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The site of synthesis of mitochondrial proteins in Krebs II ascites-tumour cells.

ABSTRACT: At 22 degrees in Earle's medium, Krebs cells synthesize proteins. After a brief ;pulse' with [(14)C]valine followed by a ;chase' of [(12)C]valine the radioactivity appears first in microsomes and is transferred after ;chase' to the cell sap. Kinetics of labelling of the mitochondrial protein are different from that of either microsomal or cell-sap protein. When Krebs cells in buffer are mixed with ribonuclease in water the nuclease penetrates the cell membrane. The ribonuclease-treated cells are still viable but have lost most of their cytoplasmic ribosomes (electron micrograph). Such cells still synthesize mitochondrial protein at near normal rate but synthesis of microsomal protein is severely inhibited. The results indicate that some mitochondrial proteins are synthesized independently of the microsome-cell-sap system.

SUBMITTER: Haldar D

PROVIDER: S-EPMC1270315 | biostudies-other | 1967 Mar

REPOSITORIES: biostudies-other

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PROTEIN SYNTHESIS IN MITOCHONDRIA. 4. PREPARATION AND PROPERTIES OF MITOCHONDRIA FROM KREBS II MOUSE ASCITES-TUMOUR CELLS.

Project description:1. Methods of disrupting Krebs II mouse ascites-tumour cells have been studied. After washing the cells free of ions with sucrose solutions, rapid disruption was obtained in sucrose by use of an Ultra-Turrax disintegrator or a Dounce homogenizer. 2. Disruption of cells after osmotic shock led to the loss of proteins, especially cytochrome c, from the mitochondria. Such losses did not occur when cells were disrupted by shear in 0.3 m-sucrose. 3. The distribution of protein, RNA, DNA, malate dehydrogenase, cytochrome c, cytochrome oxidase and succin-oxidase was measured in the various cell fractions after separation by differential centrifuging. 4. The mitochondrial fraction sedimented at 9500g was further fractionated by equilibrium sedimentation in a sucrose gradient. The distribution of protein and enzyme activity in the gradient indicated that the 9500g pellet contains other material besides mitochondria. 5. Krebs-cell mitochondria contain up to five times as much RNA as do liver mitochondria. 6. After purification by equilibrium centrifugation Krebs-cell mitochondria still contain traces of DNA.

| S-EPMC1206534 | biostudies-other

Factors controlling amino acid incorporation by ribosomes from krebs II mouse ascites-tumour cells.

Project description:1. A ribosome-cell sap system capable of supporting the incorporation of (14)C-labelled amino acids into protein has been prepared from Krebs II mouse ascites-tumour cells. The requirements of this system for optimum activity and response to added messenger RNA have been investigated. One such system has been obtained for which amino acid incorporation is almost wholly dependent on the addition of suitable messenger RNA. 2. Ribosomes of widely different but predictable activities in the cell-free system have been prepared from Krebs cells pretreated in a variety of ways. The factors in the pretreatment of the cells responsible for these differences have been investigated. 3. The structural and functional properties of these different ribosome preparations and their response to exogenous messenger RNA have been examined and are discussed in the light of modern concepts of the control of protein synthesis.

| S-EPMC1264925 | biostudies-other

Phosphorylation in vivo of non-ribosomal proteins from native 40 S ribosomal particles of Krebs II mouse ascites-tumour cells.

Project description:Four non-ribosomal proteins from native 40 S ribosomal subunits with mol.wts. of 110 000, 84 000, 68 000 and 26 000 were phosphorylated in vivo when ascites cells were incubated in the presence of [32P]Pi. The 110 000-, 84 000- and 26 000-dalton proteins are identical with phosphorylated products from native 40 S subunits after phosphorylation in vitro by a cyclic nucleotide-independent protein kinase. Phosphoserine was the major phosphorylated amino acid of the proteins phosphorylated in vivo and in vitro.

| S-EPMC1162839 | biostudies-other

Comparison of phosphorylation of ribosomal proteins from HeLa and Krebs II ascites-tumour cells by cyclic AMP-dependent and cyclic GMP-dependent protein kinases.

