Studies on the synthesis of liver proteins with particular reference to prothrombin

Goswami, Pratul (1962) Studies on the synthesis of liver proteins with particular reference to prothrombin. PhD thesis, University of Glasgow.

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Abstract

The formation of prothrombin by liver cell particles has been studied under in vitro conditions. Mitochondria, a heavy microsome, and a light microsome fractions were isolated from rat liver and were incubated in a modified bicarbonate buffer solution at pH 6.9 in an atmosphere of oxygen. The heavy microsome fraction showed an increase in prothrombin activity. This increased prothrombin activity by the heavy microsomes, lasting throughout a three-hour incubation period, was also seen if the system was incubated in an atmosphere of nitrogen up to a period of two hours. Feeding the rat with glucose before killing resulted in a considerably increased rate of formation of prothrombin by the incubated heavy microsomes. In spite of this observation, it was found that the early stages of prothrombin formation by the heavy microsome fraction was not dependent on oxidative phosphorylation, since incubation with dinitrophenol, nicotihamide-adenine dinucleotide (NAD), nicotinamide-adenine dinucleotide phosphate (NADP), and the corresponding reduced forms NADH2 and NADPH2 and adenosine-5'-triphosphate (ATP) neither inhibited nor increased the initial formation of prothrombin, though some effects after two hours of incubation were seen, but were regarded as non-specific. The increased activity of prothrombin during incubation was not due to release of preformed prothrombin bound to the microsomal vesicles, as there was no increase in activity when the microsomes were disrupted. Prothrombin formation by the heavy microsomes was, however, dependent on the ribonucleic acid of the membrane of the microsomes. Prothrombin formation was not seen if the system was incubated with ribonuclease, but this inhibitory effect of ribonuclease was not restored by addition of samples of ribonucleic acid prepared from microsomes or by pH 5 enzyme prepared from cell sap containing soluble ribonucleic acid (sRNA). Microsomal membrane, prepared by treatment of the microsomes with sodium pyrophosphate, showed an increase in prothrombin activity when incubated under the above conditions. This increment in activity was also inhibited with ribonuclease. In view of the involvement of ribonucleic acid in the formation of prothrombin activity the nucleotide composition of ribonucleic acid in the whole microsomes, nuclei, microsomal membrane and ribosomes was studied. The ribonucleic acid from the membrane of the microsomes showed an unusual micleotide composition, notably an absence of pseudouridylic acid and a high quantity of guanylic and cytidylic acids, as compared with that of the ribosomes. Absence of pseudouridylic acid was observed in the ribonucleic acid of the nuclei also. The amount of guanylic and cytidylic acids of the membrane-ribonucleic acid and ribosomal-ribomicleic acid was significantly reduced in protein-fed animals, the change of pattern being most marked in the membrane-RNA. The pseudouridylic acid content of ribosomal-RNA was found to increase significantly with the inclusion of protein in the diet. The molecular size of ribonucleic acid associated with the membrane was found to be low in comparison with that of whole microsomal-RNA and ribosomal RNA when studied by ultracentrifugal analysis and by ECTEOLA column chromatography. The uptake of 32P was initially highest in the nuclear-RNA, the maximum specific activity being attained some time between 3-8 hours after injection. The membrane-RNA showed some incorporation of 32P at one hour after administration, but little activity was found at that time in the ribosomal-RNA. The activity in both fractions (gradually increased up to 8 hours after injection of 32P, the activity in the membrane-RNA even at that period being higher than the ribosomal-RNA. At 16 hours after injection, the activity in ribosomal-RNA was now higher than in the membrane-RNA. The capacity to incorporate 32P was markedly stimulated if the rats were prefed with protein one hour before injection of isotope. From these findings it has been concluded that the prothrombin molecule can be finalised by the microsome membrane, a type of RNA associated with the membrane being essential for the process. In view of the similarity between the nucleotide composition of the membrane-RNA and nuclear-RHA, and the labelling pattern of the membrane-RNA, it is suggested that membrane-RNA arises from the nucleus and may represent a, form of messenger RNA. This concept would be consistent with electron micrograph evidence of the origin from nuclei of endoplasmic reticulum, from which the microsomes of disrupted cells are derived.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: H N Munro
Keywords: Molecular biology
Date of Award: 1962
Depositing User: Enlighten Team
Unique ID: glathesis:1962-72748
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 11 Jun 2019 11:06
Last Modified: 11 Jun 2019 11:06
URI: http://theses.gla.ac.uk/id/eprint/72748

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