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M Gruber

Publications and source records attributed to M Gruber.

At least 145 records · Page 8Linked to original sources
PubMed1976

Estradiol-induced synthesis of vitellogenin. III. The isolation and characterization of vitellogenin messenger RNA from avian liver.

The messenger RNA of the hormone-induced protein vitellogenin was isolated from the liver of estrogen-treated roosters. Starting from total polysomal RNA, the vitellogenin messenger was purified 67-fold by oligo (dT)-cellulose chromatography and sizing on a sucrose gradient. The messenger was translated in vitro into a 170 000 dalton polypeptide chain, having the immunochemical characteristics of vitellogenin. From electrophoretic and immunochemical analysis of the in vitro product of translation at least 63% of the messenger activity of the RNA preparation could be attributed to vitellogenin mRNA. Gel electrophoresis of the most purified fraction revealed residual contamination with the larger ribosomal RNA species. The molecular weight of the messenger RNA molecule, obtained by contour length measurements in the electron microscope, lies between 2.5 - 10(6) and 2.8 - 10(6).

Animals↗
PubMed1976

Localization and some properties of lysosomal dipeptidases in rat liver.

1. The rates of hydrolysis of 26 synthetic dipeptides by extracts from highly purified lysosomal fractions from rat liver at pH 5.0 and by whole liver homogenates at pH 7.4 have been determined. Extracts from the lysosomal fractions hydrolysed most peptides at a lower rate per mg protein than the homogenates, and some peptides not at all. 2. Properties of two dipeptidases present in the extracts from the lysosomal fractions, splitting Ile-Glu and Leu-Gly, respectively, were studied in greater detail. The enzyme that hydrolysed Ile-Glu was strongly activated by dithiothreitol, showed optimal activity at pH 4.5 and had a molecular weight of about 120 000. Leu-Gly dipeptidase did apparently not contain an essential thiol group and had a molecular weight of approx. 90 000. It showed maximal activity at pH 6.5. 3. After differential centrifugation of liver homogenates, Ile-Glu and Leu-Gly-splitting activities were determined in the fractions, under the optimal conditions mentioned above. The Ile-Glu-hydrolysing enzyme activity showed about the same distribution as the lysosomal marker enzyme acid phosphatase. Leu-Gly-splitting activity, however, was largely present in the cytosol fraction, with only a small peak in the lysosomal fraction. We obtained evidence that the activities present in the lysosomal fraction and in the cytosol fraction were due to different enzymes, and that one of these enzymes was localized exclusively in lysosomes. 4. It is concluded that some dipeptides originating from intralysosomal proteolysis might be split by lysosomal dipeptidases, whereas others are probably hydrolysed only in the extra-lysosomal compartment of the cell.

Acid Phosphatase↗
PubMed1976

The sequence of ppGpp and pppGpp in the reaction scheme for magic spot synthesis.

The kinetics of ppGpp (guanosine 5'-diphosphate, 3'-diphosphate) ahd pppGpp (guanosine 5'-triphosphate, 3'-diphosphate) synthesis, at the onset of amino acid starvation, and of their decay after inhibiting synthesis, were analysed in Escherichia coli. The pentaphosphate, but not ppGpp, is the first product of the stringent response to amino acid starvation. The pentaphosphate is rapidly converted with first order kinetics (t 1/2 = 6 s) to ppGpp which is broken down less rapidly (t 1/2 = 20 s).

Amino Acids↗
PubMed1976

Estradiol-induced synthesis of vitellogenin. I. The isolation of large polysomes from estrogenized rooster liver.

Conventional procedures for the isolation of polysomes, applied to estrogenized rooster liver, fail to yield polysomes containing 30 or more ribosomes, the size expected for polysomes synthesizing the estradiol-induced protein vitellogenin. A new procedure characterized by early and selective removal of cell components ribonucleases allowed the isolation of polysomes with up to 55 ribosomes. Electron microscopy was used for the determination of polysome size and showed that the large polysomes were not aggregates.

