Crystallization and preliminary crystallographic study of carnosinase CN2 from mice.

Mammalian tissues contain several histidine-containing dipeptides, of which L-carnosine is the best characterized and is found in various tissues including the brain and skeletal muscles. However, the mechanism for its biosynthesis and degradation have not yet been fully elucidated. Crystallographic study of carnosinase CN2 from mouse has been undertaken in order to understand its enzymatic mechanism from a structural viewpoint. CN2 was crystallized by the hanging-drop vapour-diffusion technique using PEG 3350 as a precipitant. Crystals were obtained in complex with either Mn(2+) or Zn(2+). Both crystals of CN2 belong to the monoclinic space group P2(1) and have almost identical unit-cell parameters (a = 54.41, b = 199.77, c = 55.49 A, beta = 118.52 degrees for the Zn(2+) complex crystals). Diffraction data were collected to 1.7 and 2.3 A for Zn(2+) and Mn(2+) complex crystals, respectively, using synchrotron radiation. Structure determination is ongoing using the multiple-wavelength anomalous diffraction (MAD) method.

Proteomic analysis of slow- and fast-twitch skeletal muscles.

Skeletal muscles are composed of slow- and fast-twitch muscle fibers, which have high potential in aerobic and anaerobic ATP production, respectively. To investigate the molecular basis of the difference in their functions, we examined protein profiles of skeletal muscles using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and two-dimensional gel electrophoresis with pH 4-7 and 6-11 isoelectric focusing gels. A comparison between rat soleus and extensol digitorum longus (EDL) muscles that are predominantly slow- and fast-twitch fibers, respectively, showed that the EDL muscle had higher levels of glycogen phosphorylase, most glycolytic enzymes, glycerol 3-phosphate dehydrogenase, and creatine kinase; while the soleus muscle had higher levels of myoglobin, TCA cycle enzymes, electron transfer flavoprotein, and carbonic anhydrase III. The two muscles also expressed different isoforms of contractile proteins including myosin heavy and light chains. These protein patterns were further compared with those of red and white gastrochnemius as well as red and white quadriceps muscles. It was found that metabolic enzymes showed a concerted regulation dependent on muscle fiber types. On the other hand, expression of contractile proteins seemed to be independent of the metabolic characteristics of muscle fibers. These results suggest that metabolic enzymes and contractile proteins show different expression patterns in skeletal muscles.

Identification and characterization of a mouse dipeptidase that hydrolyzes L-carnosine.

L-Carnosine is a bioactive dipeptide present in mammalian tissues including the central nervous system. We have recently shown that L-carnosine is involved in the regulation of energy homeostasis through the autonomic nervous system, but the mechanisms for its biosynthesis and degradation have not yet been fully elucidated. Here we report the biochemical and immunohistochemical characterization of a mammalian protein that has a 17% overall amino acid sequence homology with a Lactobacilus carnosinase, PepV. A recombinant protein expressed in E. coli has the enzymatic ability to digest L-carnosine and various other dipeptides, and this activity is inhibited by bestatin. It requires Mn2+ for enzymatic activity and its effect is reversible. Immunohistochemical analysis showed that a few neuronal populations express this protein at very high levels. It is highly expressed in the parafascicular nucleus of the thalamus, tuberomammillary nucleus of the hypothalamus and the mitral cell layer of the olfactory bulb. In addition, neuronal processes, but not cell bodies, are stained in the striatum. In all these areas, the protein did not colocalize with the glial fibrilary acidic protein. These results suggest that a peptidase that digests L-carnosine is enriched in several specific neuronal populations in the central nervous system.