Project description:Phosphorylation of eukaryotic ribosomal proteins in vitro by essentially homogeneous preparations of cyclic AMP-dependent protein kinase catalytic subunit and cyclic GMP-dependent protein kinase was compared. Each protein kinase was added at a concentration of 30nM. Ribosomal proteins were identified by two-dimensional gel electrophoresis. Almost identical results were obtained when ribosomal subunits from HeLa or ascites-tumour cells were used. About 50-60% of the total radioactive phosphate incorporated into small-subunit ribosomal proteins by either kinase was associated with protein S6. In 90 min between 0.7 and 1.0 mol of phosphate/mol of protein S6 was incorporated by the catalytic subunit of cyclic AMP-dependent protein kinase. Of the other proteins, S3 and S7 from the small subunit and proteins L6, L18, L19 and L35 from the large subunit were predominantly phosphorylated by the cyclic AMP-dependent enzyme. Between 0.1 and 0.2 mol of phosphate was incorporated/mol of these phosphorylated proteins. With the exception of protein S7, the same proteins were also major substrates for the cyclic GMP-dependent protein kinase. Time courses of the phosphorylation of individual proteins from the small and large ribosomal subunits in the presence of either protein kinase suggested four types of phosphorylation reactions: (1) proteins S2, S10 and L5 were preferably phosphorylated by the cyclic GMP-dependent protein kinase; (2) proteins S3 and L6 were phosphorylated at very similar rates by either kinase; (3) proteins S7 and L29 were almost exclusively phosphorylated by the cyclic AMP-dependent protein kinase; (4) protein S6 and most of the other proteins were phosphorylated about two or three times faster by the cyclic AMP-dependent than by the cyclic GMP-dependent enzyme.

| S-EPMC1161273 | biostudies-other

A spectrophotometric study of the secondary structure of precursor ribosomal ribonucleic acid from Krebs ascites-tumour cells.

Project description:The spectrophotometric analysis of 45S precursor rRNA shows that it contains more G and C residues than does mature 28S or 18S rRNA. The helical content and the length of double-helical segments in 45S and 28S rRNA are similar.

| S-EPMC1165829 | biostudies-other

The morphological site of synthesis of cytochrome c in mammalian cells (Krebs cells).

Project description:In Krebs ascites-tumour cells, cytochrome c is segregated in the mitochondria and the level in microsomes could not be measured. At 22 degrees in glucose-buffer Krebs cells synthesized a spectrum of proteins including cytochrome c. Mild osmotic shock in the presence of ribonuclease had little effect on incorporation of [(14)C]-leucine or [(14)C]valine into mixed mitochondrial protein but strongly inhibited synthesis of non-mitochondrial cytoplasmic proteins. Under these conditions, labelling of cytochrome c was also strongly inhibited. After pulse labelling of Krebs cells at 22 degrees for 10min. the cytcchrome radioactivity found in mitochondria was higher than in microsomes. After addition of unlabelled amino acid as ;chase' there was 137% increase in radioactivity of cytochrome c but only a 3% increase in radioactivity of whole-cell protein. It is concluded that the peptide chain of cytochome c is synthesized on cytoplasmic ribosomes. Mitochondria therefore do not have the character of self-replicating entities, but are formed by the cooperative function of messenger RNA of cytoplasmic ribosomes and, possibly, of intramitochondrial messenger derived from the mitochondrial DNA.

| S-EPMC1198412 | biostudies-other

The phosphorylation of an acidic protein of the large ribosomal subunit of Krebs II ascites cells.

Project description:The ribosomes of Krebs II ascites cells contain an acidic protein, apparently analogous to proteins L7/12 of Escherichia coli. When ascites cells were incubated with [32P]Pi, this protein became labelled, indicating that it is a phosphoprotein.

| S-EPMC1164584 | biostudies-other

A strategy to eradicate well-developed Krebs-2 ascites in mice.

