Relationship between protease activity and a sialoglycopeptide inhibitor isolated from bovine brain

Sharifi, Behrooz G.; Bascom, Charles C.; Fattaey, Heideh K.; Nash, S.; Johnson, Terry C.

doi:10.1002/jcb.240310106

Cited by 8 publications

(4 citation statements)

References 28 publications

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“…It has been shown that the sialoglycopeptide contains a unique protease activity that could not be dissociated from the inhibitory activity (Sharifi et al, 1986b). However, our data are inconsistent with the protease activity being responsible for mediating the biological effect of the sialoglycopeptide.…”

Section: Discussionmentioning

confidence: 45%

“…We have been interested in understanding the mechanisms of density-dependent regulation of cell growth for several years (Kinders et al, 1980;Kinders and Johnson, 1982;Johnson et al, 1985). We recently purified a 18,000 dalton sialoglycopeptide (PI 3.0) that inhibits protein synthesis and DNA synthesis of non-transformed but not transformed cells while not afl'ecting uptake of radiolabeled precursors (Sharifi et al, 1986a;Sharifi et al, 1986b).…”

mentioning

confidence: 99%

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Receptor occupancy by a bovine sialoglycopeptide inhibitor correlates with inhibition of protein synthesis

Bascom

Sharifi

Johnson

1986

Journal Cellular Physiology

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We have isolated from bovine cerebral cortex cells and purified to homogeneity an 18,000 dalton, pl 3.0 sialoglycopeptide that inhibits protein synthesis and DNA synthesis of nontransformed but not transformed cells without affecting uptake of radiolabeled precursors. In this paper, we examine the relationship between the binding of the sialoglycopeptide inhibitor to 3T3 cells and inhibition of protein synthesis. Binding of the sialoglycopeptide to 3T3 cells was rapid at 37 degrees C and reached a maximum at 30 min; the binding at 37 degrees C was shown to be saturable and specific. Scatchard analysis of the binding indicated that 3T3 cells contained about 2 X 10(4) receptors/cell with a dissociation constant of 1.0-1.5 nM. Several lines of evidence indicated that receptor occupancy on 3T3 cells correlated with the protein synthesis inhibitory activity of the sialoglycopeptide. A comparison of the kinetics of inhibitor binding with the kinetics of protein synthesis inhibition demonstrated that binding directly correlated with the inhibition of protein synthesis, concentration-dependent inhibition of protein synthesis directly correlated with concentration-dependent receptor occupancy, and a direct correlation was also observed between the kinetics of inhibitor dissociation from its specific cell surface receptor and the kinetics of recovery from protein synthesis inhibition.

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Section: Discussionmentioning

confidence: 45%

mentioning

confidence: 99%

Receptor occupancy by a bovine sialoglycopeptide inhibitor correlates with inhibition of protein synthesis

Bascom

Sharifi

Johnson

1986

Journal Cellular Physiology

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“…The purified 18 kD sialoglycopeptide has a PI of 3.0, is composed of a single polypeptide without subunit structure, and has a unique protease activity [12]. Although the protease cannot be physically resolved from the inhibitor, the enzymatic activity is not responsible for the biological inhibitory activity [13].…”

mentioning

confidence: 99%

“…We have recently isolated and purified, from bovine cerebral cortex cell surfaces, a sialoglycopeptide that reversibly inhibits protein and DNA synthesis of a variety of normal cells without altering the uptake of radiolabeled precursors [ 10,111. The purified 18 kD sialoglycopeptide has a PI of 3.0, is composed of a single polypeptide without subunit structure, and has a unique protease activity [12]. Although the protease cannot be physically resolved from the inhibitor, the enzymatic activity is not responsible for the biological inhibitory activity [13].…”

mentioning

confidence: 99%

Inhibition of epidermal growth factor‐stimulated DNA synthesis by a bovine sialoglycopeptide inhibitor occurs at an intracellular level

Bascom

Sharifi

Johnson

1987

J of Cellular Biochemistry

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The control of cell proliferation involves the complex interaction between growth factors and growth inhibitors. We have examined this interaction with the mitogen epidermal growth factor (EGF) and a recently purified 18 kD, pI 3, sialoglycopeptide that reversibly inhibits cellular metabolism of a variety of cells. The sialoglycopeptide was a very potent inhibitor of EGF action; 0.22 nM of the inhibitor completely blocked the mitogenic effect of 1.60 nM of EGF. The sialoglycopeptide, however, did not affect the binding of EGF to 3T3 cells. Neither the mixed affinities (0.11-1.9 nM) of binding nor the total number of receptors (50,000 receptors/cell) for EGF were altered by the addition of the sialoglycopeptide. In addition, competitive binding experiments demonstrated the specificity of inhibitor binding to 3T3 cells and also showed that EGF and the sialoglycopeptide did not share the same receptor, suggesting that the inhibitor blocked EGF action at a postreceptor, intracellular event in the signal cascade. We further demonstrated that the sialoglycopeptide had to be added within 2.5 hr after EGF to block effectively the stimulation of DNA synthesis by the growth factor, suggesting that the inhibitor blocked EGF stimulation at a relatively early step in the signal transduction mechanism.

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Purification and stability characterization of a cell regulatory sialoglgycopeptide inhibitor

Moos¹,

Fattaey²,

Johnson³

1995

J of Cellular Biochemistry

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Previous attempts to physically separate the cell cycle inhibitory and protease activities in preparations of a purified cell regulatory sialoglycopeptide (CeReS) inhibitor were largely unsuccessful. Gradient elution of the inhibitor preparation from a DEAE HPLC column separated the cell growth inhibitor from the protease, and the two activities have been shown to be distinct and non-overlapping. The additional purification increased the specific biological activity of the CeReS preparation by approximately two-fold. The major inhibitory fraction that eluted from the DEAE column was further analyzed by tricine-SDS-PAGE and microbore reverse phase HPLC and shown to be homogeneous in nature. Two other fractions separated by DEAE HPLC, also devoid of protease activity, were shown to be inhibitory to cell proliferation and most likely represented modified relatives of the CeReS inhibitor. The highly purified CeReS was chemically characterized for amino acid and carbohydrate composition and the role of the carbohydrate in cell proliferation inhibition, stability, and protease resistance was assessed.

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Relationship between protease activity and a sialoglycopeptide inhibitor isolated from bovine brain

Cited by 8 publications

References 28 publications

Receptor occupancy by a bovine sialoglycopeptide inhibitor correlates with inhibition of protein synthesis

Receptor occupancy by a bovine sialoglycopeptide inhibitor correlates with inhibition of protein synthesis

Inhibition of epidermal growth factor‐stimulated DNA synthesis by a bovine sialoglycopeptide inhibitor occurs at an intracellular level

Purification and stability characterization of a cell regulatory sialoglgycopeptide inhibitor

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