High Molecular Weight Polypeptides Research Articles

Isolation of microtubule protein from chicken erythrocytes and determination of the critical concentration for tubulin polymerization in vitro and in vivo.

Microtubule protein isolated from nucleated chicken erythrocytes was examined with respect to composition and assembly properties to determine its significance in a microtubule bundle called the marginal band. 1) The protein contains greater than 95% tubulin with small amounts of tau polypeptides and no high molecular weight polypeptides. 2) Microtubule assembly in vitro at 37 degrees C is characterized by low levels of nucleation, despite an abundance of ring oligomers at 5 degrees C, as indicated by long lag times, slow assembly rates, and microtubules that are twice as long as brain microtubules assembled under the same conditions. 3) By radioimmunoassay and sodium dodecyl sulfate gel analysis we determined that 0.6% of erythrocyte protein is tubulin of which three-quarters is in a nonextractable form and is associated with the microtubule bundle and the cell cortex. From these values the in vivo concentrations of total tubulin and tubulin dimer subunits are 2.4 and 0.7 mg/ml, respectively. The value of 0.7 mg/ml is close to the range of values of 0.1-0.6 mg/ml for the critical concentration of erythrocyte microtubule protein in vitro, suggesting that the assembly properties of tubulin in vitro and in vivo are similar.

A Messenger RNA for Tobacco Mosaic Virus Coat Protein in Infected Tobacco Mesophyll Protoplasts

AbstractThe low molecular weight tobacco mosaic virus (TMV)‐specific RNA component (LMC) was demonstrated in tobacco mesophyll protoplasts by polyacrylamide gel electrophoresis of 14C‐uridine‐labelled RNA from infected protoplasts.Free and membrane‐bound polysomes were isolated from infected protoplasts, and RNA extracted from them was analyzed. TMV‐specific RNA species including full‐length viral RNA, its replicative intermediate, and LMC were found in both free and membrane‐bound polysomes, but were present in free polysomes in much larger amounts. In particular, as much as 37 % of total LMC in protoplasts was found in free polysomes. Fractionation of polysomes by sedimentation in sucrose gradients showed that LMC is associated with small‐sized polysomes (mono‐ to tetrasomes). Polysomes of this size class produced viral coat protein in a cell‐free protein synthesizing system from rabbit reticulocytes. On the other hand, full‐length TMV‐RNA was associated predominantly with larger polysomes which produced in the cell‐free system TMV‐specific high molecular weight polypeptides but no coat protein. These results indicated that LMC, a subgenomic RNA of TMV, in fact functions in vivo as messenger RNA for viral coat protein, as has been postulated on the basis of in vitro studies.

Identity and Origin of the ATPase activity associated with neuronal microtubules. I. The ATPase activity is associated with membrane vesicles.

Microtubule protein purified from brain tissue by cycles of in vitro assembly-disassembly contains ATPase activity that has been postulated to be associated with microtubule-associated proteins (MAPs) and therefore significant for studies of microtubule-dependent motility. In this paper we demonstrate that greater than 90% of the ATPase activity is particulate in nature and may be derived from contaminating membrane vesicles. We also show that the MAPs (MAP-1, MAP-2, and tau factors) and other high molecular weight polypeptides do not contain significant amounts of ATPase activity. These findings do not support the concept of "brain dynein" or of MAPs with ATPase activity.

Purification of a HeLa cell high molecular weight action binding protein and its identification in HeLa cell plasma membrane ghosts and intact HeLa cells.

