Biospecific fractionation matrices for sequence specific endonucleases

Fractionation of several type II specific restriction endonucleases was achieved by separation on two novel biospecific matrices. The matrices are pyran, a copolymer of divinyl ether of maleic anhydride, and Cibacron Blue F3GA, a blue dye commonly used for the calibration of molecular sieves. Both compounds are insolubilized by coupling to sepharose through a cyanogen bromide linkage and in their soluble form inhibit the restriction endonucleases which we have tested. These affinity matrices can be used to obtain restriction endonucleases from crude extracts after removal of nucleic acids. They have also proven to have a high capacity when used as subsequent steps in enzyme purification. Their additional advantage is the rapid development time and reusability of columns packed with the two matrices.     

Influence of gender and castration on liver and CNS N-demethylation of morphine in rats1

Rats were injected with combinations of morphine-N-¹⁴CH₃ and morphine-6³H and the isotope content of the brain and liver was measured by combustion in a tissue oxidizer. The liver of intact male rats showed a significant increase in the ³H to ¹⁴C isotope ratio relative to the blood reflecting the existence of N-demethylation in this organ. This increase was not observed in the liver of either intact females or castrated males or females. Centrally, the hypothalamus, medial thalamus, and corpus striatum of both intact and castrated male and female rats exhibited increases in ³H to ¹⁴C isotope ratios indicating the presence of N-demethylation in these tissues. These results indicate that testicular hormones serve to increase the hepatic N-demethylation of morphine, but apparently reduce the comparable reaction in the CNS.     

The role of sodium-channel density in the natriferic response of the toad urinary bladder to an antidiuretic hormone

Summary Urinary bladders ofBufo marinus were depolarized, by raising the serosal K concentration, to facilitate voltage-clamping of the apical membrane. Passive Na transport across the apical membrane was then studied with near-instantaneous current-voltage curves obtained before and after eliciting a natriferic response with oxytocin. Fitting with the constant-field equation showed that the natriferic effect is accounted for by an increase in the apical Na permeability. It is accompanied by a small increase in cellular Na activity. Furthermore, fluctuation analysis of the amiloride-induced shot-noise component of the short-circuit current indicated that the permeability increase is not due to increased Na translocation through those Na channels which were already conducting prior to hormonal stimulation. Rather, the natriferic effects is found to be based on an increase in the population of transporting channels. It appears that, in response to the hormone, Na channels are rapidly recruited from a pool of electrically silent channels.     

Synthesis and characterization of 2′-azidoaminopterin as a potential photoaffinity label for folate-utilizing enzymes

A photoreactive analog of aminopterin, 2′-azidoaminopterin (VI), was synthesized and evaluated as a potential inhibitor and photoaffinity label of folate-utilizing enzymes. The compound was tightly bound to dihydrofolate reductase (DHFR) from escherichia coli (MB 1428) with K₁ equal to 3 × 10^?11M and to the enzyme from mouse (S-180) cells with K₁ approximately equal to 2 × 10^?10M. Dissociation constants measured by equilibrium dialysis using radioactive 2′-azidoaminopterin gave a value of K_D = 3.2 × 10^?9M for the bacterial enzyme. The presence of NADPH enhanced the affinity by more than an order of magnitude. Azidoaminopterin is also an inhibitor of thymidylate synthetase from Lactobacillus casei, competitive with methylene-tetrahydrofolate (K_i 7 × 10^?7M). Photolysis of the radioactive inhibitor in complex with DHFR from E. coli led to approximately 3% covalent incorporation of label into protein. The greater part of this attachment was nonspecific as shown by the lack of protection in the presence of methotrexate. Thymidylate synthetase from L. casei was not significantly inactivated upon photolysis in the presence of the inhibitor and deoxyuridylate. Model studies showed that photoreaction of the inhibitor led to covalent linkages with thiol, lysyl amino groups, and the hydroxyl groups of alcohols. Azidoaminopterin may be useful in labeling other enzymes of folate metabolism, although a minor photoproduct reacts nonspecifically with many proteins. The antifolate can be photoconjugated to polylysine as well as to proteins. The polylysine conjugates inhibit DHFR. Difference spectrum analysis of the photoproducts from the irradiation of the DHFR I complex indicates that water reacts efficiently with the enzyme-bound nitrene and must therefore have access to at least part of the bound p-aminobenzoyl group. This analysis suggests that azide analogs of protein ligands may be useful as reporter groups in probing the hydrophobicity of binding sites.     

