Cytomatrix synthesis in MDCK epithelial cells

Journal Article · Fri Jun 01 00:00:00 EDT 1990 · Journal of Cellular Physiology; (USA)

DOI:https://doi.org/10.1002/jcp.1041430315· OSTI ID:6643925

Mitchell, J J; Low, R B; Woodcock-Mitchell, J L ^[1]

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1. Univ. of Vermont, Burlington (USA)

Detailed information regarding the synthesis rates of individual protein components is important in understanding the assembly and dynamics of the cytoskeletal matrix of eukaryotic cells. As an approach to this topic, the dual isotope technique of Clark and Zak, was employed to measure fractional synthesis rates (FSRs) in growing and quiescent cultures of MDCK epithelial cells. Cell protein was labeled to equilibrium with (14C)leucine over several days and then pulse-labeled for 4 hours with (3H)leucine. FSRs (as percent per hour) were calculated from the 3H/14C ratio of cell extracts or individual proteins separated by two-dimensional polyacrylamide gel electrophoresis and the 3H/14C ratio of free leucine in the medium. Synthesis of total cell protein rose from approximately 1.4%/hour in quiescent cells to 3.5%/hour in the growing cultures. The latter rate was sufficient to account for the rate of protein accumulation and a low level of turnover in the growing cultures. The FSR of the buffered-Triton soluble extract was higher and the cytoskeletal FSR significantly lower than that for total protein in quiescent monolayers. This difference, however, was not observed in growing cultures. A distinct pattern of differences was seen in the FSRs of individual cytoskeletal proteins in the quiescent cultures. Vimentin synthesis was significantly lower than that of the keratins and the keratin FSRs were not obviously matched in pairwise fashion. Unexpectedly, the FSRs of alpha- and beta-tubulin diverged in quiescent cells with alpha-tubulin turnover exceeding beta-tubulin. Likewise, components of the microfilament lattice showed unequal fractional synthesis rates, myosin and alpha-actinin being faster than actin. In addition, the FSR for globular actin exceeded that of the cytoskeletal associated form.

• OSTI ID: 6643925
• Journal Information: Journal of Cellular Physiology; (USA), Vol. 143:3; ISSN 0021-9541
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
ANIMAL CELLS
BIOCHEMISTRY
PROTEINS
BIOSYNTHESIS
CARBON 14 COMPOUNDS
CELL CULTURES
DOGS
ELECTROPHORESIS
EPITHELIUM
KIDNEYS
LEUCINE
QUANTITY RATIO
TRACER TECHNIQUES
TRITIUM COMPOUNDS
AMINO ACIDS
ANIMAL TISSUES
ANIMALS
BODY
CARBOXYLIC ACIDS
CHEMISTRY
HYDROGEN COMPOUNDS
ISOTOPE APPLICATIONS
LABELLED COMPOUNDS
MAMMALS
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANS
SYNTHESIS
TISSUES
VERTEBRATES
550201* - Biochemistry- Tracer Techniques