Abstract
Hep-G2 cells, incubated with L-[35S]methionine, incorporate radioactivity into fibrinogen and several fibrinogen-related compounds. Pulse-chase experiments indicate that several of these compounds are precursors of fibrinogen and that the cells contain intracellular pools of A alpha and gamma chains which participate in the assembly of fibrinogen. The rate of synthesis of the three component chains of fibrinogen is unequal with that of the B beta chain being less than that of the A alpha and gamma chains. The sequence of events which lead to the assembly of fibrinogen was deduced by determining the appearance of the radioactive chains in each of the fibrinogen precursors and in fibrinogen at various times during a pulse-chase incubation. Fibrinogen assembly commences while nascent incomplete B beta chains are attached at polysomes. Preformed A alpha and gamma chains, drawn from the intracellular pool, combine independently to the growing B beta chains. On completion of the nascent B beta chains, newly formed B beta-A alpha and B beta-gamma complexes are released from the polysomes and enter the luminal space of the endoplasmic reticulum. Later other A alpha and gamma chains are added by ordered disulfide interaction, leading to the eventual formation of dimeric fibrinogen.
Highlights
Corporate radioactivity into fibrinogen andseveral fi- In the present study the precursors are partially identified brinogen-related compounds.Pulse-chaseexperiments indicate that several of these compounds are precursors of fibrinogen and that the cells contain intracellular pools ofAa and y chains which participate in the assembly of fibrinogen
The synthesis of the Bj3 chains lagged behind that of the A a and y chains for the initial 10 minand the rateappeared to equal that of the otherchains during the subsequent 5 min
In the formation of fibrinogen and that radioactive A a and y Precursor Forms of Fibrinogen-To determine which of the chains persist. These results suggest the presence of intracel- intracellular radioactiveproteins are precursors of fibrinogen, lular pools of both A a and y chains which participate in the Hep-G2 cellswere pulse labeled for3 min with ~ - [ ~ S ] methi
Summary
In pulse-chase experiments the pulse-labeled cells were plexes are released from the polysomes and enter the washed twice with phosphate-buffered saline and “chase” incubated luminalspaceoftheendoplasmicreticulum.Later a t 37 “Cfor various periods of time with fresh medium containing 20 other Aa and y chains are added by ordered disulfide interaction, leading to the eventual formation of dimeric fibrinogen. Forms-The cella, incubated with L-[%3]methionine, were washed with phosphate-buffered saline containing 2 mM EDTA’, 100 units per ml of Trasylol (aprotinin), 1mM PMSF, and 0.1 mM TPCK.
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