Antisera to scrapie-associated fibril protein and prion protein decorate scrapie-associated fibrils.

Scrapie-associated fibrils (SAF) are an infection-specific structure observed in the unconventional-agent diseases. Polyclonal antisera raised to scrapie proteins were used to test the antigenic relationship between purified fibrils and SAF isolated from non-protease-treated synaptosomal-mitochondrial preparations. The experimental design utilized fibrils from scrapie strain 263K-infected hamsters, scrapie strain 139A-infected mice, and scrapie strain ME7-infected mice. Preparations were examined by negative-stain immune electron microscopy and Western blot analysis of the polypeptides. Fibrils and polypeptides from each preparation reacted with a rabbit antiserum raised to each of the following: hamster 263K prion protein (PrP 27-30), hamster 263K SAF protein, and mouse ME7 SAF protein. Immune electron microscopy and Western blot analysis revealed similar antigenic relationships among the three scrapie antisera. Thus, fibrils and polypeptides can be considered to be the same in each preparation. No reactivity of the fibrils was observed with antisera raised to Alzheimer neurofibrillary tangles or a synthetic peptide of cerebrovascular amyloid. Thus, the fibrils observed in purified preparations share structural and antigenic similarities plus biochemically related peptides with SAF present in non-protease-treated preparations.     

Plasma cholesteryl ester-triglyceride transfer protein. The catalytic domain is a low molecular weight proteolipid

The lipid transfer protein complex (LTC) isolated from human plasma by immunoaffinity chromatography transfers cholesteryl esters (CE), triglycerides, and phosphatidylcholine (PC) between lipoproteins in vitro. The molecular weight of this lipid transfer catalyst in sodium dodecyl sulfate-polyacrylamide gels was 65,000. When resolved on a gel filtration column by high performance liquid chromatography (HPLC), LTC was composed of fractions of high (greater than 150,000) to low (18,000) molecular weight, although sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of each fraction revealed bands at Mr 65,000 (major) and 52,000 (minor). The CE and triglyceride transfer activity of the low Mr HPLC fraction (1049 nmol of triglyceride/mg/h and 244 nmol of CE/mg/h) was significantly greater than that of the high Mr HPLC fraction (15-27 nmol of triglyceride/mg/h and 20-30 nmol of CE/mg/h). The PC transfer activity of the HPLC fractions was not determined. LTC proteins were separated by dialysis in acidified chloroform:methanol solution into dialysand and dialysate proteins. The dialysate contained a low Mr proteolipid, designated the catalytic domain Cd, which catalyzed CE and triglyceride transfer at equivalent rates (11.0 versus 9.5 mumol/mg/h, respectively). PC transfer activity was approximately 10% of these levels (1.5 mumol/mg/h). The dialysand consisted of a protein, designated the transfer protein TP, which facilitated CE (3.4 mumol/mg/h) preferentially over triglyceride and PC (1.0 mumol/mg/h) transfer, and a catalytically inactive protein, designated the heparin-binding domain Hd. We propose a model of the LTC protein (based on catalytic activities, monoclonal antibody reactivities, and heparin-binding capacities of the isolated proteins) in which both Hd (approximately 13 kDa) and Cd (approximately 3 kDa) originate from a single lipid transfer protein, TP.     

Claudin-2 forms homodimers and is a component of a high molecular weight protein complex

Tight junctions are multiprotein complexes that form the fundamental physiologic and anatomic barrier between epithelial and endothelial cells, yet little information is available about their molecular organization. To begin to understand how the transmembrane proteins of the tight junction are organized into multiprotein complexes, we used blue native-PAGE (BN-PAGE) and cross-linking techniques to identify complexes extracted from MDCK II cells and mouse liver. In nonionic detergent extracts from MDCK II cells, the tight junction integral membrane protein claudin-2 was preferentially isolated as a homodimer, whereas claudin-4 was monomeric. Analysis of the interactions between chimeras of claudin-2 and -4 are consistent with the transmembrane domains of claudin-2 being responsible for dimerization, and mutational analysis followed by cross-linking indicated that the second transmembrane domains were arranged in close proximity in homodimers. BN-PAGE of mouse liver membrane identified a relatively discrete high molecular weight complex containing at least claudin-1, claudin-2, and occludin; the difference in the protein complex sizes between cultured cells and tissues may reflect differences in tight junction protein or lipid composition or post-translational modifications. Our results suggest that BN-PAGE may be a useful tool in understanding tight junction structure.     

The ataxia telangiectasia clastogenic factor is a low molecular weight peptide

Summary The clastogenic factor in the plasma of ataxia telangiectasia (AT) patients and in conditioned medium from AT skin fibroblast cultures is a peptide with a molecular weight in the range of 500 to 1000. No clastogenic activity could be demonstrated in extracts of cultured AT fibroblasts.     