Animals↗
PubMed1976

The role of energy-generating processes in the degradation of guanosine tetrophosphate, ppGpp, in Escherichia coli.

The effect of energy limitation on the intracellular level of guanosine 5'-diphosphate, 3'-diphosphate (ppGpp) was investigated. Glucose exhaustion, as well as uncoupling of oxidative phosphorylation, evokes an immediate increase in the ppGpp concentration. This increase is a consequence of a lowered degradation rate of the nucleotide. The level of ppGpp, which determines the rate of ribosomal RNA synthesis, is thus subject to a double control system: amino acyl-tRNA availability governs the rate of synthesis while energy metabolism governs the rate of degradation.

Dinitrophenols↗
PubMed1976

The mechanism of action of ppGpp on rRNA synthesis in vitro.

We have studied the mechanism of the specific inhibition of ribosomal RNA synthesis by ppGpp in a purified system using as templates E. coli DNA and DNA from lambdad5ilv, which carries a rRNA cistron from E. coli. Ribosomal RNA synthesis, as well as its inhibition by ppGpp, are critically salt-dependent. Of a number of guanosine phosphates tested, only pppGpp (MS II) mimicked the action of ppGpp, establishing the specificity of ppGpp. The two templates gave similar results for rRNA synthesis in all experiments. By using the initiation inhibitor rifampicin, we could show that the specific inhibition of rRNA synthesis by ppGpp is due to its effect on rRNA initiation. The somewhat variable inhibition of RNA synthesis in general by ppGpp is mainly or wholly a consequence of premature chain termination. We propose that ppGpp specifically inhibits rRNA synthesis by acting on the formation of the so-called "closed-promoter complex".

Cell-Free System↗
PubMed1975

Ribosomal and non-ribosomal RNA synthesis in vitro.

The synthesis of total and ribosomal RNA using nucleoids of Escherichia coli as template was measured; of the total RNA synthesized by endogenous RNA polymerase which only completes chains, and added RNA polymerase which initiates new chains, 50-70 and 3-5%, repectively, was rRNA. Total RNA synthesis by added enzyme, however, was 10-20 times higher than endogenous RNA synthesis; thus rRNA was synthesized at the same rate by the endogenous and the added enzyme. We conclude that the percentage rRNA in vitro is no measure of the rate of rRNA synthesis. Furthermore, it follows that the added enzyme, like the endogenous one, is packed at the physical limit on the ribosomal cistrons. Consequently, initiation of ribosomal cistrons by added enzyme was at or near the maximal rate possible for this system in which the elongation rate is 10-20% of that in vitro. When RNA synthesis was assayed at various ratios of RNA polymerase to phenol-extracted DNA, the amount of rRNA made per DNA, which is a measure of the frequency of transcription of ribosomal cistrons, varied. The ratio of rRNA synthesis relative to total RNA synthesis also varied, but in a different way, again leading to the conclusion that this ratio, as determined in vitro, does not reflect the efficiency of transcription of the ribosomal cistrons.

DNA↗
PubMed1975

Rapid uptake by liver sinusoidal cells of serum albumin modified with retention of its compact conformation.

The clearance from the blood and the conformation of serum albumin modified by nitroguanidination and labeled with 125-I have been studied. Like formaldehyde-denatured albumin, but in contrast to native albumin, the nitroguanidinated derivative is rapidly cleared from the blood and taken up in lysosomes of liver sinusoidal cells. Although 94% of the free amino groups were blocked by nitroguanidination, we could not detect significant conformational changes using gel filtration, determination of reducible disulfide groups, and titration of tyrosine residues. It is concluded that extensive denaturation is no prerequisite for the uptake of albumin derivatives in liver sinusoidal cells. It is suggested that the nitroguanidinated protein, in contrast to native albumin, is bound on membrane receptors of sinusoidal cells. The nitroguanidino groups themselves might be bound on these receptors, but it seems equally possible that the blocking of positive charges of the albumin molecule or minor, local conformational changes of the protein are sufficient for the binding on the receptors.

Acid Phosphatase↗