The high molecular weight protein (HMWP) which was previously observed to be a major component of the actin based gels formed by incubating cytoplasmic extracts of HeLa cells at 25 degrees C [Weihing, R. R. (1977) J. Cell Biol. 75, 95-103] has now been purified by gel filtration of 0.6 M KCl extracts of precipitated gels. A few hundred micrograms of HMWP, which is about 90% pure, can be isolated from 4 X 10(9) cells. HMWP can gel muscle actin and cross-link it into filament bundles. Its subunit molecular weight is 250 0000, its Stokes radius is 125 A, and its sedimentation coefficient is 9 S. A native molecular weight of 480 000 was calculated by using the latter two parameters, and therefore the native molecule is a dimer. Its amino acid analysis is nearly indistinguishable from that of macrophage actin binding protein and of mammalian and avian filamins. All of these findings indicate that HMWP is homologous to the latter proteins. However, HeLa cell HMWP and avian filamin must differ in their primary sequences because their partial peptide maps are distinct and because an antiserum against HMWP reacts only weakly with filamin. For studies on the intracellular location of HMWP, a goat antiserum against purified HMWP was prepared and characterized and then used to localize HMWP in suspension grown cells. The technique of immunoblotting revealed that the antiserum reacted virtually exclusively with the high molecular weight polypeptide that comigrates with HMWP in cell lysates and in ZnCl2-stabilized plasma membrane ghosts prepared from HeLa cells [Gruenstein, E., Rich, A., & Weihing, R. R. (1975) J. Cell Biol. 64, 223-234] and that it did not react with rabbit myosin heavy chain, microtubule proteins (MAPS and tubulin) from HeLa cells and calf brain, or the proteins of human erythrocyte ghosts including spectrin. Suspension-grown cells which were stained with the antiserum by the technique of indirect immunofluorescence showed bright fluorescence at the rim of the cells and less intense generalized fluorescence. If preimmune serum or immune serum treated with HMWP was substituted for the immune serum, then staining at the rim was not observed, but the generalized fluorescence was only slightly reduced; unpermeabilized cells were not stained. These results indicate that HMWP is a component of the cortical cytoplasm of HeLa cells. Possible functions of cortical HMWP are discussed briefly.

Plastid gene expression in a yellow-green leaf mutant of Petunia hybrida

We have analyzed the morphology and gene expression in plastids of a yellow-green leaf mutant of Petunia hybrida (E 5059). Under normal light intensities (20,000 lx), yellow-green leaves develop with a typical proplastid morphology (few membranes, incomplete stacking). When such plants are grown under low light intensities (3,000 lx), the newly formed leaves are green. The plastids in these green leaves have a wild-type like chloroplast morphology (thylakoids and grana structure). Pre-existing green leaves remain green in 20,000 lx, indicating that chlorophyll is not degraded. An analysis of polypeptides synthesized in isolated plastids from the yellow-green and green leaves of this mutant plant shows several differences. In the yellow-green leaf plastids only a very small amount of the large subunit of ribulose-1,5-bisphosphate carboxylase (RuBPCase) is present, while in green plastids this polypeptide is present in much higher amounts. Hybridization experiments indicated that in plastids from the yellow-green leaves the mRNA coding for the large subunit polypeptide is present in much lower amounts than in plastids from the 3,000 lx green leaves of this mutant or in chloroplasts from wild type plants. These results indicate regulation at the mRNA level. Furthermore, in yellow-green leaf plastids eleven polypeptides are present with high molecular wieght (higher than 67,000 d). Five of them are synthesized by the yellow-green leaf plastid itself. Such high molecular weight polypeptides are also synthesized by proplastids isolated from white petunia cell suspension cultures, but are not synthesized by 3,000 lx green leaf plastids, or by isolated normal leaf chloroplasts. These results indicate that the synthesis of these polypeptides is specific for the proplastid stage of chloroplast biogenesis.

Solubilisation and characterisation of wheat gluten proteins: correlations between the amount of aggregated proteins and baking quality.

AbstractA comparison was made of the effectiveness of various solvents in extracting protein from wheat gluten. The best proved to be 0.055m cetyltrimethyl ammonium bromide‐6m urea‐0.01m acetic acid. When extracts were separated on columns of controlledpore glass, with a nominal exclusion limit of 1.2 × 106 daltons for molecules in the extended form, two peaks were obtained termed F1 and F2. F1 was excluded from the column and contained material which was stable in the absence of reducing agent but was markedly reduced in size in the presence of 2‐mercaptoethanol. SDS‐PAGE showed that F1 was enriched in high molecular weight polypeptides (about 100 000 daltons) and also contained polypeptides with molecular weights in the range 30 000–45 000. F2 consisted almost entirely of polypeptides with molecular weights of less than 50 000. The ratio of F1 :F2 in different varieties varied from 0.1 to 0.58 and this variation was correlated with the NIAB potential breadmaking quality score. However, there did not appear to be any difference between the total amounts of high molecular weight polypeptides in a poor and a good breadmaking variety.

