Biomedical subjects

M Ambili

Publications and source records attributed to M Ambili.

7 recordsLinked to original sources

PubMed2003

Structure-function relationship in serine hydroxymethyltransferase.

Serine hydroxymethyltransferase (SHMT), a pyridoxal-5'-phosphate (PLP)-dependent enzyme catalyzes the tetrahydrofolate (H(4)-folate)-dependent retro-aldol cleavage of serine to form 5,10-methylene H(4)-folate and glycine. The structure-function relationship of SHMT was studied in our laboratory initially by mutation of residues that are conserved in all SHMTs and later by structure-based mutagenesis of residues located in the active site. The analysis of mutants showed that K71, Y72, R80, D89, W110, S202, C203, H304, H306 and H356 residues are involved in maintenance of the oligomeric structure. The mutation of D227, a residue involved in charge relay system, led to the formation of inactive dimers, indicating that this residue has a role in maintaining the tetrameric structure and catalysis. E74, a residue appropriately positioned in the structure of the enzyme to carry out proton abstraction, was shown by characterization of E74Q and E74K mutants to be involved in conversion of the enzyme from an 'open' to 'closed' conformation rather than proton abstraction from the hydroxyl group of serine. K256, the residue involved in the formation of Schiffs base with PLP, also plays a crucial role in the maintenance of the tetrameric structure. Mutation of R262 residue established the importance of distal interactions in facilitating catalysis and Y82 is not involved in the formaldehyde transfer via the postulated hemiacetal intermediate but plays a role in stabilizing the quinonoid intermediate. The mutational analysis of scSHMT along with the structure of recombinant Bacillus stearothermophilus SHMT and its substrate(s) complexes was used to provide evidence for a direct transfer mechanism rather than retro-aldol cleavage for the reaction catalyzed by SHMT.

Binding Sites↗

PubMed1999

Modulation of neutral matrix metalloproteinases of involuting rat mammary gland by different cations and glycosaminoglycans.

The synthesis and regulation of the matrix metalloproteinases (MMPs) are important factors contributing to the involution of mammary gland. In order to understand the role of these MMPs in involution and in remodeling of the mammary gland, the different MMPs (130K, 68K, and 60K gelatinases) were partially purified by gel filtration and affinity chromatography over gelatin Sepharose and subjected to kinetic analysis. Comparative analysis of the different gelatinases showed that the 130K that appears at the early involuntary phase and the constitutive 68K enzyme are more specific for Col IV of the basement membrane, while the inducible 60K that appeared at the later phase of involution degraded Col I more efficiently. These neutral proteinases required Ca2+/Zn2+ for their activity and the analysis of cation dependence revealed that Ca2+ at 10 mM concentration and above completely inhibited the enzyme. The 60K was active at very low concentration of Zn2+ (5 microM); but at higher concentration of Zn2+ (2 mM), where the 68K and 130K were active, the 60K gelatinase was inhibited, indicating a difference in the cation dependence of these enzymes. Chondroitin sulfate A and chondroitin sulfate C caused inhibition of the 130K, 68K, and 60K, while hyaluronic acid and heparin did not show any effect, suggesting that the chondroitin sulfate proteoglycan that decorates collagen in the ECM can modu late the activity of the collagenases in vivo. These results suggest that the 130K gelatinase expressed during the early phase of involution degraded Col IV of the basement membrane, making the 60K gelatinase formed at a later stage of involution more accessible to its preferred substrate (Col I of the underlying stroma), highlighting the role of these MMPs in mammary gland involution.

Animals↗

PubMed1999

Matrix metalloproteinase in mammary gland remodeling-modulation by glycosaminoglycans.

Mammary gland which undergoes proliferation, differentiation and involution in adult life is a useful model system to study the role of extracellular matrix (ECM) in regulating tissue specific functions. The involution that follows weaning results in the suppression of casein gene expression, collapse of alveolar structures and degradation of basement membrane as evidenced by biochemical analysis of matrix components like proteoglycans and collagen. Differential expression of three different MMPs viz. 130 K, 68 K and 60 K with varying specificity to Col IV of basement membrane and Col I of stroma, their selective inhibition by TIMP and proteoglycans and modulation by estrogen highlight the importance of these in the remodeling of the ECM in the mammary gland. The inhibition of these MMPs by glycosaminoglycans, particularly CS and change in the concentration of CS at different stages of mammary gland development suggests the existence of a novel mechanism for the regulation of the activity of MMPs at extracellular sites.

