Carboxypeptidase E activity is deficient in mice with the fat mutation. Effect on peptide processing

Area of Science:

• Biochemistry
• Neuroscience
• Genetics

Background:

• Carboxypeptidase E (CPE) is crucial for peptide hormone and neurotransmitter biosynthesis.
• Mice with the 'fat' mutation exhibit reduced CPE activity in the pituitary and pancreatic islets.
• Previous research identified a point mutation in the cpe gene in these mice.

Purpose of the Study:

• To investigate CPE-like activity and peptide processing in various tissues of C57BLKS/LtJ-Cpefat/Cpefat mutant mice.
• To determine the specific carboxypeptidases responsible for residual activity in mutant mice.
• To elucidate the role of CPE in the processing of specific peptide hormones, such as Leu-enkephalin.

Main Methods:

• Assay of CPE-like enzyme activity in tissue homogenates.
• Sensitivity testing using p-chloromercuriphenyl sulfonate.
• Substrate affinity chromatography to assess binding properties.
• Enzyme activity and immunoreactivity analysis of purified brain enzymes.
• Quantification of Leu-enkephalin levels and enkephalin-containing peptides.

Main Results:

• The majority of CPE-like activity in mutant mice is not due to CPE and shows different sensitivities and binding properties.
• Purified enzyme activity from mutant brain is distinct from CPE.
• Leu-enkephalin levels are significantly reduced in mutant mouse brain.
• High molecular weight enkephalin-containing peptides are elevated in mutant mouse brain.
• Treatment with carboxypeptidase B restores Leu-enkephalin levels in mutant brain extracts.

Conclusions:

• CPE is completely inactive in Cpefat/Cpefat mice.
• Residual CPE-like activity in mutant mice is attributed to other carboxypeptidases, potentially carboxypeptidase D.
• CPE plays a vital role in efficient peptide hormone processing, particularly in conjunction with endopeptidases.
• Other carboxypeptidases contribute to peptide processing, but CPE is essential for optimal enkephalin peptide maturation.