Density distribution, characterization and comparative aspects of plasma lipoproteins in the salamander, Pleurodeles waltii

1. 1. Blood plasma from a Urodele amphibian, Pleurodeles waltii, has been found to contain very-low density, low-density and high-density lipoproteins (VLDL, LDL and HDL, respectively). HDL and LDL predominated (concentration range 72–114 and 51–101 mg/dl) with lesser quantities of VLDL (23–51 mg/dl). 2. 2. Following isolation by density gradient ultracentrifugation, the physicochemical properties (morphology, particle size, hydrated density, electrophoretic mobility, and chemical composition) of the three major classes resembled, but were not identical with, those of the corresponding fractions in normal human plasma. 3. 3. The protein moieties of VLDL and LDL consisted mainly of polypeptides of high molecular weight ( Mr > 60,000 ), which in their solubility were akin to human apo-B: smaller amounts of another protein migrated to the position of human apo-C-III in basic polyacrylamide gels. 4. 4. The major HDL apolipoproteins were of Mr 31,000 and 27,000, with minor amounts of 13,000, 61,000 and 76,000 components; in alkaline, urea-containing polyacrylamide gels, these major proteins migrated faster than the major components (apo-AI and apo-AII) of human HDL. 5. 5. Immunochemically, lipoproteins with pre-β-, β- and α-mobility were detected in the VLDL, LDL and HDL, respectively. 6. 6. Salamander VLDL and LDL were found to share an antigenic site(s) with the LDL of trout, chicken and guinea-pig, suggesting the presence of an apo-B-like protein. 7. 7. The chemical compositions and immunochemical reactions of salamander lipoproteins indicate that their structure and metabolism resemble that of other amphibia, reptiles and fish rather more closely than that of most mammals and birds.

Binding of HeLa spectrin to a specific HeLa membrane fraction.

From 30-40 g of Hela-S3 cells grown in suspension, 0.25-0.50 mg of spectrin has been purified by conventional biochemical procedures starting from a low ionic strength extraction at alkaline pH of crude Hela membranes. Hela spectrin consists in its native form of a tetramer alpha 2 beta 2 of two high molecular weight polypeptides (240,000 and 230,000 daltons). Three different populations of Hela membranes depleted of both spectrin and actin have been prepared on discontinuous sucrose gradients. Surprisingly, spectrin will reassociate with only the heavier membrane fraction. This reassociation is specific for Hela spectrin, since three other purified Hela proteins as well as human erythrocyte spectrin do not reassociate under the same conditions. This binding is not due to the presence of traces of actin still present in the membrane fraction since two Hela actin-binding proteins (filamin I and II) do not show any significant binding to this fraction. The nature of the membrane-binding site for Hela spectrin is discussed.

Cytoplasmic dynein of the sea urchin egg. II. Purification, characterization and interactions with microtubules and Ca-calmodulin.

Purification of cytoplasmic dynein from unfertilized sea urchin eggs was performed in the presence of protease inhibitors to avoid proteolysis throughout the purification procedure, which comprised several chromatographies, including a calmodulin-Sepharose 4B affinity column chromatography. This is the first report of the purification of cytoplasmic dynein to near homogeneity. The purified fraction was composed of a single high molecular weight polypeptide and some minor low molecular weight polypeptides. The high molecular weight polypeptide comigrated with flagellar dynein A beta chain from sperm on SDS-polyacrylamide gels. There was no polypeptide stainable with periodic acid-Schiff reagent (PAS) in the purified cytoplasmic dynein fraction. Cytoplasmic dynein showed characteristics quite similar to those of axonemal dynein, i.e. high substrate specificity for ATP and inhibition by low concentrations of vanadate, though its Ca-ATPase activity showed almost the same dependence on the concentration of either divalent cations or KC1 as the Mg-ATPase activity. The purified enzyme seemed to possess functional form as judged from its properties: 1) pH dependence of the ATPase activity, 2) dependence of the ATPase activity on MgCl2 and KCl concentration, 3) Km for Mg-ATP, and 4) binding to flagellar doublet microtubules. Cytoplasmic dynein bound to calmodulin-Sepharose 4B only in the presence of Ca2+, and was eluted with EGTA. Furthermore, the ATPase activity was enhanced 6-fold by calmodulin in a Ca2+-dependent manner. The activation by calmodulin was prevented by a stoichiometric amount of trifluoperazine.

