ABSTRACT
De novo protein synthesis and the heat-shock response during different stages of bacterial culture of Mycobacterium smegmatis LR222 were investigated. A discontinuance in the increase in number of colony forming units occurred at mid-exponential phase of growth. This coincided with a plateau in the ATP content of the culture, a reduction in the synthesis of exponential phase proteins (58, 30.5, and 20 kDa), a transitory synthesis of a 32 kDa protein and the induction of stationary-phase proteins (48, 46, 31, 25, and 20 kDa). The response to heat shock showed a growth-phase dependency, with the highest fold-induction of the 75 kDa (DnaK) protein occurring during the transitory cessation in the increase in CFU, while the greatest increase of the 95 kDa, 66 kDa (GroEL), and approximately 17 kDa (a doublet) proteins occurred during stationary phase. The approximately 17 kDa doublet was resolved into four polypeptides by two-dimensional electrophoresis. Mass spectrometric analysis of the sequence of one polypeptide (named Hsp17-2, 16.8 kDa) revealed significant homology to a conserved, 16.2 kDa, hypothetical protein of unknown function in Mycobacterium tuberculosis H37Rv. The increased synthesis of Hsp17-2 in response to heat shock suggests that it may represent a new low molecular weight heat shock protein.
Subject(s)
Bacterial Proteins/isolation & purification , Heat-Shock Proteins/isolation & purification , Mycobacterium smegmatis/metabolism , Adenosine Triphosphate/metabolism , Amino Acid Sequence , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Colony Count, Microbial , Crystallins/biosynthesis , Electrophoresis, Polyacrylamide Gel , Heat-Shock Proteins/chemistry , Heat-Shock Proteins/metabolism , Humans , Molecular Sequence Data , Molecular Weight , Mycobacterium smegmatis/growth & developmentABSTRACT
Carboxypeptidases are proteases that cleave single amino acids from the carboxy termini of proteins or peptides. In addition to degradative functions in the gut, carboxypeptidases activate or inactivate bioactive peptides such as angiotensin, bradykinin, and endothelin I. Using differential display PCR, we cloned a novel carboxypeptidase expressed in human macrophages but not in other leukocytes. The 476-amino-acid gene product has a putative signal sequence but no transmembrane domain and has striking sequence similarity to serine carboxypeptidases, a large family of enzymes in eukaryotes. Only one serine carboxypeptidase, lysosomal protective protein, has previously been reported in mammals. Among known proteins, this gene is most similar (43% amino acid identity) to vitellogenic carboxypeptidase, a serine carboxypeptidase expressed in mosquito ovaries. Therefore, we have named this new gene carboxypeptidase, vitellogenic-like (CPVL). In addition to monocyte/macrophage-rich sources such as spleen, leukocytes, and placenta, CPVL mRNA is abundantly expressed in heart and kidney, suggesting a separate role for CPVL outside the immune system. The CPVL gene contains at least 13 exons spread over more than 150 kb on human chromosome 7p14-p15. An affinity-purified polyclonal antiserum recognized a protein of approximately 57 kDa in macrophage lysates, but not in lysates from lymphocytes, neutrophils, or monocytes. CPVL protein expression was induced during maturation of monocytes into macrophages. Possible functions for CPVL in macrophages include digestion of phagocytosed particles in the lysosome, participation in an inflammatory protease cascade, and trimming of peptides for antigen presentation.
Subject(s)
Carboxypeptidases/genetics , Macrophages/enzymology , Amino Acid Sequence , Base Sequence , Blotting, Northern , Cell Line , Cells, Cultured , Cloning, Molecular , DNA, Complementary/chemistry , DNA, Complementary/genetics , Female , Gene Expression , HeLa Cells , Humans , Jurkat Cells , Molecular Sequence Data , RNA, Messenger/genetics , RNA, Messenger/metabolism , Sequence Alignment , Sequence Analysis, DNA , Sequence Homology, Amino Acid , Tissue Distribution , Tumor Cells, Cultured , U937 CellsABSTRACT
Tumour necrosis factor alpha (TNF alpha) has been reported to play a key role in the pathogenesis of sepsis and chronic inflammatory diseases, including rheumatoid arthritis and atherosclerosis, suggesting that agents which inhibit TNF alpha production may have therapeutic utility for the treatment of such conditions. Production of TNF alpha by LPS (lipopolysaccharide)-stimulated murine, rat and human heparinized blood was investigated. LPS (1-100 micrograms/ml) caused a similar concentration- and time-dependent stimulation of TNF alpha production by rat and human blood, achieving levels of 750-5000 U/ml (L929 bioassay) at 6 h. In contrast, TNF alpha production by LPS-stimulated murine blood was poor and variable (0-150 U/ml). Dexamethasone and pentoxifylline caused a concentration-dependent inhibition of TNF alpha production by LPS-stimulated human and rat blood with IC50s of 0.26 +/- 0.05 and 73.0 +/- 26.4 microM for human and 5.7 +/- 1.8 nM and 20.6 +/- 8.0 microM for rat blood, respectively. Therefore, LPS-stimulated rat and human, but not murine, blood are suitable systems for the detection and evaluation of inhibitors of TNF alpha production.
