds to the c-kit receptor. The predicted amino acid sequence of isolated KL-specific cDNA clones suggests that KL is synthesized as an integral transmembrane protein. Linkage analysis maps the KL gene to the Sl locus on mouse chromosome 10, and KL sequences are deleted in the genome of the Sl mouse. These results indicate that the Sl locus encodes the ligand of the c-kit receptor, KL.

Mycobacterium tuberculosis expresses methionine sulphoxide reductases A and B that protect from killing by nitrite and hypochlorite.

Methionine sulphoxide reductases (Msr) reduce methionine sulphoxide to methionine and protect bacteria against reactive oxygen intermediates (ROI) and reactive nitrogen intermediates (RNI). Many organisms express both MsrA, active against methionine‐(S)‐sulphoxide, and MsrB, active against methionine‐(R)‐sulphoxide. Mycobacterium tuberculosis (Mtb) expresses MsrA, which protects ΔmsrA‐Escherichia coli from ROI and RNI. However, the function of MsrA in Mtb has not been defined, and it is unknown whether Mtb expresses MsrB. We identified MsrB as the protein encoded by Rv2674 in Mtb and confirmed the distinct stereospecificities of recombinant Mtb MsrA and MsrB. We generated strains of Mtb deficient in MsrA, MsrB or both and complemented the mutants. Lysates of singly deficient strains displayed half as much Msr activity as wild type against N‐acetyl methionine sulphoxide. However, in contrast to other bacteria, single mutants were no more vulnerable than wild type to killing by ROI/RNI. Only Mtb lacking both MsrA and MsrB was more readily killed by nitrite or hypochlorite. Thus, MsrA and MsrB contribute to the enzymatic defences of Mtb against ROI and RNI.

A sensitive fluorometric assay for amino acid oxidases

Abstract An assay for amino acid oxidases is described which is capable of measuring 0.005 μg of enzyme or the oxidation of 0.5 nmole of amino acid. It involves the coupled oxidation of scopoletin by hydrogen peroxide catalyzed by peroxidase and the fluorometric determination of the scopoletin remaining. By the use of this method, it is shown that glycine is a substrate of l -amino acid oxidase.

[218a] Assay of amino acid oxidase

Publisher Summary This chapter discusses assay of amino acid oxidase. Conditions are given in the chapter for the determination of the L -amino acid oxidase of snake venoms and the D -amino acid oxidase of mammalian kidney and liver. Although the principles are the same for both, experimental details differ because of differences in the stability, pH optimum, and other properties of the two types of enzyme. For example, FAD must be added in the D -amino acid oxidase assay, because it dissociates readily from the enzyme, whereas in the case of L -amino acid oxidase the prosthetic group is so tightly bound that the addition of free FAD in the assay mixture is unnecessary. The spectrophotometric assay described in chapter is useful over a wide range of enzyme concentrations and is convenient as a general procedure, for example, for following enzyme purification, assaying fractions from a column, or observing changes in activity resulting from enzyme modifications.

The amino acid sequence of bovine thymus prothymosin α

Prothymosin alpha has been purified from calf thymus and its amino acid sequence determined. It contains 109 amino acid residues and closely resembles human prothymosin alpha, with only two substitutions, glutamic acid for aspartic acid at position 31 and alanine for serine at position 83. This is in contrast to six differences between rat and bovine prothymosins, including four substitutions and two deletions. The structural similarity of the bovine and human polypeptides makes the former a good candidate for studies on the evaluation of the biological activities of prothymosin alpha in human systems.

Biochemical studies of a patient with hereditary hepatorenal tyrosinemia: evidence of glutathione deficiency.

ABSTRACT: Metabolic and enzymatic studies in a patient with hereditary tyrosinemia demonstrated for the first time a deficiency of erythrocyte and hepatic glutathione. Markedly decreased hepatic fumarylacetoacetate hydrolase activity was demonstrated in this patient. The activities of hepatic enzymes not involved in tyrosine metabolism were also determined. Assay of mixed function oxidase activity demonstrated low levels of aryl hydrocarbon hydroxylase and 7-ethoxycoumarin deethylase, suggesting decreased hepatic detoxification capacity. 5-Aminolevulinic acid dehydratase activity was undetectable. Succinylacetone (4,6- dioxoheptanoic acid), an abnormal metabolic product secondary to fumarylacetoacetate hydrolase deficiency was found in serum and urine. Succinylacetone was demonstrated to inhibit 5-aminolevulinic acid dehydratase in vitro, as did the urine, plasma, and red cell Iysates of the patient.

Renal γ-glutamyl transpeptidases: Structural and immunological studies

Mammalian kidney gamma-glutamyl transpeptidases are compared with respect to subunit size, amino-terminal sequences of the two subunits, immunological, and some catalytic properties. The species-related variation in the apparent molecular weight of the subunits has been shown to be primarily due to the extent and nature of protein glycosylation. Using antibodies raised against the native enzymes and isolated sodium dodecyl sulfate-treated subunits, it is shown that the transpeptidases share some antigenic determinants. Some of these determinants in the highly glycosylated transpeptidase subunits can be detected by the antibodies only upon deglycosylation of the subunits. The amino-terminal sequences of the subunits exhibit considerable homology, in agreement with the immunological data. Thus, there are two segments of identity (3 and 5 residues in length, respectively) in the first 17 amino-terminal residues of the heavy subunits of rat, bovine, dog, and human kidney transpeptidases (papain-solubilized). Of particular interest is the finding of 91 to 96% identity in the first 23 amino-terminal residues of the small subunit of these transpeptidases. The small subunit contains the gamma-glutamyl binding site of the enzyme. There are three segments of identity (7, 6, and 8 residues in length, respectively) in the first 23 residues, each separated by either a Ser or an Ala residue. The first 7 amino-terminal residues of the small subunit in all four species are identical, indicating a high degree of specificity in the proteolytic processing of the common, single-chain precursor of the two subunits. Differences noted between transpeptidases in their relative acceptor specificity and in their susceptibility to inactivation by the glutamine antagonist, AT-125 (acivicin), must reflect subtle structural differences in their active center domains.