A new retinoid-binding protein with a low molecular weight

Cellular retinoid-binding proteins and nuclear receptors may mediate the intracellular transport and the action of retinoids in the control of differentiation and tumorigenesis. We report a new retinoid-binding protein (Ret BP) with a molecular size of 4,000 that binds retinol, retinoic acid, and some of their derivatives. Purification of Ret BP from chick skin cytosol involved DEAE-Sephadex, Sephadex G-100, and Mono Q column chromatography. The Ret BP-retinoid complex eluted at 195 mM NaCl during Mono Q column chromatography using a 0-300 nM NaCl gradient. Superose-12 column chromatography indicated a molecular size of 4,000 for Ret BP. The binding protein showed a pI of 6.8 on electrofocusing in ampholines of pH 3-10. Ret BP may act as an affinant for retinoids in the cell, and may serve to dispense the ligands to their respective functionally active sites.     

Ceroid lipofuscinosis in sheep. II. The major component of the lipopigment in liver, kidney, pancreas, and brain is low molecular weight protein

Previous studies on the lipopigment from the livers of sheep affected with ceroid lipofuscinosis showed that the disease does not involve a defect in lipid metabolism or abnormal lipid peroxidation and that most of the lipopigment was proteinaceous. In this study, lipopigment was isolated from liver, kidney, pancreas, and brain of affected sheep without the use of proteolytic enzymes. Lipopigment from all tissues was two-thirds protein. Modified silver staining after sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed a major band of Mr = 14,800, heterogeneous material between Mr = 5,000 and 9,000, and a major band of Mr = 3,500. These compounds did not stain for RNA or carbohydrate and were digested by a nuclease-free protease as expected for protein. They are not normal lysosomal proteins. Lipopigment levels of dolichol, ubiquinone, and cholesterol were consistent with the lipopigment being protein-enriched lysosome-derived cytosomes. The presence of the Mr = 3,500 proteins in whole affected tissue homogenates distinguished them from homogenates of normal tissues. It was concluded that low Mr proteins are specifically stored in ovine ceroid lipofuscinosis and that the ceroid lipofuscinoses may result from inherited defects in lysosomal protein catabolism.     

Actin polymerizability is influenced by profilin, a low molecular weight protein in non-muscle cells

We have previously isolated and crystallized a complex from calf spleen, containing actin and a smaller protein which we call profilin. In this paper we describe some properties of this complex, and show that association with profilin is sufficient to explain the persistent monomeric state of some of the actin in spleen extracts; moreover, spleen profilin will recombine with skeletal muscle actin to form a non-polymerizable complex resembling that isolated from spleen. Profilin is not restricted to spleen, but is found in a variety of other tissues and tissue-cultured cell lines. We propose that reversible association of actin with profilin in the cell may provide a mechanism for storage of monomeric actin and controlled turnover of microfilaments.     

A 25,000 molecular weight protein constituent of human amyloid fibrils related to amyloid protein AA

Amyloid fibrils from a patient with diffuse amyloid disease are dissociated in 6 m guanidine hydrochloride and fractionated by gel chromatography. Two major components are separated on Sepharose 6B. Both proteins are characterized by chromatography, immunodiffusion, discontinuous gel electrophoresis, amino acid tryptic peptide mapping and amino acid sequence analysis. The smaller of the two components is typical of the known protein AA by size (8400 daltons), amino acid composition and a 30-residue N-terminal sequence. The larger of the components (25,000 daltons) undergoes electrophoresis as a single band and appears unaffected by thiol reduction. It differs from protein AA in amino acid content and by its tryptic peptide map, although it contains an N-terminal amino acid sequence identical to protein AA when carried to 20 residues. Treatment of this larger component by mild acid hydrolysis results in the release of the 8400-dalton protein AA. Fractionation after guanidine hydrochloride treatment of this particular amyloid fibril preparation is compared to the fractionation of a typical secondary amyloid preparation that contains only protein AA as the major component. The origin and relationship of the 8,400- and 25,000-dalton protein components is discussed.     

Increased synthesis of a low molecular weight protein in vincristine-resistant cells

A 19,000-dalton peptide (pI = 5.7) that is synthesized in increased amounts in vincristine-resistant Chinese hamster cells ($\text{DC-3F}\text{VCRd-5}$) has been identified by two-dimensional gel electrophoresis. Reduced amounts of the protein were present in a revertant line of $\text{DC-3F}\text{VCRd-5}$, and only trace amounts were detected in control DC-3F cells. A similar protein (M_r = 19,000; pI = 5.7) was also found in a vincristine-resistant mouse line. Two vincristine-resistant human neuroblastoma cell lines likewise contained elevated levels of a low molecular weight acidic protein. Increased biosynthesis of the 19,000-dalton polypeptide in $\text{DC-3F}\text{VCRd-5}$ cells coincides with the presence of a homogeneously staining region, HSR, on a metaphase chromosome.     

