Bovine Lens Leucine Research Articles

Sulfhydryl content of bovine eye lens leucine aminopeptidase. Determination of the reactivity of the sulfhydryl groups of the zinc metalloenzyme, of the enzyme activated by Mg2+, Mn2+, and Co2+, and of the metal-free apoenzyme.

The reactivity of the sulfhydryl groups of leucine aminopeptidase from bovine eye lens has been studied by carboxymethylation with iodoacetate of the native enzyme (Zn2+-Zn2+), of the enzyme activated with magnesium (Zn+-Mg2+) or manganese (Zn2+-Mn2+), the enzyme incubated with cobalt (CO2+-Co2+), and the metal-free apoenzyme. The reactivity of the sulfhydryl groups was also determined in the presence of denaturing agents with or without reducing agents. All seven half-cystines per leucine aminopeptidase subunit are in the sulfhydryl form. In the native and the Zn2+-Mn2+ enzyme, only one of the cysteines (residue 344) reacts readily with iodoacetate. In the Zn2+-Mg2+ enzyme, two cysteines react (residues 344 and 412) and in the Co2+-incubated enzyme only one (residue 412). The metal-free apoenzyme has at least three reactive cysteines (residues 344, 412, and 429). The data suggest that sulfhydryl groups are involved in the binding of metal ions.

Leucine Aminopeptidase from Bovine Eye Lens

Specific modification of bovine eye lens leucine aminopeptidase by the substrate-like and radioactive tagged diazonium peptide inhibitor, L-(2,3-3H)Phe(pN2+)-L-Phe, was investigated using the technic of affinity and differential labelling. The degree of labelling and the loss of activity increased with excess of the label. A competitive inhibitor diminished the degree of azo-coupling and also the degree of inactivation. First results of tryptic digestion of labelled LAP are given.

The suitability of bovine-lens leucine aminopeptidase for sequence analysis and limited hydrolysis of polypeptides.

In the present paper we report findings showing the suitability of recrystallized bovine‐lens leucine aminopeptidase as a tool for peptide chemistry.The enzyme is homogeneous with respect to hydrodynamic, chromatographic and electrophoretic separation criteria. It is not contaminated by endopeptidases.The insulin B‐chain (S‐sulfonate) served as model substrate for sequence studies. The initial progress curves of the liberation of the first six amino acids resulted in a sequence expected from the known primary structure. The complete resistance of imido bonds towards the action of leucine aminopeptidase has been proved.Many commercially available protein preparations are found to be contaminated by endopeptidases. We developed an endopeptidase‐detection method in which the leucine aminopeptidase serves as a sensitivity amplifier.