Some properties and function of ribosomal proteins from the seeds of Pinus lambertiana

• Main Author: Ko, Thong-Sung
• Other Authors: Biochemistry and Nutrition
• Format: Others
• Language: en_US
• Published: Virginia Polytechnic Institute 2019
• Subjects: LD5655.V856 1970.K6; Ribosomes
• Online Access: http://hdl.handle.net/10919/94524

Functional roles of ribosomal proteins (r-proteins) on the structural properties of ribosomes from Pinus lambertiana were studied. For this purpose, r-proteins in the ribosome were dansylated with dansyl chloride (l-dimethylaminonaphthalene-5-sulfonyl chloride) dispersed on celite (DNS-Cl-celite); sedimentation properties, ribonuclease sensitivity, and the temperature-absorbance profile, of the dansylated ribosomes (DNS-ribosomes) were compared with normal ribosomes; the isoelectric focusing patterns of proteins removed (supernatant fraction) during washing procedures and those of proteins remaining (ribosomal pellet fraction) were investigated; characteristics of extracted r-proteins were examined. Ribosomal proteins in the ribosome were dansylated, leaving the r-RNA unreacted. Prepared dansyl ribosomes (DNS-ribosomes) had the same sedimentation patterns as the normal ribosomes. The dissociation and reassociation properties of ribosomes were not altered by the dansylation at various magnesium concentrations. In the temperature-absorbance (260 nm) profiles, dansylation of the ribosome brought about a lowering of the Tm by 2° and a 0.057 increase of the slope of the differentiated melting curve at the Tm. The DNS-ribosome was more sensitive to ribonuclease digestion than the normal ribosome, under all conditions tested. From the above observations, it was concluded that: The DNS-ribosome may be useful in the study of the functional role of r-proteins in the protein synthesis processes; the r-protein may be an important factor for the maintenance of the conformation of the r-RNA in the ribosome; ribosomal proteins may protect the r-RNA from ribonuclease digestion. In the case of normal ribosomes, the 40S subunit was the most sensitive to ribonuclease, while the 80S was the most resistant to the ribonuclease. As the magnesium concentration was lowered, this difference in ribonuclease sensitivity between ribosomal units decreased. More than 20% of the total protein of the crude ribosome could be detached in the washing processes without causing a destruction of the structural integrity of the ribosome as evidenced by sedimentation analysis. Proteins most readily removed were those whose pI's lay in the range of 6.3 - 7.5. The most basic (pI, 8 - 8.5) and the most acidic (pI, 4.5) protein were the least detachable, as shown by the isoelectric focusing patterns of removed proteins. The proteins removed from the ribosomes during each washing process, contained a higher content of acidic amino acids than basic amino acids, whereas the proteins remaining in the ribosome contained a higher content of basic amino acids than acidic amino acids. The neutral pI's associated with the detached proteins, instead of acidic pI's as would have been expected from the amino acid data, were probably due to the presence of many amide groups, as suggested by the high ammonia peaks from these proteins,and the constant ratio of the content of acidic amino acids to ammonia. All of the isoelectric focusing bands, from proteins which were removed during washing (removed protein), corresponded to bands which remained with the ribosomal particle. Previous work had shown that the disc electrophoresis patterns of the removed proteins were different from those proteins remaining in the ribosome. Although the primary structure of the removed proteins are the same as those remaining in the ribosome (same pI values), there may be conformational and aggregational differences between these two groups of proteins (different disc gel bands). This speculation, with the observation that the integrity of the ribosomal structure is not destroyed by the detachment of proteins, may lead to the theory that r-proteins are composed of multiple copies of fundamental subunit proteins, and that the protein component patterns in isoelectric focusing might represent the patterns of pI's of these fundamental subunit proteins. The physicochemical properties of r-proteins in ribosomes might be different depending upon their positions in the ribosome. Characteristics of pine seed r-proteins were typical in their electrophoretic and charge heterogeneity, amino acid composition, sedimentation properties, M̄w, spectrophotometric properties. As NH₂-terminal amino acids, arginine, alanine, and lysine were detected. === Ph. D.