Ultrastructural studies of scrapie-associated fibrils and prion protein from hamster brains infected with scrapie

We report here about the purification of prion protein 27-30 (PrP 27-30) and scrapie-associated fibrils (SAF) from hamsters infected with the 263K strain of scrapie. Ultrastructural analysis of fractions from scrapie-infected brains revealed numerous fibrils measuring approximately 20 nm in diameter and 100-200 nm in length. The substructure of these fibrils consisted of protofilaments which were usually straight and rarely helically arranged. We conclude that the electron microscopic appearance of SAF depends much on the purification scheme.     

A cytoskeletal protein with a molecular weight of 100 kD is a component of the endosomes participating in receptor-mediated endocytosis

Our previous paper (Rodionov et al., 1985) reported production of monoclonal antibodies RN-17 reacting in cultured fibroblasts with a protein having a molecular weight of 100 kD. Immunofluorescence and immunoelectron microscopy showed that this protein was a component of microtubules, intermediate filaments and coated vesicles. We challenged a possibility whether these coated vesicles containing the 100 kD protein may take part in the receptor-mediated endocytosis. alpha 2-Macroglobulin conjugated with fluorescein isothiocyanate or 20 nm colloidal gold particles was used as a marker of the receptor-mediated endocytosis. Mouse embryo fibroblasts or Swiss 3T3 cells were incubated with labeled alpha 2 M, fixed and "stained" with DN-17 antibody, and the distribution of alpha 2 M and 100 kD protein was examined within the same cells. In both cell lines the endocytic vesicles contained 100 kD protein and alpha 2 M. Therefore 100 kD protein is a component of endocytic vesicles. Probably this protein mediates microtubule-dependent transport of endocytic vesicles in the cells.     

Purification and properties of a low molecular weight protein factor of mitochondrial energy-linked functions.

1. A soluble protein with a molecular weight of 11-12-10(3) has been isolated from bovine-heart mitochondria, which stimulates the following ATP-dependent reactions of submitochondrial particles treated with 0.6 mM EDTA and 1 M NH4OH: reverse electron transfer from succinate to NAD, transhydrogenation from NADH to NADP, and ATP-Pi exchange. The factor has no effect on the NADH oxidase, succinate oxidase and ATPase activities of the particles. 2. The stimulatory effect of the factor in the ATP-dependent reduction of NAD by succinate is 12 mumol-min-1-mg-1 of the factor protein. However, the NH4OH-EDTA treated particles are saturated for maximal activation of the above reaction by very small amounts of the factor (about 20-40 mug factor per mg particle). 3. Electrophoresis of the factor preparation on polyacrylamide gels showed a single protein band plus a nonprotein material which moved at the dye front and was weakly stained with Coomassie Blue. The protein was shown to be required for activation of the particles; whether the fast-moving, nonprotein material is also required is not known. 4. The factor is inhibited by mercurials and N-ethylmaleimide. The former, but not the latter, inhibition is completely reversed by 1,4-dithiothreitol. 5. The NH4OH-EDTA treated particles are also stimulated by rutamycin up to about 0.1 nmol of rutamycin per mg particle; higher rutamycin concentrations inhibit. Depending on the particle preparation, the factor stimulates up to about 3 nmol per mg particle, but does not inhibit at higher concentrations. In addition, under certain conditions in which appropriate concentrations of rutamycin fail to stimulate the particles, the factor still does.     

The anchoring filament protein kalinin is synthesized and secreted as a high molecular weight precursor.

Kalinin, a recently characterized novel protein component of anchoring filaments, has been shown to be involved in keratinocyte attachment to culture substrates and to dermis in vivo, and to exist in keratinocyte-conditioned culture medium in two heterotrimeric forms of 440 and 400 kDa (Rousselle, P., Lunstrum, G.P., Keene, D.R., and Burgeson, R.E. (1991) J. Cell Biol. 114, 567-576). This study demonstrates that kalinin is initially synthesized in a cell-associated form estimated to be 460 kDa. By second dimension reduced electrophoresis, V8 protease digestion, and immunoblot analysis, we demonstrate that the cell form contains nonidentical subunits of 200, 155, and 140 kDa. The 440-kDa medium form is derived from the cell form by extracellular processing of the 200-kDa subunit to 165 kDa, a step which also occurs in skin organ culture. The 400-kDa form is derived from the 440-kDa form by extracellular processing of the 155 kDa-subunit to 105 kDa. The cell form is secreted by keratinocytes, deposited onto culture substratum, and is the form which facilitates attachment and adhesion of growing and spreading keratinocytes. It is also the form initially synthesized in skin organ culture. Kalinin purified from tissue, which appears to facilitate epithelial-mesenchymal cohesion in vivo, is closely related to the 400-kDa medium form purified from culture.