Histidine Metabolism and Function.

Histidine is a dietary essential amino acid because it cannot be synthesized in humans. The WHO/FAO requirement for adults for histidine is 10 mg · kg body weight-1 · d-1. Histidine is required for synthesis of proteins. It plays particularly important roles in the active site of enzymes, such as serine proteases (e.g., trypsin) where it is a member of the catalytic triad. Excess histidine may be converted to trans-urocanate by histidine ammonia lyase (histidase) in liver and skin. UV light in skin converts the trans form to cis-urocanate which plays an important protective role in skin. Liver is capable of complete catabolism of histidine by a pathway which requires folic acid for the last step, in which glutamate formiminotransferase converts the intermediate N-formiminoglutamate to glutamate, 5,10 methenyl-tetrahydrofolate, and ammonia. Inborn errors have been recognized in all of the catabolic enzymes of histidine. Histidine is required as a precursor of carnosine in human muscle and parts of the brain where carnosine appears to play an important role as a buffer and antioxidant. It is synthesized in the tissue by carnosine synthase from histidine and ß-alanine, at the expense of ATP hydrolysis. Histidine can be decarboxylated to histamine by histidine decarboxylase. This reaction occurs in the enterochromaffin-like cells of the stomach, in the mast cells of the immune system, and in various regions of the brain where histamine may serve as a neurotransmitter.

Production of recombinant rat hepatic histidase / Producción de histidasa recombinante de hígado de rata

Rat liver histidase was expressed in E. coli by using a PCR product of the coding sequence obtained from the rat liver cDNA of histidase cloned in the expression vector pRSET. The construct (pRSET-HAL) produced a fusion protein containing a tail of polyhistidine. The expression product was purified with a resin containing Ni+ that retains proteins with polyhistidine fragments. pRSET-HAL was analyzed by restriction enzyme mapping and by sequencing confirming the correct orientation and nucleotide sequence. Native rat liver histidase was also purified and it had a Mr of 72 kDa. An antiserum against native histidase was obtained in rabbit. Western blot analysis revealed one band of 72 kDa observed in membranes containing purified histidase or rat liver high speed supernatants. The same antiserum also detected in cell lysates of E. coli transformed with the pRSET-HAL plasmid a single band of 74 kDa of the recombinant histidase before cleavage with enterokinase. After the proteolysis, the Western blot analysis showed a single band of approximately 72 kDa. Kinetic analysis of the recombinant histidase showed similar Km and Vmax compared with native histidase.

La histidasa de hígado de rata se expresó a partir del producto de amplificación por PCR de la región codificante del DNAc de la histidasa, el cual se clonó en el vector de expresión pRSET. La construcción (pRSET-HAL) permitió la producción de una proteína de fusión que contenía una cola de polihistidinas. El producto de expresión se purificó con una resina que contiene Ni+, la cual retiene proteínas con fragmentos de polihistidina. El pRSET-HAL se analizó por medio de un análisis con enzimas de restricción y por secuenciación de DNA para confirmar su correcta orientación así como tambièn su secuencia nucleotídica. La histidasa nativa hepática fue tambièn purificada y tuvo un Mr de 72 kDa. A partir de la enzima purificada nativa se preparó un antisuero contra la histidasa en conejo. El análisis por Western blot mostró una sola banda de 72 kDa en membranas que contenían histidasa purificada o en la fracción citosólica de hígado de rata. El mismo anticuerpo tambièn detectó en lisados de E. coli transformados con el plásmido pRSET-HAL una sola banda de 74 kDa correspondientes a la histidasa recombinante antes de su hidrólisis con enterocinasa. Despuès de la proteólisis, el análisis por Western blot mostró una sola banda de aproximadamente 72 kDa correspondiente a la histidasa. Análisis cinèticos de la histidasa recombinante mostraron Km y Vmax similares a las observadas con la histidasa nativa.