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Biomedical subjects

M D Maxwell

Publications and source records attributed to M D Maxwell.

12 recordsLinked to original sources
PubMed1992

Identification of transglutaminase substrates in HT29 colon cancer cells: use of 5-(biotinamido)pentylamine as a transglutaminase-specific probe.

A biotinamine probe, 5-(biotinamido)pentylamine, was used for biotin-labeling of proteins in HT29 colon cancer cell extracts by endogenous transglutaminase activity. The biotin-labeled protein substrates were isolated and recovered by avidin-affinity chromatography. The proteins were separated using SDS-polyacrylamide gel electrophoresis, electroblotted onto a polyvinylidene difluoride membrane, visualized using Coomassie blue, cut out, and sequenced. Amino acid sequence data identified human fructose-1,6-bisphosphate aldolase A, an intracellular protein, as a substrate for cellular transglutaminase.

Amines↗
PubMed1991

Localization of cellular transglutaminase on the extracellular matrix after wounding: characteristics of the matrix bound enzyme.

Extending our previous observation that tissue transglutaminase (TGase) binds to extracellular matrix (ECM) fibronectin, we report here that endogenous tissue TGase is localized on the adjacent ECM after puncture wounding embryonic human lung fibroblasts (WI-38). The bound TGase persisted at the wound site for many hours, demonstrated by immunofluorescence and by catalytic activity using an overlay assay. The binding characteristics of TGase with ECM were studied further by the addition of exogenous TGase to cell monolayers and monitoring by immunofluorescence or overlay catalytic activity assays. Binding occurred equally well at 4 degrees C or 37 degrees C. Prior incubation of exogenous TGase with guanosine 5'-triphosphate (GTP), guanosine 5'-diphosphate (GDP), or adenosine triphosphate (ATP) had little effect on the amount bound to matrix, but prior treatment with calcium, magnesium, strontium, or manganese ions enhanced binding 2- to 3-fold. The Ca(++)-dependent change was a concentration-dependent effect on soluble exogenous TGase, rather than an effect on ECM. Immunofluorescent techniques showed that binding of exogenous TGase to ECM was prevented by prior mixing with fibronectin or collagen, but not with several other ECM components, including laminin, elastin, chondroitin sulfate, heparan sulfate, and hyaluronic acid. ECM-bound TGase was released by 2 M potassium thiocyanate (KSCN) treatment but was not released by treatment with a variety of amino acids, salts, reducing agents, glycerol, or other chaotropic agents.

Adenosine Triphosphate↗
PubMed1987

Cellular transglutaminase has affinity for extracellular matrix.

Cellular transglutaminase (TGase) was demonstrated as an intracellular enzyme by immunofluorescence in WI-38 cells. Following cell membrane perturbation by Triton X-100 treatment, TGase was bound to the extracellular matrix and was found to coexist with fibronectin as visualized by immunofluorescence microscopy. The binding of TGase to the cell matrix was blocked by anti-fibronectin antibody. Exogenous sources of soluble TGase were transferred to the extracellular matrix of an untreated or methanol fixed cell. The experimental data indicated that "particulate bound" TGase is a consequence of soluble TGase binding to the extracellular matrix following cell rupture.

Extracellular Matrix↗
PubMed1983

Enhanced transglutaminase activity in transformed human lung fibroblast cells after exposure to sodium butyrate.

The low level of transglutaminase activity in virus-transformed human embryonic lung fibroblasts (WI-38 VA13A) increased markedly when cells were exposed to sodium butyrate. The effect of sodium butyrate was time- and concentration-dependent and fully reversible. Transformed cells exposed for 5 days to 1 mM sodium butyrate had fewer cells, showed an 8-10 fold higher transglutaminase activity, and stained more abundantly for transglutaminase and pericellular fibronectin than control cells when examined by indirect immunofluorescence. Non-transformed cells (WI-38) showed only a 2-4-fold increase in transglutaminase activity when treated in a similar manner. Studies with metabolic inhibitors revealed the increase in activity was the result of synthesis of new protein. Kinetic studies showed the affinity of putrescine for the enzyme was essentially unchanged but the number of active sites increased 9-fold following exposure to sodium butyrate. Enhanced transglutaminase activity returned to control levels within 7 days after subculture and sodium butyrate removal. These findings suggest that sodium butyrate offers a potential model system to understand better the role of transglutaminase in cells in culture; particularly growth control in transformed cells.

Acyltransferases↗
PubMed1975

Demonstration of acid phosphatase-containing vacuoles in hyphal tip cells of Sclerotium rolfsii.

A lysosomal system was demonstrated in hyphal tip cells of Sclerotium rolfsii by light and electron microscopy observations of the sites of acid phosphatase activity visualized by a modified Gomori lead nitrate method. The cytochemical reaction product was found to be present in numerous vacuoles, each aout 0.5 mum in diameter, which were seen as chains of spheres when viewed with the light microscope. They usually did not occur in the first 30 to 40 mum of the hyphal tip cell, but were concentrated in a zone extending from 30 to 200 mum from the hyphal apex. As shown by the electron microscope, the vacuoles were sometimes interconnected by narrow channels. Acid phosphatase reaction product was also occasionally localized in vacuoles of the older hyphal cells, but never in apical vesicles, lipid bodies, or microbodies. It is proposed that this vacuolar system may orginate from the endoplasmic reticulum.

Acid Phosphatase↗