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Published on: November 23, 2016
Studies on the soluble and membrane-bound amino acid 2-naphthylamidases in pig and human epidermis
Abstract:
1. Membrane-bound (particulate) and soluble amino acid 2-naphthylamidases (EC 3.5.1.-) were present in subcellular fractions of epidermis from pig and human. 2. The particulate enzymes exhibited Michaelis-Menten kinetics, with Km 5.1x10(-5) (pig) and Km 7.3x10(-5)M (human) for the substrate L-leucine 2-naphthylamide. They were inhibited by puromycin and partially inhibited by EDTA. They did not require heavy metals and were not inhibited by thiol-group-blocking agents. Their pH optima were 7.0 (human) and 6.6 (pig). The particulate enzyme from pig epidermis retained 50% activity after 30 min at 70 degrees C. 3. The soluble amino acid 2-naphthylamidases gave sigmoidal curves for reaction velocity versus substrate concentration, and the kinetic data suggested that there was positive co-operativity between binding sites. This co-operativity was lost after treatment with 0.1mM-p-hydroxymercuribenzoate and the enzymes showed first-order kinetics at low substrate concentrations. The soluble enzymes were inhibited by puromycin and by thiol-group-blocking agents and activated by dithiothreitol. They were inactivated above 60 degrees C and lost activity on storage, but this could be restored with dithiothreitol. 4. The amino acid 2-naphthylamidases of human epidermis were much more active (2.5 times) towards L-alanine 2-naphthylamide than towards the commonly used substrate L-leucine 2-naphthylamide. 5. The kinetics of both the solube and particulate enzymes from epidermis of some elderly patients with either diabetes or ischaemia showed some differences from the kinetics of enzymes from healthy epidermis from younger individuals.
Insights
This study characterizes amino acid 2-naphthylamidases in human and pig epidermis. Differences in soluble and particulate enzyme kinetics were observed, with potential implications for age-related skin conditions.
Area of Science:
- Biochemistry
- Enzymology
- Dermatology
Background:
- Amino acid 2-naphthylamidases are enzymes found in various tissues, including the epidermis.
- Understanding their properties is crucial for comprehending skin physiology and pathology.
Purpose of the Study:
- To isolate and characterize membrane-bound (particulate) and soluble amino acid 2-naphthylamidases from pig and human epidermis.
- To investigate the kinetic properties, substrate specificity, and stability of these enzymes.
- To explore potential differences in enzyme kinetics in elderly patients with diabetes or ischemia.
Main Methods:
- Subcellular fractionation to isolate particulate and soluble enzyme fractions.
- Enzyme kinetic assays using L-leucine 2-naphthylamide and L-alanine 2-naphthylamide as substrates.
- Determination of kinetic parameters (Km, Vmax), pH optima, and thermal stability.
- Assessment of inhibition by puromycin, EDTA, and thiol-group-blocking agents, and activation by dithiothreitol.
Main Results:
- Particulate enzymes followed Michaelis-Menten kinetics, while soluble enzymes exhibited sigmoidal kinetics indicative of positive co-operativity.
- Soluble enzymes were sensitive to thiol modification and required reducing agents for stability.
- Human epidermal enzymes showed higher activity towards L-alanine 2-naphthylamide compared to L-leucine 2-naphthylamide.
- Differences in enzyme kinetics were noted in elderly patients with diabetes or ischemia.
Conclusions:
- Distinct kinetic and biochemical properties differentiate particulate and soluble amino acid 2-naphthylamidases in the epidermis.
- The observed differences in enzyme characteristics may be relevant to understanding epidermal function and age-related diseases.
- Further research is warranted to elucidate the specific roles and regulatory mechanisms of these enzymes in skin health.
