Co-purification and characterization of ATP-sulfurylase and adenosine-5'-phosphosulfate kinase from rat chondrosarcoma

The two sulfate-activating enzymes, ATP-sulfurylase (EC 2.7.7.4) and adenosine-5'-phosphosulfate kinase (adenylylsulfate kinase, EC 2.7.1.25), were each purified about 2000-fold from crude rat chondrosarcoma homogenate. Throughout a purification protocol which included Sephacryl S-300 gel filtration, DEAE-Sephadex ion exchange, hydroxylapatite, and ATP-agarose affinity chromatography, these two activities consistently co-purified. ATP-sulfurylase and adenosine-5'-phosphosulfate kinase each showed a pH optima of 7.0-7.4 and a bimodal temperature optima of 46 and 52-54 degrees C. Both activities preferred Mg2+ as their divalent cation source over Mn2+, Co2+, or Zn2+. The apparent Km values determined for adenosine 5'-phosphosulfate in both assays was 1-5 microM; the Km for pyrophosphate in the sulfurylase reaction was 40 microM and for ATP in the kinase reaction was 5 mM. Gel electrophoresis indicated major bands at Mr = 160,000 in nondenaturing systems and 35,000-37,000 and 60,000 under dissociative conditions, whereas gel filtration of the most highly purified fractions yielded a coincident peak in the molecular weight range 260,000.     

A binding assay for serine hydroxymethyltransferase

A sensitive assay for measuring serine hydroxymethyltransferase activity has been developed, based on the binding of N5,N10-[14C]methylene tetrahydrofolate (THF) to DEAE-cellulose paper. The complete assay requires THF, pyridoxal 5'-phosphate, [14C]serine, and enzyme. The reaction is stopped by streaking an aliquot of the reaction mixture onto a square of DEAE-cellulose paper, washing the paper with water to remove unreacted serine, drying the paper, and counting the bound N5,N10-[14C]methylene-THF. To determine that the labeled product was N5,N10-methylene-THF, unlabeled formaldehyde, which exchanges with the labeled methylene carbon, was added after the product had accumulated; 2 min after the addition of formaldehyde the amount of labeled product was reduced by 50%, and by 85% after 10 min. In addition, glycine, which reverses the reaction, and hydroxylamine, which reacts with the methylene carbon, reduced the number of counts bound to the paper. Binding of product to the filter is proportional to both enzyme concentration and assay time. No counts were retained on phosphocellulose filters. This assay represents a new and simple method for measuring serine hydroxymethyltransferase activity, which can be used to measure enzyme activity in tissue homogenates and for screening large numbers of samples.     

Implications of the human genome initiative for the primary care physician

... Despite the need for physicians to be knowledgeable about and open to advances in genetic technology, little is known about the level of preparedness of primary care physicians to offer new genetic tests. Evidence suggests that several barriers exist to physicians adopting genetic tests. These include lack of knowledge, inability to interpret probabilistic information, low tolerance for uncertainty, negative attitudes about their responsibility for genetic counseling and testing, lack of confidence in their clinical skills, and unfamiliarity with ethical issues raised by testing. This paper will explore some of these barriers in further depth, discuss the ethical impact of physician unpreparedness on both patient care and the diffusion of genetic tests, and describe a study that is currently underway to investigate some of these issues.     

Identification of the cardiac and circulating form of atriopeptin in rabbit

Analysis of peptides purified from high and low molecular weight fractions of rabbit atrial extracts indicates that the sequence of the first 30 residues of rabbit atriopeptigen exhibits 80% homology with the rat peptide, and that the low molecular weight rabbit peptide (28 residues) is identical to rat atriopeptin 28 (AP 28). The effects of infused 1-deaminoarginine8-vasopressin (dAVP) and phenylephrine, volume expansion, and water immersion on AP release into the circulation of the rabbit was studied. Neither dAVP, nor water immersion elevated right atrial pressure (RAP) or plasma AP levels in the anesthetized rabbits. Phenylephrine induced a sustained increase in systemic blood pressure and right atrial pressure which was accompanied by elevated plasma AP immunoreactivity which appeared to be identical to rat AP-28 on HPLC. There is obviously a preferential conservation of the AP sequence, since the C-terminal peptide is exactly the same in rabbit, rat and mouse and differs from human, dog, cow and pig only by the single substitution of an isoleucine for a methionine residue.     

