Microsomal nucleoprotein particles from pea seedlings

Ultracentrifugal analysis of an extract of pea epicotyls, previously freed of debris and larger particles by centrifugation at 40,000 g for 10 minutes, has revealed the presence of a major component which possesses a sedimentation coefficient of 74 S. This component constitutes about 25 per cent of the TCA-precipitable material in the clarified epicotyl extract and is estimated to make up 1 to 2 per cent of the dry weight of the original tissue. In size, chemical composition, and morphology, the 74 S component resembles the nucleoproteins of the microsomes from animal tissues. The 74 S component of pea epicotyl extracts has been purified by repeated cycles of differential centrifugation to yield a preparation which is 80 per cent homogeneous in the analytical ultracentrifuge. It has been found to contain 30 to 37 per cent RNA as judged by a variety of analytical techniques. Approximately 55 per cent of the weight of the material is protein and a further 4.5 per cent phospholipide. Electron micrographs of air-dried specimens of the purified preparation show the 74 S constituent to be flattened spheres with an average height of 180 A and an average diameter of approximately 280 A. The molecular weight of the 74 S particles is computed from sedimentation, viscosity, and partial specific volume data to be 4.5 million ± 10 per cent in agreement with the value estimated from electron micrographs. The 74 S or microsomal component of pea epicotyls is rapidly aggregated in the presence of low concentrations of Mg ions or by somewhat higher concentrations of Ca or K salts. ATP on the contrary causes resolution of electrolyte-induced microsomal aggregates with simultaneous degradation of the particles to an ultracentrifugally inhomogeneous mixture of lower molecular weight materials

The Incorporation of Leucine-C14 into Microsomal Particles and other Subcellular Components of the Pea Epicotyl

Incorporation of leucine-C14 into subcellular fractions of the apical section of pea seedlings has been studied as a function of the length of incubation. The specific activity of the microsomes was higher than that of the supernatant for short but not for long incubations, in agreement with observations on other systems. In this developing tissue the nuclei and especially the mitochondria appear to incorporate amino acid very rapidly. An insoluble fraction of the microsome pellet, which is presumably a liponucleoprotein complex, was found to possess, after 1 hour of incubation, a specific activity much greater than that of the purified microsomal particles or the supernatant fraction. Ninety-eight per cent of the leucine-C14 in the purified microsomal particles has been shown to possess bound amino groups, presumably in peptide linkages, by the DNP-end group method. These particles liberate but little peptide or protein of very high specific activity when they are destroyed by removal of Mg or by hydrolysis of RNA. Microsomal particles were fractionated into an RNA fraction and five protein fractions by means of density gradient centrifugation. By this method 95 per cent of the RNA can be separated from 90 per cent of the protein of the particle. Furthermore, the RNA fraction has been shown to contain very little protein of high specific activity. A particular protein fraction which contains the remaining 5 per cent of the RNA, possessed after 1 hour of incubation a specific activity 2 to 9 times higher than the protein of the other fractions

THE ISOLATION OF MYXOMYOSIN, AN ATP-SENSITIVE PROTEIN FROM THE PLASMODIUM OF A MYXOMYCETE

1. A procedure has been developed for the preparation of an active concentrate from the myxomycete, Physarum polycephalum. This concentrate responds with a lowered viscosity to the addition of small amounts of ATP. The preparation recovers in viscosity, and the process may be repeated. 2. In the most active concentrates, 75 per cent of the non-dialyzable material moves as a single boundary both in the descending limb in electrophoresis and in the ultracentrifuge. It contains about 10 per cent ribonucleic acid, which is at least in part reversibly bound to the protein. 3. The active material has been designated myxomyosin because of its origin and its similarity to actomyosin in ATP response

