A new method using ‘proteinase K’ to prevent mRNA degradation during isolation from HeLa cells

Wiegers, U.; Hilz, Helmuth

doi:10.1016/0006-291x(71)90632-2

Cited by 107 publications

(28 citation statements)

References 9 publications

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“…Susceptibility to degradation by the nonspecific proteolytic enzyme proteinase K may be also demonstrated (Fig. 2, lane D) (17). This confirms the protein nature of Mr 30,000.…”

supporting

confidence: 69%

Spermidine is bound to a unique protein in early sea urchin embryos.

Canellakis¹,

Bondy²,

Infante³

1985

Proc. Natl. Acad. Sci. U.S.A.

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Spermidine is rapidly taken up and becomes bound to protein during the very early hours of sea urchin embryogenesis. During the first 6 hr after fertilization of freshly obtained sea urchin eggs (Strongylocentrotus purpuratus), which are incubated in the presence of exogenous 13H]-spermidine, up to 7% of the total cell-associated spermidine appears uniquely as spermidine. bound in macromolecular form. This unique protein containing spermidine migrates as a single radioactive band in gel electro phores It has a Mr of :30,000 and is readily distinguishable from the protein initiation factor eIF-4D, which has a Mr of 18,000, the only other identifiable protein known to date to be posttranslationally modified by polyamines.In very early sea urchin (Strongylocentrotus purpuratus) embryogenesis we have discovered a protein containing intact, covalently bound spermidine. Putrescine, spermidine, and spermine, collectively referred to as the parent polyamines, are essential for the life of a cell and their intracellular concentrations are elevated during rapid cell division and growth (1-3). In addition, agents known to interfere with polyamine levels and metabolism block growth and differentiation (4). Yet, until recently, it has been difficult to assign particular roles to these small aliphatic amines, which are the most abundant endogenous cellular cations. The finding of hypusine, a polyamine metabolite, as a covalent component of the eukaryotic protein translation initiation factor eIF-4D was, to our knowledge, the first example of a polyamine derivative in a specific macromolecule (5). We now find that intact spermidine is present as a posttranslational modification of a unique protein with a Mr of =30,000, an example of an intact polyamine bound covalently to a specific macromolecule.The cellular uptake of [3H]spermidine by freshly fertilized sea urchin eggs and its distribution between the trichloroacetic acid (CCl3COOH)-soluble and CCl3COOH-insoluble fraction of cells are presented in Fig.

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“…Susceptibility to degradation by the nonspecific proteolytic enzyme proteinase K may be also demonstrated (Fig. 2, lane D) (17). This confirms the protein nature of Mr 30,000.…”

supporting

confidence: 69%

Spermidine is bound to a unique protein in early sea urchin embryos.

Canellakis¹,

Bondy²,

Infante³

1985

Proc. Natl. Acad. Sci. U.S.A.

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“…However, as expected for isoleucyl peptides, the natural product and isoleucylleucine both developed their ninhydrin colours much slower than leucylisoleucine. By proteinase K [25] (6 h, 37 "C three additions of enzyme) the pentapeptide was preferentially split into leucylasparagine (RF 0.36) and serylisoleucylleucine (RF 0.74). A cleavage like that effected by thermolysin also occurred but to a minor extent only.…”