Kidney Stones

The prevalence of kidney stones has steadily risen during this century; passage of a calculus and a positive family history increase the probability of recurrence. Findings from recent studies on the cause of renal calculi have stressed crystallization and crystal aggregation of stone minerals from supersaturated urine, rather than excessive organic matrix. Absence of normal urine inhibitors of calcium salts is also stressed. Formation of calcium oxalate stones is the major problem. Therapy with decreased calcium and oxalate intake, thiazides, phosphate salts and allopurinol in various combinations has substantially decreased the prevalence of recurrent stones. The rationale for the use of allopurinol is that uric acid salts enhance the tendency for calcium oxalate to crystallize from supersaturated urine. The hypercalciuria seen in 30 percent to 40 percent of patients with oxalate stones is usually caused by intestinal hyperabsorption of calcium. Although patients with uric acid calculi constitute only a small fraction of those in whom stones form, they represent a group in whom good medical therapy, based on sound physiologic principles, has proved extremely successful. Renal tubular syndromes lead to nephrocalcinosis and lithiasis through hypercalciuria, alkaline urine and hypocitraturia, the latter an inhibitor of calcium salt precipitation. Recent advances in surgical techniques are discussed, including the rationale for removing staghorn calculi. The ileal ureter and coagulum pyelolithotomy deserve special emphasis.     

Purification and characterization of α-l-fucosidase from the liver of a fucosidosis patient

Intact viable 13762 mammary-adenocarcinoma ascites cells hydrolyse added ATP. The localization of hydrolysis product and inactivation by the slowly penetrating chemical reagent diazotized sulphanilic acid indicate that this ATPase is at the external surface of the cell. A number of features differentiate this enzyme from mitochondrial, myosin and cation-transport ATPases. It is stimulated by either Ca2+ or Mg2+ and has little or no activity in their absence. It is insensitive to ouabain, oligomycin and azide. It is the major ATPase activity found in homogenates of gently disrupted 13762 cels. The ATPase activity is inhibited at high substrate concentrations and shows an apparent stimulation by concanavalin A in isolated membranes, but not in intact cells. The stimulation by concanavalin A results predominantly from a release from substrate inhibition.     

The use of cell lethal mutations in the study of Drosophila development

Cell-lethal mutations are currently being used to study the role of cell death in the development of normal and mutant phenotypes, and in highly detailed studies of pattern formation. Histological studies of certain “position-specific” cell-lethals suggest that programmed cell death may be important in the development of normal imaginal discs, and that many mutations may be producing an abnormal phenotype by amplifying this normal process. Somatic mosaic studies using temperature-sensitive cell-lethals have provided much information about the development of pattern duplications, including the number of cells involved, their growth rate, and compartmentalization during duplication. Further work in this area may, when combined with genetic analyses of the loci involved, reveal much about the nature of the processes controlling pattern determination and differentiation.     

The effect of proteinases on phenylalanine ammonia-lyase from the yeast Rhodotorula glutinis.

Phenylalanine ammonia-lyase (EC 4.3.1.5) of the yeast Rhodotorula glutinis was rapidly inactivated by duodenal juice. It was susceptible to chymotrypsin and subtilisin and to a lesser extent trypsin. Initial proteolysis of the enzyme by chymotrypsin and trypsin resulted in cleavage of the monomeric subunit (75 000 Mr) into a large (65 000 Mr) and a small (10 000 Mr) peptide. The small peptide was rapidly degraded. The 65 000-Mr fragment was resistant to prolonged incubation with chymotrypsin, but was degraded by trypsin under the same conditions. Phenylalanine ammonia-lyase was cleaved into several polypeptides by subtilisin, the 65 000-Mr peptide being totally absent. The N-terminal region of the enzyme was contained in the 65 000-Mr fragment, as was the dehydroalanine moiety, the prosthetic group. Active-site-binding ligands protect the enzyme from inactivation by the three proteinases, and peptide-bond cleavage by trypsin and chymotrypsin. Several chemical modifications were performed on phenylalanine ammonia-lyase. Some decreased its antigenicity, and ethyl acetimidate decreased the rate of degradation of the 65 000-Mr peptide by trypsin. The modification did not protect the enzyme from proteolytic inactivation of the enzymic activity. These observations are discussed in terms of the structure of phenylalanine ammonia-lyase and site of action of the proteinases.