The presence of high molecular weight aggregates in the protein bodies of developing endosperms of wheat and other cereals

Protein bodies have been isolated from developing grain of wheat, barley and rye. The protein has been solubilised in a solvent consisting of 0·01- M acetic acid, 6- M urea and 55-m M cetyltrimethylammonium bromide and chromatographed on columns of controlled pore glass. In each case a proportion of the protein was eluted in the excluded volume of the column, indicating the presence of multimeric aggregates in the protein bodies. In all three cereals these aggregates contained an enhanced proportion of high molecular weight polypeptides and certain sulphur-rich prolamins. The proportion of aggregated material in wheat protein bodies was similar to that in gluten preparations and two-dimensional electrophoretic analyses showed that protein bodies and gluten consisted of almost identical polypeptides. We therefore suggest that the aggregates of gluten proteins, which have been implicated as important in determining bread-making quality, are laid down in protein bodies shortly after synthesis in the developing seed. Similar studies have also been carried out with maize protein bodies. Although these contained some aggregated proteins they did not appear to contain the very large complexes observed in the other three cereals.

Structural genes on the Y chromosome of Drosophila melanogaster.

Testis proteins of Drosophila melanogaster deficient for six different Y-chromosome regions were fractionated by means of a sodium dodecyl sulfate/polyacrylamide gel system designed to separate high molecular weight polypeptides (Mr, greater than 200,000). Analysis of the banding patterns indicates that the three regions containing fertility genes kl-2, kl-3, and kl-5 are responsible for three different high molecular weight polypeptides. Several observations indicate that these polypeptides are structural components of the sperm axoneme. They are present in seminal vesicles, which are highly enriched for mature sperm. They are first detected during development at a time when the first spermatids are elongating. Finally, deletion of either kl-5 or kl-3 leads to the absence of the outer dynein arm of the peripheral doublets of the axoneme. Although absence of the kl-2 region eliminates the third polypeptide, an associated structural defect in the axoneme has yet to be identified. The three polypeptides are in the Mr 300,000-350,000 range, and their mobilities are similar to those of dynein polypeptides from Chlamydomonas axonemes. Experiments using dosage variation and a temperature-sensitive sterile mutation in kl-5 suggest that the Y-chromosome regions contain the coding sequences for the polypeptides.

Formation and identification of cytoskeletal components from liver cytosolic precursors.

Liver cytosol forms a macroscopic fibrillary network in the presence of low concentrations of MgCl2. This process represents the generation of 3- to 11-nm filaments from soluble precursors, involving selectively at least 12 major polypeptides. Similar polypeptides are enriched in the detergent-insoluble fraction from hepatocytes, suggesting that they may be important constituents of the native cytoskeleton. AcA 34 gel-permeation chromatography resolves the cytosol into three independently "polymerizing" peaks: A, B, and C. The formation of filaments follows biphasic kinetics in peaks B and C, whereas peak A lacks the slow phase. Filament formation in all three systems is inhibited by 1-15 mM inorganic phosphate, 10 mM NaF, or 10 mM sodium molybdate. The polymerization of peak C only is inhibited by 0.2-2 mM ATP. CaCl2 (1-100 microM) has no apparent regulatory effect. Two-dimensional polypeptide analysis and peptide mapping show that actin is a major component of peak C, while peaks A and B contain prominent polypeptides that may be related to intermediate filament subunits. In addition, all three systems contain two or three high molecular weight (greater than 170,000) polypeptides that may participate in modulating and extending the filament network. The filaments from peaks A and B are soluble in 8 M urea and reform on removal of the urea in the presence of 5 mM MgCl2. The polypeptide composition remains constant through three such cycles.

Purification and characterization of a highly purified human factor VIII consisting of a single type of polypeptide chain.

Human factor VIII was purified 350,000-fold (relative to plasma) from a commercial factor VIII concentrate. The procedure used standard protein separation techniques and was performed in the absence of protease inhibitors. The product has a specific activity of 4,900 units/mg, an activity-to-antigen ratio of 75:1 (unit/unit) and no more than 0.1% von Willebrand protein. Electrophoresis of the reduced protein in a denaturing polyacrylamide gel showed a single major band of Mr 100,000. Procoagulant activity was eluted from a nondenaturing gel after electrophoresis in the region of the single major band. Thrombin converted the Mr 100,000 polypeptide to a polypeptide of Mr 75,000. The procoagulant activity was increased 10-fold by thrombin or factor Xa and was completely inhibited by activated protein C or factor VIII inhibitor plasma. This factor VIII preparation consists of a single high molecular weight polypeptide chain and has the highest specific activity thus far reported for human factor VIII.