Section: T H E Amino-acid Compositions and Xequences Of The Three Chymentioning

confidence: 99%

Structure of the Porcine Vasoactive Intestinal Octacosapeptide

Mutt

Said

1974

European Journal of Biochemistry

386

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The amino acid sequence of the porcine vasoactive intestinal octacosapeptide is His-Ser-AspAla-Val-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-~ln-Met -Ala -Val -Lys -Lys -Tyr -Leu -AsnSer-Ile-Leu-Asn-NH,. Its amino acid residues 1, 2, 6 and 7, counted from the N-terminus, are identical to those in the corresponding positions in both porcine glucagon and secretin. The residues in positions 3, 12, 13, 14 and 23 are identical to those in secretin, but not in glucagon, and position 10 is occupied by a tyrosyl and position 28 by an asparaginyl residue, like in glucagon but not in secretin. If in addition to identical amino acid residues also chemically similar residues, such as isoleucine in position 26 of the octacosapeptide, as compared to leucine in secretin and glucagon, are taken into consideration then the similarity between these three polypeptides is still more evident.At a more remote level there is some structural resemblance also between these peptides and the other four peptides from the intestinal wall, cholecystokinin-pancreozymin, motilin, the gastric inhibitory peptide and substance P, the structures of which are known.The elucidation of the structure of the octacosapeptide was facilitated by the finding that pancreatic kallikrein preferentially cleaved only one of the three bonds in its N-terminal cyanogen bromide heptadecapeptide that are susceptible to cleavage with trypsin.In a previous paper [l] we reported that the porcine vasoactive intestinal octacosapeptide, like secretin and like glucagon, has an N-terminal histidylseryl structure. The complete amino acid sequence of the polypeptide has now been elucidated. Partial sequences have already been reported in publications on its synthesis [2,3] and the complete sequence too has recently been disclosed in a preliminary communication by Bodanszky et al. on the completion of the synthesis [4]. The present paper gives the evidence for the proposed sequence. MATERIALS AND METHODSPorcine vasoactive intestinal octacosapeptide was prepared as described earlier [l].Pancreatic kallikrein (sialidase -treated pig pancreatic kallikrein R, batch H 376) was a gift from Professor E. Werle, Munich. Tosyl-lysyl-chloromethyl ketone-treated chymotrypsin, proteinase K and L -(1 -tosylamido -2 -pheny1)ethyl chloromethylketone-treated trypsin, and carboxypeptidase A treated with diisopropyl phosphofluoridate from Worthington.L-Aspartic acid diamide -HCl, hemihydrate, was obtained from Cyclo Chemical, isoleucylleucine and leucylisoleucine from Fox Chemical Company and cyanogen bromide from Eastman Kodak.L-Asparagine-tert-butyl ester-HC1, L-aspartic aciddi-tert-butyl ester-HC1, tert-butyloxycarbonyl-L-asparagine p-nitrophenyl ester, and tert-butyloxycarbonyl-L-aspartic acid-p-tert-butyl-a-p-nitrophenyl ester were obtained from either Fox Chemical Company or from Bachem (Liestal, Switzerland).The sources and qualities of the other materials used have been described previously, as have the methods for paper chromatography and paper electrophoresis of peptides and for their h...

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“…Against hemo globin it is about 6 times more active than pronase and about 3 times more active than bovine trypsin [6], Furthermore, proteinase K has been used extensively to digest proteins or inactivate ribonucleases [7][8][9][10], To minimize the risk that this enzyme might damage RPE cells, we used the lowest concentration (0.1 %) of proteinase K that was effective in isolating RPE cells from intact rat eyeballs.…”

Section: Discussionmentioning

confidence: 99%

“…One of the serine proteases, proteinase K, is a so-called strong protease [6] that has been used to extract mRNA from cells [7], for rapid purification of ferritin from red blood cells [8] or of lipopolysaccharide from bacteria [9] and to establish the sequence of amino acids [10]. Proteinase K has also been used in ophthal mologic research, to isolate the retina and RPE cells from neonatal mice [11],…”

Section: Introductionmentioning

confidence: 99%

A Rapid Method for Isolation of Retinal Pigment Epithelial Cells from Rat Eyeballs

et al. 1995

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A new, simple method for isolating retinal pigment epithelial (RPE) cells from rat eyeballs was developed: incubation of the eyeballs in 0.1% proteinase K solution. This method yielded an average of 4 × 10⁴ viable RPE cells from each Sprague-Dawley rat eyeball in 1 h. These RPE cells were hexagonal and had cytokeratin in their cytoplasm and numerous microvilli on their surface, similar to RPE cells evaluated in vivo. The results of this study thus show that our method for incubating eyeballs in proteinase K can provide good-quality RPE cells in sufficient quantities for study in a short time.

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A new method using ‘proteinase K’ to prevent mRNA degradation during isolation from HeLa cells

Cited by 107 publications

References 9 publications

Spermidine is bound to a unique protein in early sea urchin embryos.

Spermidine is bound to a unique protein in early sea urchin embryos.

Structure of the Porcine Vasoactive Intestinal Octacosapeptide

A Rapid Method for Isolation of Retinal Pigment Epithelial Cells from Rat Eyeballs

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