Chemical modification of gastric microsomal potassium-stimulated ATPase

Selective chemical modification was used to examine amino acid residues that might be critical for the operation of the gastric K⁺-stimulated ATPase. Modification of amino groups with the fluorigenic reagent 2-methoxy-2,4-diphenyl-3-dihydrofuranone resulted in selective inhibition of the K⁺-stimulated ATPase and H⁺-transporting activities of the gastric microsomes, while the Mg²⁺-ATPase was not affected. Half-maximal inhibition occurred at about 3 μg 2-methoxy-2,4-diphenyl-3-dihydrofuranone/ml at pH 8.5. ATP provided complete protection against inhibition; the apparent K_m for ATP protection was about 50 μM. Nucleotide selectivity for protection was ATP > ADP > ITP > GTP > CTP > AMP. Sodium dodecyl sulfate gel electrophoresis of the reacted microsomes showed that virtually all the fluorescent label was on the M_r 100 000 peptide band, a very small peptide, and aminolipids. In the presence of ATP there was about 75% reduction in the fluorescent label on the M_r 100 000 peptide, but no change in the labeling of the other components. The arginine specific reagent, butanedione, inhibited Mg²⁺-ATPase and K⁺-ATPase activities, with the former being much less reactive. Similar to 2-methoxy-2,4-diphenyl-3-dihydrofuranone, ATP provided complete protection from butanedione treatment. It is concluded that amino and guanidino groups are critical to the function of the K⁺-ATPase and may be actually at the ATP binding site.     

Multiple effect of hydroxylamine on mushroom tyrosinase

Mushroom tyrosinase is affected by hydroxylamine (NH₂OH) in several ways. At relatively low concentrations (up to 33 mM) NH₂OH shortens the lag period of tyrosine hydroxylation. The o-dihydroxyphenolase activity of mushroom tyrosinase is slightly stimulated by short exposure to relatively low concentrations ofNH₂OH (1.5 mM). Relatively high concentrations ofNH₂OH (above 20 mM) inhibit the o-dihydroxyphenolase activity of the enzyme and lowers the extent of final pigment production. Preincubation of mushroom tyrosinase with different concentrations ofNH₂OH for different times results in the inactivation of the enzyme. The rate of inactivation occurred much faster under anaerobic than under aerobic conditions. It was also found that NH₂OH changes the spectra of o-quinones prepared chemically or of products formed during the oxidation of o-dihydroxyphenols by mushroom tyrosinase. These spectral changes were attributed to the formation of oximes (mono- or dioximes) as a result of an interaction between o-quinones and NH₂OH. The apparent inhibition exerted by NH₂OH on the o-dihydroxyphenolase activity of mushroom tyrosinase is, in part, due to spectral changes in pigmented product formation and, in part, due to the inactivation of the enzyme by NH₂OH.     

Calcium inhibition of rat liver microsomal calcium-dependent ATPase

Measurement of the inward rate of Ca2+ transport by rat liver microsomes under conditions of varying free intravesicular Ca2+ (1 microM to 5 mM) revealed that inward transport rate is maximum at low intravesicular Ca2+, and that transport rate decreases with an apparent inhibition constant of about 250-350 microM as intravesicular Ca2+ accumulates. This relationship is confirmed by measurement of Ca2+-dependent ATPase activity; activity is greatest when intravesicular Ca2+ is 1 microM, is lower when intravesicular Ca2+ is 60 microM, and is minimum when intravesicular Ca2+ is 5 mM. Unexpectedly, the ratio of Ca2+ transport rate to Ca2+-dependent ATP hydrolysis rate appears to be significantly greater than 2:1.     

The mutagenic effect of hydroxylamine onMycobacterium phlei strain PA

The mutagenic effect of 0.05m and 1m HA onMycobacterium phlei PA was investigated. To establish the mutagenic effect the inactivating effect was studied under the same experimental conditions. Hydroxylamine at a higher concentration (1m) exhibited relatively high mutagenic effect. This was indicated by about 100-fold and 10-fold higher frequency of INH^r and STM^r mutants, respectively (as compared with spontaneous mutations) and induction of auxotrophic mutants. On the other hand, the mutagenic effect of 0.05m hydroxylamine was low under the same experimental conditions. The inactivating effect of a higher HA concentration (1m under given experimental conditions) was considerably higher when using the given model microorganism than that of the lower one (0.05m under the same experimental conditions). This finding does not agree with literature data obtained in other experimental models.