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Leucine Aminopeptidase (LAP)

(L-Leucyl-peptide hydrolase) Leucine aminopeptidase (LAP) is a proteolytic enzyme which hydrolyzes the peptide bond adjacent to a free amino group. It is called leucine aminopeptidase because it rapidly catalyzes the hydrolysis of leucine containing peptides. However, it also catalyzes the hydrolytic release of other amino acids located at the N-terminal end of various peptides and proteins. The enzyme from porcine kidney has been extensively studied. It has a molecular weight of 255,000 and it consists of four subunits each having one atom of zinc. (Himmelhock, S.R., Arch. Biochem. Biophys., 134, 597, 1969).

Determination of microsomal leucine amino peptidase activity in serum is of clinical significance, since serum LAP levels are elevated in obstructive jaundice, liver cirrhosis, liver carcinoma and also during the later part of pregnancy (Methods of Enzymatic Analysis, Bergmeyer, H.U. ed., Vol. I, 26, 1975, Academic Press, New York). Leucine amino-peptidase is also extensively used in determination of the amino acid sequence of proteins and peptides.

1.Source:	Porcine Kidney
Form:	Ammonium Sulfate
Solubility:	Soluble in distilled water or dilute buffer
Stability:	Stable when stored at 4°C. Do not freeze
Activity (approx.):	>100 U/mg protein
Protein (approx.):	90% (biuret)
Catalog No.:	089B0100

$5.00/mg

2.Source:	Porcine Kidney Microsomes
Form:	Freeze-dried
Solubility:	Soluble in distilled water or dilute buffer
Stability:	-20° C; -4° F
Activity (approx.):	20 U/mg protein
Protein (approx.):	90% (biuret)
Catalog No.:	152A0020

$.50/U

That amount of enzyme which catalyzes the hydrolysis of one micromole L-leucinamide per minute at 25°C at pH 8.5.

The assay method is based on the one described by Binkley and Torres (Binkley, F. and Torres, C., Arch. Biochem. Biophys., 86, 201, 1960). The rate of decrease in absorbance at 238 nm, resulting from the hydrolysis of L-leucinamide, is proportional to the catalytic activity of the enzyme.

0.5 M Tris-HCl buffer, pH 8.5.
0.025 M Maganese chloride.
0.0625 M Magnesium chloride.
0.06 M L-leucinamide-HCl (11 mg/ml) in buffer.
Leucine aminopeptidase (enzyme) solution. Dissolve in buffer to a concentration of 1000-2000 U/ml.

Set spectrophotometer (equipped with strip chart recorder and temperature control) at 238 nm and 25°C.
Enzyme treatment. In a test tube pipette the following:
Manganese chloride 0.4 ml
Tris buffer 2.5 ml
Enzyme (LAP) 0.1 ml

Incubate the mixture at 40°C for about 60 minutes. An aliquot from this treated enzyme mixture will be used in the assay system.
In a quartz cuvette, place the following in the amounts indicated:
Tris buffer 0.20 ml
Magnesium chloride 0.20 ml
L-leucinamide-HCl 2.50 ml

Place cuvette in spectrophotometer at 25°C for 5 minutes. Record absorbance at 238 nm (blank).
To initiate the reaction, add 0.10 ml of treated enzyme (step 2) to the cuvette. Follow the reaction by recording the decrease in absorbance at 238 nm for 5-8 minutes.
Calculate E238nm/min

Activity (U/mg) =	(ΔE_238nm/min)(Total Vol.)(Enz. Diln.)
	(0.011)(Enz. Vol.)(mg Enz./ml)

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