Project description:We describe the strategy, which allows curing experimental mice engrafted with Krebs-2 ascites. The strategy is based on the facts that i) Krebs-2 tumor-initiating stem cells (TISCs) are naturally capable of internalizing fragments of extracellular double-stranded DNA (dsDNA); ii) upon delivery into TISCs, these dsDNA fragments interfere with the on-going DNA repair process so that TISCs either die or lose their tumorigenic potential. The following 3-step regimen of therapeutic procedures leading to eradication of Krebs-2 ascites is considered. Firstly, three timed injections of cyclophosphamide (CP) exactly matching the interstrand cross-link (ICL) repair phases that lead to synchronization of ascites cells in late S/G2/M. Secondly, additional treatment of ascites 18 hours post each CP injection (at NER/HR transition timepoint) with a composite dsDNA-based preparation interfering with the NER and HR repair pathways, so that tumorigenic properties of ascites cells are compromised. Thirdly, final treatment of mice with a combination of CP and dsDNA injections as ascites cells undergo apoptotic destruction, and the surviving TAMRA+ TISCs arrested in late S/G2/M phases massively enter into G1/S, when they regain sensitivity to CP+dsDNA treatment. Thus, this regimen assures that no viable cells, particularly Krebs-2 TISCs, remain.

| S-EPMC4905495 | biostudies-literature

Response of methylenetetrahydrofolate levels to methotrexate in Krebs ascites cells.

Project description:Levels of methylenetetrahydrofolate in Krebs ascites cells subsequent to transplantation and the effects of methotrexate on these levels have been measured. To directly measure methylenetetrahydrofolate in tissue extracts, the cofactor was incorporated into a covalent ternary complex with thymidylate synthase and 3H-labelled fluorodeoxyuridine monophosphate. A 3-4-day lag preceded rapid growth of the tumour cells, and this same kinetic behaviour was observed for methylenetetrahydrofolate levels in the tumour cells. Liver and kidney tissue from the same animals also showed an increase in methylenetetrahydrofolate over the same time period. The impact of methotrexate on methylenetetrahydrofolate in the tumour cells depended upon concentration and the post-transplantation time at which treatment was initiated. Levels of methylenetetrahydrofolate in the tumour cells were most sensitive to the drug at the beginning of the rapid growth phase and were more sensitive to a given level of methotrexate in the presence of phospholipids. A slight but significant increase in methylenetetrahydrofolate occurred in some cases in response to the presence of methotrexate.

| S-EPMC1152504 | biostudies-other

STUDIES ON PROTEIN AND NUCLEIC ACID METABOLISM IN VIRUS-INFECTED MAMMALIAN CELLS. THE FORMATION OF A VIRUS-SPECIFIC ANTIGEN IN KREBS II ASCITES-TUMOUR CELLS INFECTED WITH ENCEPHALOMYOCARDITIS VIRUS.

Project description:1. Krebs II mouse ascites-tumour cells infected with encephalomyocarditis virus were found to contain, in addition to mature virus, a virus-specific protein antigen. An assay, based on the ability of this antigen to block the neutralization of purified virus by its specific antiserum, was developed. 2. This antigen was present both in the culture fluid 17 hr. after the infection of cells with virus and intracellularly, where its titre increased at a time when viral capsid protein was being synthesized. Within the cell, it was mostly localized in the soluble cell sap. 3. In contrast with virus, the antigen did not agglutinate sheep erythrocytes, and its immunological properties were destroyed by digestion with trypsin. Ribonucleic acid was not detected in concentrated preparations of the antigen, nor was the titre of antigen affected by ribonuclease. 4. The antigen had a sedimentation coefficient (20 degrees ) of approx. 14s, and its diffusion coefficient, determined by the method of Allison & Humphrey (1960), was 3.2x10(-7) cm.(2)sec.(-1). The particle weight of the antigen was hence 420000+/-40000. 5. The capsid protein from purified encephalomyocarditis virus could be degraded by treatment with ethanolamine into a protein of sedimentation coefficient (20 degrees ) of approx. 4s. The 14s antigen, when similarly treated, yielded a protein of similar size. However, no such smaller antigen was detected in virus-infected cells. 6. It is concluded that the non-haemagglutinating antigen represents a polymeric form of the basic viral capsid-protein molecule and that it is synthesized in the cytoplasm of infected cells. It may be either an intermediate or a by-product in the process of viral capsid-protein synthesis.

| S-EPMC1206515 | biostudies-other

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