Probing the subunit structure of cow anterior lens capsule with the detergent, sodium dodecyl sulfate

Independent of solubilization temperature and presence of reducing agent, the bulk of anterior lens capsule dissolved in buffered sodium dodecylsulflate (SDS). The electrophoretic pattern of solubilized capsule varied with extent of solubilization and presence of reducing agent, but not with the presence of proteolytic enzyme inhibitors and sonication during capsule preparation. When the capsule was partially solubilized, high molecular weight polypeptides predominated. Increased solubilization resulted in formation of lower molecular weight bands. Analysis of conversion of cystine to cysteine and control experiments with standard proteins suggest that electrophoretic band multiplicity is due to incomplete cleavage of disulfide bonds and cleavage of peptide bonds. Reducing conditions resulted in cleavage of polypeptides with molecular weights ranging from 2000 to 37000. Amino acid analyses showed the initial extraction of collagenous and non-collagenous protein.After extensive extraction, a non-collagenous cystine-rich protein remains insoluble. These data are consistent with the idea that the lens capsule contains two types of non-collagenous glycoproteins. The insoluble non-collagenous cystine-rich protein accounts for as much as 4% of the membraneproteins. Since most of its cystine residues were converted to S-carboxymethylcysteine, its insolubility must be accounted for by bonds other than disulfide bonds. In vivo, it is likely that this is a bridging membrane component to which other components are bound by disulfide bonds.

Light and benzylaminopurine induce changes in ultrastructure and gene expression in plastids of Petunia hybrida cell cultures

The regulatory effect of light and the cytokinin 6-benzylaminopurine (BA) on the plastid ultrastructure and plastid DNA gene expression is studied in white and mutant green cell suspension cultures of Petunia hybrida. By electron microscopy we show that both light and 6-benzylaminopurine induce the formation of thylakoid membranes and grana structures in plastids of the green cultures. For membrane formation in plastids of white cultures, light in combination with BA is required. Light and benzylaminopurine also influence the plastid DNA gene expression. By in-organello protein synthesis with isolated plastids we show that light as well as benzylaminopurine affects the synthesis of plastid DNA encoded proteins. A characteristic effect of benzylaminopurine on plastids from white and green cultures is the reduction in the synthesis of the CFI subunits of 55,000 and 57,000 D, and the reduction in the synthesis of large polypeptides with a molecular weight higher than 67,000 D. In contrast to benzylaminopurine, light only affects the DNA gene expression of plastids from white cell cultures, that are in a very early stage of plastid development. Light stimulates the synthesis of polypeptides with a molecular weight of 84,000, 70,000 and 46,000 D which are encoded by cpDNA in these white culture plastids. In green cell cultures both plastids with a etioplast-like phenotype and with a chloroplast like morphology synthesize similar polypeptides, resulting in the same polypeptide pattern. Our results indicate that qualitative differences in plastid DNA gene expression as an effect of light do occur but only in plastids at very early stages of chloroplast development. We observe a gradual reduction in the number of high molecular weight polypeptides at later stages of chloroplast development. This suggests that these large polypeptides are characteristic for plastids at an early developmental stage.

Characterization of excretory-secretory antigens of Fasciola hepatica.

Twenty-one day old Fasciola hepatica were recovered from the livers of infected mice and cultured for 5-7 days in a serum-free medium containing either [14C]leucine, [14C]isoleucine or [35S]methionine, or in a medium containing [14C]leucine and serum from sheep vaccinated with excretory-- secretory (ES) antigens of juvenile F. hepatica. All three labelled amino acids were incorporated into fluke proteins. Labelled proteins also appeared in the culture medium. Three major polypeptides detected in the culture media had apparent molecular weights of 26,000, 24,000 and 23,000. All were immunoprecipitated from [14C]leucine-labelled culture medium using antisera against fluke somatic antigens raised in rabbits or from sheep vaccinated with ES antigens of juvenile F. hepatica. A polypeptide of molecular weight 27,000 was also prominent in the culture medium when [14C]isoleucine was used. This polypeptide was present was a minor component when [14C]leucine and [35S]methionine were included in the culture media; it did not appear to be immunoprecipitated by the above antisera from [14C]leucine-labelled culture medium. In the presence of serum from vaccinated sheep, the ES antigens formed immune complexes which contained the polypeptides mentioned above, together with several higher molecular weight polypeptides. Additionally, a number of minor bands of varying molecular weight were present. After micro-Ouchterlony gel immunodiffusion, 2 precipitin lines formed between the labelled ES antigens and antisera. Electrophoresis of these indicated that the 23,000, 24,000 and 26,000 Dalton labelled polypeptides were present in each. The higher molecular weight and the 27,000 Dalton labelled polypeptides were also present in one of the lines.