Isoelectric focusing in polyacrylamide gels.

A method for the separation of proteins by isoelectric focusing in polyacrylamide gels was developed in our laboratory as an analytical tool for the study of L-amino acid oxidase isozymes.l.2 Although we also used the sucrose gradient method of Vesterberg and Svenssons successfully for separating the isoenzymes, that method had a number of drawbacks for our purpose. In the first place, our samples were often too small to apply to the available large-scale sucrose gradient columns. Another problem was the limitation in the number of experiments that could be carried out in any given time because of the lengthy procedure, involved in preparing the sucrose gradient, allowing the separation to take place, collecting the fractions, and analyzing them first for pH, then for protein, and then for enzymatic activity. We thought that for many applications, polyacrylamide gel would provideo a suitable substitute for a sucrose gradient as an anticonvection medium in isoelectric focusing. It appears that the time was right for such an idea because it was conceived independently in several other laboratories around the world.4-10 In this paper, we would like to indicate the advantages of this method and to illustrate some of its applications, particularly to the L-amino acid oxidases of snake venom. For analytical purposes, the use of polyacrylamide gel has several distinct advantages over the sucrose gradient method. (1) One of these is that the separation takes less time. We found in our experiments that about 9 hr at 120 V was optimal for a 10-cm gel. By using a higher voltage and appropriate cooling, it is possible to reduce this time even further. (2) Only very simple equipment is needed. An apparatus similar to that used by Davis11 for disc dectrophoresis is perfectly suitable. Such equipment is inexpensive and is now available in most biochemical laboratories. (3) In addition, the small amounts of carrier ampholytes needed places the method within reach of laboratories with very limited budgets. (4) The gels can be analyzed for protein, enzymatic activity, or radioactivity, just as in disc electrophoresis. For protein staining, however, it should be noted that some of the dyes commonly employed, such as amido black, may also stain the carrier ampholytes. Thus, unless the ampholytes are thoroughly washed out, such dyes sometimes given rise to a dense background, making observation of the protein bands difficult. This problem may be avoided by using Coomassie blue, which we found to be suitable for staining proteins in focused gels without the necessity of removing the carrier ampholytes. The protein bands may be fixed and stained simultaneously by immersing the gel for a few hours in a solution containing 5% trichloroacetic acid, 5% sulfosalicylic acid, 18% methanol, and 0.02% Coomasie blue.12 Destaining may be accomplished by washing the gels in water or in dilute acetic acid, but this is not necessary for the bands to become visible. (5) Very small samples, of the order of a few micrograms of protein. can be easily detected, either by protein or enzymatic staining. (6) Many samples can be run simultaneously in the same apparatus for comparative purposes. (7) The technique may be com-

Transport into brain of buthionine sulfoximine, an inhibitor of glutathione synthesis, is facilitated by esterification and administration of dimethylsulfoxide

Buthionine sulfoximine (BSO), an inhibitor of glutathione synthesis, is poorly transported into the brain of adult mice, and only a slight decrease (approximately 10%) in the level of brain glutathione is found 30-60 min after intraperitoneal administration of BSO. When BSO is given as the ethyl ester, the brain level of BSO increases substantially after 5-15 min, and the glutathione level decreases by about 25% after 30-60 min. When BSO or its ester is given in 15% dimethylsulfoxide solution the brain levels of BSO are increased significantly and the brain glutathione levels are decreased by 20-35%. These observations suggest procedures that may be useful in decreasing the glutathione levels of the brains of adult animals. The finding that administration of BSO ethyl ester led to about a 25% decrease in the brain level of glutathione within 15 min suggests that a fraction of brain glutathione turns over very rapidly and may therefore be of special physiological significance.

Bovine parathymosin: Amino acid sequence and comparison with rat parathymosin

Parathymosin has been purified from calf liver and its primary sequence established, except for a segment containing approximately 11 amino acid residues in the central part of the polypeptide chain. Bovine parathymosin contains approximately 101 amino acid residues and shows 90% identity with rat parathymosin, with substitution of Glu for Asp at positions 21, 57, and 58, Asp for Glu at positions 60 and 63, and Ala for Val at position 77. Three non-conservative substitutions were Ala for Thr at position 81, Leu for Arg at position 78, and Val for Lys at position 79. The replacement at the last two positions of a pair of basic by hydrophobic amino acid residues may account for differences in chromatographic behavior observed for the bovine and rat polypeptides. Analysis of the NH2-terminus employed a new deblocking procedure which was also employed to analyze rat parathymosin, requiring correction of the previously published NH2-terminal sequence for that polypeptide.