Eveline Bruenger

A new method for purification of mature elastin

The isolation of elastin from various tissues has depended upon its chemical inertness and resistance to hydrolysis. However, the majority of the procedures used to obtain a purified product are drastic enough that breakdown of elastin occurs. This communication describes a new method for the isolation of elastin with less degradation than existing methods. The tissue is milled through a 40 mesh screen in a Wiley Mill. It is extracted with guanidinium chloride, autoclaved, and then treated with formic acid-cyanogen bromide. A purified product is obtained and quantitation of NH2-terminal residues shows five to ten times less end-groups than elastin extracted with currently existing methods.

Tropoelastin purification: Improvements using enzyme inhibitors

Abstract The true monomerix size of porcine aortic tropoelastin is 72,000–74,000 daltons. The newly modified isolation procedure we describe gives meterial predominantly of this molecular size with improved yields. The protein is extremely susceptible to proteolysis and steps must be taken during isolation to prevent this occurrence. The addition of di-isopropylfluorophosphate, N -ethylmaleimide, and EDTA to the initial extraction buffer has proven effective. Lower molecular weight components which are elastin-like are always isolated along with the high molecular weight material. In the absence of the inhibitors, these smaller components are greatly increased. Alanine enrichment as revealed by NH 2 -terminal analyses of the degraded material suggests that the tissue proteinases which produce these are elastase-like.

Identities and Phylogenetic Comparisons of Posttranscriptional Modifications in 16 S Ribosomal RNA from Haloferax volcanii

Small subunit (16 S) rRNA from the archaeonHaloferax volcanii, for which sites of modification were previously reported, was examined using mass spectrometry. A census of all modified residues was taken by liquid chromatography/electrospray ionization-mass spectrometry analysis of a total nucleoside digest of the rRNA. Following rRNA hydrolysis by RNase T1, accurate molecular mass values of oligonucleotide products were measured using liquid chromatography/electrospray ionization-mass spectrometry and compared with values predicted from the corresponding gene sequence. Three modified nucleosides, distributed over four conserved sites in the decoding region of the molecule, were characterized: 3-(3-amino-3-carboxypropyl)uridine-966,N 6-methyladenosine-1501, andN 6,N 6-dimethyladenosine-1518 and -1519 (all Escherichia coli numbering). Nucleoside 3-(3-amino-3-carboxypropyl)uridine, previously unknown in rRNA, occurs at a highly conserved site of modification in all three evolutionary domains but for which no structural assignment in archaea has been previously reported. NucleosideN 6-methyladenosine, not previously placed in archaeal rRNAs, frequently occurs at the analogous location in eukaryotic small subunit rRNA but not in bacteria.H. volcanii small subunit rRNA appears to reflect the phenotypically low modification level in the Crenarchaeota kingdom and is the only cytoplasmic small subunit rRNA shown to lack pseudouridine.

5S rRNA modification in the hyperthermophilic archaea Sulfolobus solfataricus and Pyrodictium occultum.

The 5S rRNAs from Sulfolobus solfataricus and Pyrodictium occultum were digested to nucleosides and analyzed using directly‐combined HPLC/mass spectrometry. P. occultum 5S rRNA contains two modified nucleoside species, N4‐acetylcytidine (ac4C) and N4‐acetyl‐2′‐O‐methylcytidine (ac4Cm). Oligonucleotides were generated from P. occultum 5S rRNA by RNase T1 hydrolysis, and their molecular weights were determined using electrospray mass spectrometry and compared with those predicted from the P. occultum 5S RNA gene sequence. Deviation in mass between expected and observed molecular weights permitted ac4Cm to be located at position 35, in the nonanucleotide CAA‐CACC[ac4Cm]G, and the ac4C in one or both of two (C,U)G trinucleotides. 2′‐O‐Methylcytidine is unambiguously characterized in S. solfataricus 5S rRNA, confirming earlier tentative assignments at the analogous sequence position (Stahl, D. A., Luehrsen, K. R., Woese, C. R., and Pace, N. R. (1981) Nucleic Acids Res., Vol. 9, pp. 6129‐6137; Dams, E., Londei, P., Cammarano, P., Vandenberghe, A., and De Wachter, R. (1983) Nucleic Acids Res. Vol. 11, pp. 4667–4676). Potential effects of the presence of ac4C and ac4Cm on thermal stabilization of 5S rRNA in thermophiles are discussed.— Bruenger, E., Kowalak, J. A., Kuchino, Y., McCloskey, J. A., Mizushima, H., Stetter, K. O., Grain, P. F. 5S rRNA modification in the hyperthermophilic archaea Sulfolobus solfataricus and Pyrodictium occultum. FASEB J. 7: 196‐200; 1993.