The amino terminus of the yeast F1-ATPase beta-subunit precursor functions as a mitochondrial import signal

The ATP2 gene of Saccharomyces cerevisiae codes for the cytoplasmically synthesized beta-subunit protein of the mitochondrial F1-ATPase. To define the amino acid sequence determinants necessary for the in vivo targeting and import of this protein into mitochondria, we have constructed gene fusions between the ATP2 gene and either the Escherichia coli lacZ gene or the S. cerevisiae SUC2 gene (which codes for invertase). The ATP2-lacZ and ATP2-SUC2 gene fusions code for hybrid proteins that are efficiently targeted to yeast mitochondria in vivo. The mitochondrially associated hybrid proteins fractionate with the inner mitochondrial membrane and are resistant to proteinase digestion in the isolated organelle. Results obtained with the gene fusions and with targeting-defective ATP2 deletion mutants provide evidence that the amino-terminal 27 amino acids of the beta-subunit protein precursor are sufficient to direct both specific sorting of this protein to yeast mitochondria and its import into the organelle. Also, we have observed that certain of the mitochondrially associated Atp2-LacZ and Atp2-Suc2 hybrid proteins confer a novel respiration-defective phenotype to yeast cells.     

M13 procoat inserts into liposomes in the absence of other membrane proteins

Procoat, the precursor form of the major coat protein of coliphage M13, assembles into the Escherichia coli inner membrane and is cleaved to mature coat protein by leader peptidase. This assembly process has previously been reconstituted using lipids and purified leader peptidase in a cell-free protein synthesis reaction (Watts, C., Silver, P., and Wickner, W. (1981) Cell 25, 347-353; Ohno-Iwashita, Y., and Wickner, W. (1983) J. Biol. Chem. 258, 1895-1900). We now report that procoat can also cross a liposomal membrane composed of only purified phospholipids; leader peptidase is not needed to catalyze insertion. When procoat is synthesized in vitro in the presence of liposomes with encapsulated chymotrypsin, the procoat inserts spontaneously through the membrane and is degraded. The protease was shown by several criteria to be in the lumen of the liposomes. These results demonstrate that the precursor form of an E. coli integral membrane protein can cross a membrane without the aid of leader peptidase or any other membrane proteins.     

Filter mesh size and food particle uptake by Daphnia

Food size selection of four Daphnia, species (D. magna, D. hyalina, D. galeata, D. pulicaria) was investigated using spherical plastic beads as artificial food and with small bacteria. The size of the particles ranged from 0.1 to 35 m with special emphasis to the particle diameters between 0.1 and 1 m. In one set of experiments a mixture of differently sized particles was offered as food suspension and the selectivity of filtering was determined by comparing the size spectrum of the particles found in the gut contents with the spectrum in the food suspension. In a second series of experiments suspensions of uniformly sized particles were offered to single animals and their feeding activity was observed directly. In both types of experiments the mesh sizes of the filtering apparatus of the respective animals studied were measured after the experiments by, scanning electron microscopy. The mean sizes of the filter meshes were about 0.4–0.7 m. In all experiments the size of the particles found in the gut or those which caused high feeding activities were larger than the smallest mesh sizes of the filters. As a consequence simple mechanical sieving provides a sufficient explanation for the mechanism of particle retention of the filtering process in Daphnia. D. magna was found to feed with high efficiency on suspended freshwater bacteria, the residual species investigated showed low filtering efficiencies when bacteria were offered as food.The present study was supported by Deutscher Akademischer Austauschdients