OBSERVATIONS ON AN ATP-SENSITIVE PROTEIN SYSTEM FROM THE PLASMODIA OF A MYXOMYCETE

1. Extracts of the plasmodia of the myxomycete, Physarum polycephalum, exhibit reversible decreases in viscosity in response to the addition of ATP under appropriate conditions. The protoplasm material prepared by extraction with KCl solution can apparently exist in either a high or a low viscosity state. As prepared, it is in the low viscosity condition. Rapid and extensive increases in viscosity of the extract are brought about by addition of AMP, inorganic phosphate, or, under certain conditions, of ATP. Only after the high viscosity state has been attained does addition of appropriate quantities of ATP cause a reversible decrease in viscosity. 2. The active principle of crude plasmodial extracts may be concentrated by fractional precipitation with ammonium sulfate and is found in the fraction precipitated between 30 and 40 per cent saturation. This material possesses a higher viscosity than does the original crude extract and is apparently in the high viscosity state since the addition of ATP causes an immediate reversible decrease in viscosity. 3. The ATP-sensitive fraction of myxomycete plasmodia possesses a viscosity which is dependent upon its previous thermal treatment. Extracts incubated at 0° for a period of a few hours increase greatly in viscosity when they are returned to 24.5°. This increased viscosity is structural in nature, is destroyed by mechanical agitation of the solution, and may be reversibly destroyed by addition of ATP. 4. It is suggested that the ATP-responsive protein of myxomycete plasmodia may be related to sol-gel transformations which have been observed in intact plasmodia and may participate in the protoplasmic streaming of the intact organism. This suggestion is based upon the following facts: (a) the protoplasmic streaming of myxomycete plasmodia is increased by microinjection of ATP; (b) the gel portion of the cytoplasm at the site of the microinjection of ATP is extensively converted to the sol state. The changes in structure of the intact cytoplasm are thus similar in nature to the changes exhibited in response to ATP by the purified ATP-sensitive protein. 5. The ATP-sensitive protein of myxomycete plasmodia appears to undergo reversible aggregation to form a high viscosity state. The function of ATP is to break down the aggregates thus formed. Since a specific ATPase activity is associated with the purified material, added ATP is gradually destroyed and recovery of viscosity attends the spontaneous reconstitution of aggregates

Establishment of a bioassay system for cancer risk assessment in energy technology

Separate abstracts were prepared for 20 papers in this report. For several years the Department of Energy (DOE), Office of Health and Environmental Research (OHER), has supported a research program aimed at developing new experimental approaches for the improvement of cancer risk assessments. The central issue is to overcome the organizational, species and other barriers that make it difficult to extrapolate laboratory-based data to predict risk to man. Most of the participants at the meeting are involved in research aimed at understanding the mechanism(s) of chemical carcinogenesis. Complex mixtures of chemicals are associated with many energy technologies. DOE's initial program emphasis focused on semi-applied research aimed at quantitative evaluation of carcinogenic activity of complex materials. Since much progress has been made in DOE integrated technology-specific chemical-biological characterization studies, the number and kinds of chemicals of concern has been reduced to a relatively few well-defined classes. Although the classes of compounds seem to be unique to some of the synfuel technologies, they are quite similar to compounds of general interest, for example, poly-nuclear aromatic hydrocarbons. Special emphasis was placed on molecular and cellular dosimetry as one of the key requirements for quantitative comparison of effects at the cell level in vivo and in vitro. Although it is relatively easy to measure cell, tissue, organ and whole organism doses associated with radiation exposures, we are just learning how to do this for chemical agents. Several methods have been developed in the past several years which can be used

Basic mechanism in carcinogenesis. Progress report, May 1974--June 1975. [Cultured mammalian cells]

Non

Histone Biology and Chemistry

Recent advances in the biophysics and biochemistry of the nucleic acids have clearly shown that these are the molecules entrusted by nature with the task of storage and transfer of genetic information. Rapid developments in the status of the coding problem provide us with the promise that the day is near when we shall have full understanding of the genetic language. Attention is gradually being focused upon the mechanisms which control the transcription of the genetic message, and which, by the exertion of such control, bring about organized biological activity of the cell and orderly development of a single cell into a multicellular organism. What substances in the cell could be expected to serve in the control of genetic activity; to program the transcription and expression of the genetic language