Determination of isopentenyl diphosphate and farnesyl diphosphate in tissue samples with a comment on secondary regulation of polyisoprenoid biosynthesis

A double-isotope dilution procedure is described for the determination of two isoprenoid precursors, isopentenyl and farnesyl diphosphate. Recovery of each is determined by the addition of the appropriate radioactive diphosphate to the tissue sample. After partial purification, each is coupled by a prenyltransferase with a cosubstrate of known specific activity. The products, doubly labeled farnesyl and geranylgeranyl diphosphates, are cleaved to the parent alcohols by alkaline phosphatase. The resulting polyprenols are isolated by reversed-phase thin-layer chromatography and their radioisotopic content is determined. The levels of these precursors have been measured in livers of rats and mice that have been maintained on several different diets. The concentration of each was about 0.5 mumol/g wet tissue and varied as much as 10-fold under the different test conditions. The levels of isopentenyl diphosphate isomerase, farnesyl diphosphate synthetase, and squalene synthetase were also measured in these animals. The changes in levels of these enzymes, in conjunction with the variation in substrate concentrations, are such that they could substantially influence the rate of cholesterol synthesis in liver.

The synthesis of a photolabile detergent and its use in the isolation and characterization of protein

Abstract A new ionic detergent sodium 4-(3,3-dimethyl-1-oxotridecyl)benzenesulfonate similar to sodium dodecyl sulfate has been synthesized which is photodegradable to sodium p -acetyl-benzenesulfonate and a simple olefin mixture upon irradiation in aqueous solution with light 300 nm and above in wavelength. This photodegradable detergent can be used to solubilize many proteins and provide information as to the molecular weight and subunit composition of proteins by electrophoresis. The removal of this detergent by photolysis results in no apparent damage to protein.

Structural studies of alanine- and lysine-rich regions of porcine aortic tropoelastin

Abstract Lysine residues in tropoelastin are the precursors of the cross-links of mature elastin. Trypsin digestion releases the lysine as two major peptide classes, either very large (20–100 residues) or very small (3–4 residues), in roughly equal numbers. The small peptides are mainly Ala-Ala-Lys and Ala-Ala-Ala-Lys, (L. B. Sandberg, N. Weissman, and W. R. Gray, Biochemistry , 10 (1971) 52). Further digestion of the large peptides by subtilisin produced a mixture of many small fragments. The lysine-containing fragments were analyzed by ion-exchange chromatography on Aminex A-5 resin. They showed a marked enrichment in alanine, suggesting that the association of lysine residues with alanine-rich regions is very extensive. It is proposed that such regions may be α-helical in structure.

Prenylated proteins: demonstration of a thioether linkage to cysteine of proteins.

Prenylated amino acid fragments have been isolated from prenylated proteins of Chinese hamster ovary cells. Gel-exclusion chromatography indicates that these proteins are modified by two different prenyl groups. The prenyl-amino acid fragments are labeled by 35S from cysteine, and this bond is cleaved by Raney-Ni, proving that the prenyl residue is linked to protein via a thioether to cysteine. Hydrazinolysis has been used to demonstrate that the cysteine is carboxy terminal.

Prenyltransferase and squalene synthetase in livers of neonate rats

The liver of the newly born rat has approximately the same capacity for cholesterol biosynthesis as that of the adult animal. However, during nursing, the ability to synthesize cholesterol diminishes markedly during the early neonate period and by the end of the second week has essentially vanished. The level of the regulatory enzyme of cholesterol biosynthesis, 3-hydroxy-3-methylglutaryl-CoA reductase, closely follows this pattern (Hahn, P. and Walker, B. (1979) Can. J. Biochem. 57, 1216-1219). In contrast, we have found that two other enzymes of cholesterol biosynthesis, prenyltransferase and squalene synthetase, undergo changes in activity that provide three maxima - one on birth, one during midnursing, and one on weaning. Possible explanations for this pattern are presented.

pCAP-based peptide substrates: the new tool in the box of tyrosine phosphatase assays.

Robust, facile high throughput assays based on non-peptidic probes are available to detect the enzyme activity of protein tyrosine phosphatases. However, these assays cannot replace the use of peptide-based probes in many applications; for example when a closer mimic of the physiological target is desired or in substrate profiling expeditions. Phosphotyrosine peptides are often used in these assays, but their use is complicated by either poor sensitivity or the need for indirect detection methods, among other pitfalls. Novel peptide-based probes for protein tyrosine phosphatases are needed to replace phosphotyrosine peptides and accelerate the field of tyrosine phosphatase substrate profiling. Here we review a type of peptidic probe for tyrosine phosphatases, which is based on the incorporation of the phosphotyrosine-mimic phosphocoumaryl amino propionic acid (pCAP) into peptides. The resulting fluorogenic pCAP peptides are dephosphorylated by tyrosine phosphatases with similar efficiency as the homologous phosphotyrosine peptides. pCAP peptides outperform phosphotyrosine peptides, providing an assay that is as robust, sensitive and facile as the non-peptidic fluorogenic probes on the market. Finally the use of pCAP can expand the range of phosphatase assays, facilitating the investigation of multiphosphorylated peptides and providing an in-gel assay for phosphatase activity.