The nature of the breakdown of thyroglobulin

Impiombato, F. S. Ambesi; Pitt‐Rivers, Rosalind

doi:10.1016/0014-5793(71)80410-6

Cited by 23 publications

(3 citation statements)

References 14 publications

(5 reference statements)

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“…The molecular size distribution of reduced 27 S iodoprotein and thyroglobulin was indistinguishable. Similar patterns have been obtained with hog thyroglobulin (Ambesi Impiobato and Pitt-Rivers, 1971;Rolland and Lissitzky, 1972;Itagaki Fig. 3.…”

Section: Resultssupporting

confidence: 59%

Subunit structure of 27 S thyroid iodoprotein.

et al. 1976

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Section: Resultssupporting

confidence: 59%

Subunit structure of 27 S thyroid iodoprotein.

et al. 1976

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“…Experimentalists employing plasma albumin preparations should be alert to possible artifacts resulting from the proteinase contaminants. Albumin is certainly not unique in this regard since there are increasing examples of nominally pure proteins which are found to have at least trace amounts of proteinases associated with them (Pringle, 1970;Diezel et al, 1972; Ambesi-Impiombato and Pitt-Rivers, 1971). In our experience one cannot rely on any conventional purification procedure to remove all such contamination, since the proteinases appear to be rather tightly bound to the albumin.…”

Section: Discussionmentioning

confidence: 94%

Limited hydrolysis of bovine plasma albumin at neutral and alkaline pH catalyzed by associated proteinases

Aoki¹,

Foster²

1975

Biochemistry

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Proteinase contaminants in some plasma albumin samples have previously been shown to produce cleavage of the albumin molecule at acid pH. The F conformer, existing at pH 3.8, is cleaved near erisidue number 400 to yield a large N-terminal fragment of approximately 46,000 daltons. No cleavage was found at pH above approximately 4.4. It is shown in this paper that the proteinase contaminants are active over a broad pH range from 2.5 to 11.4 provided conditions are such as to induce some breakdown of the native conformation of the albumin molecule. Addition of Tris-borate buffer (0.1 M) at pH 7.5-9 is sufficient to permit cleavage. At pH near 9 this occurs predominantly 42,000 and 27,000 daltons. Near neutral pH substantial cleavage occurs in 4-8 M urea solution or in the presence of sodium dodecyl sulfate (AD110 complex). Under these conditions there are two large fragments (42,000 and 47,000 daltons) and essentially two small ones (20,000-27,000 daltons). Under conditions where there is no cleavage at 38-40 degrees, substantial cleavage results at 50-65 degrees but enzyme inactivation also occurs toward the top of this range. The alkaline activity is inhibited by soybean trypsin inhibitor but not by pepstatin; the reverse is true of the low pH activity. Cleavage at neutral or alkaline pH under the various conditions occurs primarily at X-Leu bonds while the low pH activity was already shown to occur at X-Phe. These facts suggest the presence of at least two enzymes. There is surprisingly little pH dependence over the range 7.5-9 in any of the media examined, even though albumin is known to undergo a significant conformational change in this range, the N leads to B transition. This transition is thought to be essentially a tertiary change with little loss of helix content. It is suggested that loss of native secondary structure, especially uncoiling of helical regions, is crucial to permit attack by these enzymes.

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“…Subsequently, analysis of the reduction products of thyroglobulin by polyacrylamide-gel electrophoresis has revealed a multiplicity of fragments, which probably results from the inherent instability of reduced thyroglobulin even at -20°C, as first noted by Pierce et al (1965) and confirmed by Charlwood et al (1970). This instability is due to autolysis of thyroglobulin subunits by a factor that, so far, has not been separable from the thyroglobulin molecule during its purification (Ambesi-Impiombato & Pitt-Rivers, 1971;Pitt-Rivers, 1975;Rolland & Lissitzky, 1976). Nevertheless, a general pattern has emerged of rather large subunits from reduced thyroglobulin ;Nissley et al (1969) were able to separate and purify two major components from their reduction products of bovine thyroglobulin with mol.wts.…”

mentioning

confidence: 93%