Animals↗

PubMed1998

60K gelatinase involved in mammary gland involution is regulated by beta-oestradiol.

Cell matrix interactions are critical in the expression and maintenance of differentiated functions in mammary gland. Matrix metalloproteinases (MMPs), by acting on different matrix components, contribute to the remodelling of extracellular matrix. Of the three major gelatinases found in rat mammary gland at different stages of ontogeny, 60K gelatinase, a Ca2+ dependent neutral MMP, seems to be involved in involution, as it appears at the late stage of involution. Further investigations on its regulation by hormones which influence the mammary gland function were carried out. Administration of beta-oestradiol caused the appearance of 60K gelatinase on the 2nd day of involution, while in untreated controls this activity was absent. On treatment of mammary epithelial cells of the 2nd day involuting tissue in culture with beta-oestradiol, the 60K gelatinase activity appeared, while the untreated controls did not show the activity. The effect of beta-oestradiol was studied further by metabolic labelling of the epithelial cells from the 2nd day involuting tissue. A concentration dependent increase in the amount of radiolabelled 60K gelatinase was found on treatment with beta-oestradiol. The upregulation of the 60K gelatinase activity in vivo was also found by immunocytochemical staining of the beta-oestradiol treated tissues. The effect of beta-oestradiol appears to be specific for 60K as the activity of other gelatinases (130K and 68K) in the mammary gland were not affected. Furthermore, a drastic regression of the mammary gland as evidenced by histochemical analysis and a marked decrease in the milk protein production in beta-oestradiol treated tissues indicated the onset of early involution. These results indicate that the 60K gelatinase which is upregulated during involution or on induction of early involution may play a key role in remodelling of extracellular matrix in mammary gland and further that this enzyme is subject to modulation by beta-oestradiol.

Animals↗

PubMed1998

60K gelatinase in involuting rat mammary gland is produced as a 90K proenzyme.

The matrix metalloproteinases appear to play a key role in mammary tissue remodeling during involution. By immunoprecipitation and immunoblot using antibodies against 60K gelatinase which appears during involution a 90K polypeptide has been identified as its inactive proenzyme in the early involuting rat mammary gland. 90K polypeptide was isolated from the second day involuting rat mammary gland by immunoaffinity chromatography. On proteolytic digestion, the inactive 90K polypeptide was converted to active gelatinase. Primary cultures of mammary epithelial cells secreted the 90K polypeptide. These results indicated that the 60K inducible MMP involved in mammary gland involution and remodeling is produced as a 90K proenzyme which is activated by proteolysis in the extracellular sites.

Animals↗

PubMed1998

Assay of matrix metalloproteinases in substrate impregnated gels in multiwells.

A zymographic method for the assay of matrix metalloproteinases in substrate impregnated gels in multiwells has been developed for the analysis of a large number of samples at a time. Enzyme was copolymerized with 300 microliters of 10% acrylamide impregnated with gelatin substrate and incubated for 16 hr. The gels were stained with coomassie blue, destained with water and the intensity measured in a densitometer. This method was tested with pure bacterial collagenase and three different gelatinases purified from rat mammary gland. The characteristics of these enzymes such as cation dependence, inhibition and concentration dependence have been examined by this method.

Animals↗

PubMed1997

Characteristics of a 60K gelatinase involved in rat mammary gland involution.

In order to study the role of matrix degrading enzymes in modulating cell matrix interaction, an understanding of the characteristics and regulation of their activity is useful. A number of matrix degrading metalloproteinases are involved in modulating the cell-ECM interactions during the involutory phase of mammary gland resulting in its remodelling. Zymographic studies showed that three types of gelatinases (60K, 68K and 130K) occur during the different phases of involution. The 60K gelatinase which appeared on the fifth day of involution has been purified by affinity chromatography over gelatin sepharose. Zymographic and radiolabelled substrate digestion studies at different pH and in presence of different cations showed that the activated form of this gelatinase is a Ca2+ dependent neutral matrix metalloproteinase capable of cleaving collagen I and collagen IV. Immunocytochemical studies showed that the enzyme is localised at pericellular/extracellular sites.

Animals↗