Joachim Kruppa

Polyacrylamide gel electrophoresis: Reaction of acrylamide at alkaline pH with buffer components and proteins☆

The chemical reaction of monomeric acrylamide with primary, secondary, and tertiary amines, used as buffer components in polyacrylamide gel electrophoresis systems, was investigated in the basic pH range. Adduct formation proceeded for several minutes up to weeks, depending on the reactivity of the amino groups. A pH shift in the reaction mixture due to an altered pK value of the reaction product was observed. However, a few primary amines (tris(hydroxymethyl)aminomethane, 2-amino-2-methyl-1,3-propanediol) and secondary amines 3-([2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino)-1-propanesulfonic acid, 3-(dimethyl(hydroxymethyl)methylamino)-2-hydroxypropanesulfonic acid) showed negligible shifts of pH. They are, therefore, useful as components in the polymerization mixture; whereas some tertiary amines showing complete pH stability as well (e.g., triethanolamine) are not suitable, as they acted as accelerators of gel polymerization. Acrylamide can also covalently bind to proteins by reacting with the epsilon-amino group of lysine residues, especially. Bovine serum albumin, having an acidic isoelectric point, and the basic protein cytochrome c were treated with different acrylamide concentrations at alkaline pH yielding modified protein molecules with altered electrophoretic mobilities in different polyacrylamide gel electrophoresis systems. This reaction gave rise to artifacts in alkaline polyacrylamide gels and isoelectric focusing systems when residual acrylamide monomers were still present in the gel matrix after the polymerization process ceased.

Human ribosomal protein S3a: cloning of the cDNA and primary structure of the protein.

The amino acid (aa) sequence of human ribosomal protein S3a (hRPS3a) was deduced partially from the nucleotide sequence of the corresponding cDNA and confirmed by direct aa sequencing from the N terminus of the purified hRPS3a protein. The cDNA clone was isolated from a cDNA expression library in the pEX vector using antibodies. The hRPS3a protein has 263 aa and its calculated M(r) is 29 813.

Fractionation of biologically active messenger RNAs by HPLC gel filtration

Mixtures of high molecular weight RNAs have been separated on a preparative scale by HPLC gel filtration. The fractionation limit of RNAs was around 42S in non-denaturing solvent systems, but decreased to approximately 16S under denaturing conditions in the presence of 6M urea. The use of denaturing elution buffers improved resolution considerably. Translational activity of globin mRNA and of mRNAs of Vesicular Stomatitis Virus was retained after gel permeation chromatography.

Dephosphorylation of one 40S ribosomal protein in MPC 11 cells induced by hypertonic medium

Summary Mouse myeloma cells (MPC 11) were grown at a high cell density in the presence of [32P] orthophosphate. The tonicity of the medium was raised by 100 mM NaCl in half of the suspension culture after 4 hrs of incubation. The proportions of messenger-free ribosomes and polysomes from salt-treated and control cells were quantitated after separation by sucrose gradient centrifugation and the four ribosomal preparations were analysed by two-dimensional gel electrophoresis. Two ribosomal phosphoproteins, S3 and L28 (nomenclature of Martini and Gould; ref. 1) were detected by autoradiography in all four ribosomal protein patterns. Quantitation of 32P-incorporation revealed that a positive correlation exists between the degree of phosphorylation of S3, and the amount of ribosomes engaged in protein synthesis. S3 was highly phosphorylated in polysomes of control cells, only slightly phosphorylated in polysomes of salt-treated cells and almost completey dephosphorylated in messenger-free ribosomes of salt-treated cells. The 32Pi-incorporation of L28 was much less effected by the hypertonic medium.

Virus-Induced Shut-Off of Host Specific Protein Synthesis

Infection of cells by viruses is often followed by a rapid decline in the synthesis of host cell specific macromolecules, predominantly of protein and RNA (1–3). This phenomenon, called shutoff, was first intensively analyzed in phage infected bacteria. It was shown that adsorption of the phage protein shell (protein ghost) to E. coli leads to a rapid change in membrane permeability followed by cessation of macromolecular synthesis and death of the host cell 5). Even soluble fractions of the phage coat are able to kill E. coli cells (6). Membrane leakiness is rapidly repaired in productively infected E. coli due to phage coded proteins.