Translational properties of a non-polyadenylated oligo(U)-containing RNA fraction from wheat embryos

A non-polyadenylated oligo(U)-containing RNA (poly(A) −·oligo(U) + RNA) fraction was isolated from wheat embryo cytoplasm and its properties were compared with those of polyadenylated RNA (poly(A) + RNA) from the same source. Both RNA preparations were highly heterogeneous and effectively stimulated [ 14C]leucine incorporation in a wheat germ cell-free translation system. Electrophoretic patterns of the translation products appearing in the non-polyadenylated RNA- and polyadenylated RNA-supplemented translation assays, respectively, differed from each other. The non-polyadenylated RNA-specific translation products included, in particular, a series of high molecular weight polypeptides. It is concluded that a specific class of non-polyadenylated oligo(U)-containing mRNA species (other than histone mRNAs) occurs in the wheat embryo cells.

Chick embryo DNA polymerase alpha. Polypeptide components and their microheterogeneity.

DNA polymerase alpha was purified from a chick embryo extract by ammonium sulfate fractionation and successive column chromatographies. The final preparation had a specific enzyme activity of 39,000 units/mg of protein with activated calf thymus DNA as a template.primer. Electrophoresis in nondenaturing polyacrylamide gel showed that the preparation contained two proteins, one of which was associated with DNA polymerase activity. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the electrophoretically pure enzyme gave two clusters of polypeptide bands. One was composed of 3-4 polypeptides with similar electrophoretic mobilities corresponding to Mr = 130,000-155,000, and the other consisted of four distinct polypeptide bands corresponding to Mr = 59,000, 56,000, 54,000, and 51,000. Two-dimensional tryptic peptide mapping analysis of these polypeptides after 125I-iodination indicated that the structures of all four Mr = 51,000-59,000 polypeptides were very similar. In addition, polypeptides in the Mr = 145,000-155,000 region had almost identical structure with those in the Mr = 130,000-140,000 region. No significant structural homology was observed between the high molecular weight polypeptides and low molecular weight polypeptides. These findings indicate that the four polypeptides of Mr = 51,000-59,000 are generated by minor modification(s) of a single polypeptide. Similarly, the heterogeneity of the high molecular weight polypeptides is brought about by minor modification(s). Thus, chick embryo DNA polymerase alpha is basically composed of two kinds of subunit polypeptides.

Insect vitellins: Identification, purification, and characterization from eight orders

AbstractVitellins were identified, purified, and analyzed from insects representing eight orders. The structures and polypeptide constituents of vitellins of Hyalophora cecropia, Tenebrio molitor, Rhodnius prolixus, Forficula auricularia, Periplaneta americana, and a mayfly were found to have common features. The native proteins had Mr of 385,000–470,000 (385–470 K) and were composed of high (100–180 K) and low (47–84 K) molecular weight polypeptides in equimolar proportions. The vitellins of Apis mellifera, a sphecid wasp, and Aedes aegypti, however, had lower Mr (200–350 K) and were composed of only large polypeptides (170–190 K). The higher Diptera form a distinct third group with vitellins made up entirely of small polypeptides of about 50 K.

Mitochondrial damage in galactosamine-induced liver intoxication in rats.

Mitochondria isolated from livers of rats which received D-galactosamine (375 mg/kg body wt., four times) demonstrated a marked decrease in respiratory control ratios, the ADP/O ratios, and state 3 respiration rates and an increase in state 4 respiration rates. The aberration was profound with site I being altered prior to sites II and III. Quantitation of phospholipids revealed a reduction of total phospholipids per mg protein with decreases in phosphatidylcholine and phosphatidylethanolamine contents. Caldiolipin was the only phospholipid which remained unaltered. Fatty acid composition was altered in these phospholipids; caldiolipin was altered most severely, showing reductions in linoleic and arachidonic acids, and an elevation in saturated fatty acids and in some other small components of fatty acids. In phosphatidylethanolamine, palmitic acid decreased, whereas stearic and docosahexenoic acids increased. These changes were smaller in phosphatidylcholine fatty acids. These mitochondria were also characterized by an altered composition in high molecular weight polypeptide components. By experiments with normal mitochondria in vitro, galactosamine, but not other aminohexoses, was proved to be an uncoupling agent of the oxidative phosphorylation system. Electron microscopic observation demonstrated that both in vivo and in vitro treatments with galactosamine induced marked disorganization of mitochondrial structures. These results suggest that mitochondrial damage is also included in galactosamine-induced hepatic lesion.