Robert A. Resnik

Aminopeptidase of the ocular lens I. Metal-ion requirements and synergistic activation

Abstract A quantitative assay for aminopeptidase was developed, using the chromogenic substrate, l -leucyl-β-naphthylamide. The enzyme from beef lens was purified about 200-fold and its properties were studied. The enzyme was activated by Co 2+ , Mg 2+ , or Mn 2+ , but maximal activity was observed only in the simultaneous presence of Co 2+ and Mn 2+ , or Co 2+ and Mg 2+ . This effect was termed synergistic activation. The activation was rapid even in crude homogenates, requiring generally less than 10 min. The metal-ion effects were shown to involve activation of the enzyme rather than the substrate. Distinct species differences were noted in the susceptibility of the lens enzyme to synergistic activation, with beef, hog, and sheep lenses possessing similar properties. The enzyme was very sensitive to inhibition by heavy-metal ions, reagents for sulfhydryl groups, and chelating agents. Exhaustive treatment of the enzyme with EDTA led to irreversible loss of activity, but if the chelator was removed before this stage was reached, most of the activity could be restored by the activation with Co 2+ plus Mn 2+ . Ca 2+ , Fe 2+ or Ni 2+ activated the enzyme partially, but could not replace Co 2+ or Mn 2+ for the synergistic effect. KCN stimulated the enzyme at 5.10 −5 −5.10 −3 M concentration, even after the peptidase had been freed of contaminating metals by treatment with EDTA. Co 2+ or Mn 2+ were able to afford the enzyme considerable protection against inhibition by mercurials, whereas Mg 2+ was not. The inhibition of the peptidase by sodium p -hydroxymercuribenzoate could be reversed by cysteine, glutathione, or mercaptoethanol. It appeared probable that Co 2+ is bound through a sulfhydryl group at the active site on the enzyme. Reexamination of the purified leucine aminopeptidase from hog kidney revealed that, after treatment with EDTA, it also exhibited some ability to be synergistically activated, and to be susceptible to inhibition by mercurials.

The oxidation of C14-labeled glucose and lactate by the rabbit cornea☆

Abstract The glucose oxidation of whole cornea was studied, using C 14 -labeled glucose and lactate. In addition, the effects of large metabolic pools on the radiochemical yield were observed. The results indicate that the citric acid cycle is active and confirm the importance of the shunt pathway. Denuded cornea retains one-third of its ability to oxidize lactate but loses 90 % of its capacity to oxidize glucose. Evidence is presented which indicates that the cornea may metabolize both glucose and lactate simultaneously.

The reaction of alloxan with glutathione and protein

Abstract The reaction of alloxan with GSH to form compound 305 has been studied. A direct correlation was found between the presence of a carbonyl group at position 5 and the reaction with GSH. The structure of compound 305 is discussed.

The copper complex of alloxanic acid

Abstract It seems unlikely that copper does complex with alloxan in view of the lability of alloxan with increase in pH and its transformation to alloxanic acid in alkaline solution. Hypsochromic and hyperchromic shifts in the spectrum of copper in the presence of alloxan and alloxanic acid are induced by increases in pH.

Extrinsic cotton effects of acridine orange bound to native, denatured and formylated deoxyribonucleic acid☆

Abstract Optical rotatory dispersion curves and absorption spectra were obtained for acridine orange complexes formed by native DNA, heat denatured DNA and formaldehyde-treated DNA over a wide range of the ratio of anionic sites to dye. With regard to the controversy over whether induced optical activity does or does not occur when denatured DNA is used, the evidence appears to favor the view that denaturation, regardless of the method used, modifies but does not prohibit the appearance of extrinsic Cotton effects.

Aminopeptidase of the ocular lens II. Substrate specificity

Abstract Evidence was sought on the identity of the lens enzyme hydrolyzing l -leucyl-β-naphthylamide with the soluble kidney leucine aminopeptidase (EC 3.4.1.1). During purification, leucinamidase activity was concentrated along with leucyl-β-naphthylamidase activity. The rate of hydrolysis of l -tryptophyl-β-naphthylamide also increased slightly upon purification, while that of l -ga-glutamimyl-β-naphthylamide remained constant, and that of l -alanyl-β-naphthylamide decreased markedly. Among several species examined, the l -leucyl compound was hydrolyzed more rapidly than the l -alanyl-β-naphthylamide only by beef, hog and sheep lenses. In six others the rate of l -alanyl-β-naphthylamide hydrolysis was 2 to 3 times that of l -leucyl-β-naphthylamide. In the ungulate-lens homogenates, l -α-glutamimyl-β-naphthylamide was less readily hydrolyzed compared with the other species. Synergistic activation by two metal ions was found with all substrates examined, and this strongly indicates that the effects of the metal ions were on the enzymes rather than on the substrates. By spectrophotometric assay the purified beef-lens enzyme was found capable of hydrolyzing l -alanyl- l -leucine twice as rapidly as l -leucinamide, while l -leucylglycine and l -alanyl- l -valine were cleaved at about the same rate as the standard substrate for aminopeptidase. Examination of kidney homogenates confirmed the findings for the lens enzyme in that hydrolysis of l -tryptophyl-β-naphthylamide appeared to be associated with that of l -leucyl-β-naphthylamide. l -α-glutaminyl-β-naphthylamide may be split by the same enzyme, but l -alanyl-gb-naphthylamide hydrolysis is due to a different enzyme.

Extrinsic cotton effects of acridine orange bound to apurinic acid

Abstract Optical rotatory dispersion curves and absorption spectra were obtained for acridine orange complexes formed by apurinic acid which was prepared from calf thymus DNA. The effects of ionic strength, of dilution and of the variation of the ratios of anionic sites to dye were examined. The analogies between the Cotton effect of the apurinic acid complexes and that of the complexes formed by native or denatured DNA and poly-α, L-glutamic acid were pointed out.