Intracellular formation of two soluble glycoproteins in BHK cells infected with vesicular stomatitis virus serotype New Jersey

Infection of BHK 21 cells with VSV serotype New Jersey gave rise to three intracellular viral glycoproteins: the membrane-integrated G protein and the two soluble glycoproteins Gs and Gss which lacked the cytoplasmic and transmembrane domains as was deduced from limited chemical cleavage of the glycoproteins by hydroxylamine. Both soluble glycoproteins were completely protected by the microsomal membrane against proteolytic digestion. The soluble glycoproteins were formed in the endoplasmic reticulum because both were fully endo H sensitive after a 5-min pulse with [35S]methionine. Protease inhibitors and lysosomorphic agents had no effect on the yield of Gs and Gss. Tunicamycin treatment of VSV-infected cells reduced extensively viral particle maturation without affecting significantly the release of Gs and Gss. Two other glycosylation inhibitors, swainsonine and deoxynojirimycin did not decrease virus particle formation and secretion of both soluble glycoproteins. Since the glycosylation inhibitors showed a differential effect on the processing and transport of the glycoproteins a precursor-product relationship between G protein and soluble glycoproteins is highly unlikely. Both soluble glycoproteins were also synthesized in vitro in a reticulocyte lysate without microsomal membranes when primed with RNA extracted from VSV-infected cells or with newly transcribed mRNA from nucleocapsids in a coupled transcription system. Thus, proteases localized in the lumen of the ER seemed to be not essential for the generation of both soluble glycoproteins.

Chemical Synthesis of Cap Analogues: P1,P2-Dinucleoside-5′-diphosphates

Abstract A procedure was developed for the chemical synthesis of P1,P2-dinucleoside-5′-diphosphates (N1(5′)pp(5′)N2) on a nanomolar scale Reaction conditions for activating purine-5′-monophosphates (pA, pG, and pm7G) by 1,1′-carbonyldiimidazole were studied and optimized in respect to solvents and amount of activating reagent used. Various dinucleoside-5′-diphosphates were synthesized in 62-98% yield by incubating activated and non-activated purine-5′-monophosphates. Two unexpected by-products were formed by competition reactions: the imidazolidate of the non-activated nucleotide and the corresponding symmetrically substituted dinucleoside-5′-diphosphate. A mechanism is proposed to explain the observed side reactions.

Localization and direct quantitation of 3H-labeled proteins and RNAs in slab gels by a new detection system

The distribution patterns of 3H-labeled proteins and RNAs in dehydrated polyacrylamide and agarose gels have been recorded with a newly developed instrument: the Linear Analyzer which has a position-sensitive detector that counts a whole gel lane simultaneously. The high sensitivity makes the Linear Analyzer a suitable instrument for direct quantitation of tritium labeled macromolecules in gels without pretreatment by impregnating reagents. The very low detection limit of the Linear Analyzer reduces substantially the time of evaluation in comparison to the relatively long exposure times needed in fluorography. The resolution of radioactivity profiles monitored by the Linear Analyzer reaches the quality attained by fluorography.

MEMBRANE MEDIATED AMPLIFICATION OF TRANSLATIONAL CONTROL IN EUKARYOTES: A PLEIOTROPIC EFFECT

Tissue culture cells respond to amino acid starvation, virus infection and exposure to hypertonic medium with a reduced rate of polypeptide chain initiation. This response is accompanied by changes in the phosphorylation state of certain ribosomal proteins and by the activation and/or release of a low molecular weight mediator which inhibits protein synthesis when added to cell-free extracts. The inhibitor is reversibly bound to ribosomes and decreases the ability of ribosomes to participate in the formation of functional initiation complexes. A preliminary list of some of the biochemical properties of this low molecular weight mediator is given below.

A rapid procedure for the production and purification of vesicular stomatitis virus.

Ehrlich ascites tumour(EAT) cells were infected with vesicular stomatitis virus (VSV) within the peritoneal cavity of mice and used to prepare milligram quanities of purified virus. The protein pattern, endogenous transcriptase activity, and specific infectivity of this virus were comparable to viral preparations from HeLa and BHK cells. A total of 2-4 x 10(11) p.f.u. of VSV were obtained routinely from one mouse. The initial high concentration of VSV grown in EAT cells in the peritoneal cavity of mice facilitates virus isolation and allows its purification by a single gradient centrifugation step.