George E. Palade

Protein blotting: Principles and applications

Twenty-eight years ago, Smithies demonstrated that a starch gel could serve as a molecular sieve through which zone electrophoresis of proteins could be carried out (95). Since then, repeated innovations and improvements have brought the technique of protein separation by gel electrophoresis to the state we know today. The introduction of polyacrylamide gels (8 1) and of discontinuous buffer systems (20,75), the use of SDS’ to solubilize and disaggregate protein complexes prior to electrophoresis (102), and the addition of SDS to discontinuous buffer systems (59,69) have all been major contributions to the development of this procedure, now one of the most widely used analytical and preparative tools in cellular and molecular biology. Problems in gel electrophoresis. Initially, the main objective of these techniques was to monitor visually the homogeneity or heterogeneity of a protein preparation and to follow the appearance or disappearance of a particular “band” throughout a given experimental procedure. One-dimensional gels were found to be quite adequate when relatively simple protein samples, e.g., viruses ( 102), bacteriophages (59,100) erythrocyte ghost membranes (25), were analyzed. But, as more complex systems demanded greater resolving

Electrophoretic transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to a positively charged membrane filter

Abstract Zeta-bind, a positively charged nylon membrane, was tested as an immobilizing matrix for the electrophoretic transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels. It was found that Zeta-bind has a considerably greater capacity than does nitrocellulose for protein binding. Because of this property, more efficient elution of proteins from gels can be used (by omitting methanol from transfer buffers). The procedure described is more amenable to quantitation than usual nitrocellulose-based transfer. Antibody or lectin overlay techniques are also more sensitive on Zeta-bind than on nitrocellulose.

Intracellular sorting and polarized cell surface delivery of (Na+,K+)ATPase, an endogenous component of MDCK cell basolateral plasma membranes

Madin Darby Canine Kidney (MDCK) cells grown on polycarbonate filters in a two-chamber culture system were used to study the postsynthetic sorting of the alpha-subunit of the (Na+,K+)ATPase, an important native protein of the MDCK cell basolateral plasmalemmal domains. The N-azidobenzoyl derivative of ouabain (NAB-ouabain) and anti-ouabain antibodies were used in pulse labeling experiments to monitor the arrival of newly synthesized molecules of (Na+,K+)ATPase at the apical and basolateral cell surfaces. The results show that newly synthesized alpha-subunits bind NAB-ouabain and become substrates for immunoprecipitation only when this compound is present in the basolateral chamber. No more than 10% of the (Na+,K+)ATPase synthesized during the pulse period could appear at the apical surface without being detected by our assay. Thus, sorting of this native protein is effected intracellularly prior to its direct insertion into the basolateral plasmalemmal domain. Passage through an acidic compartment is not required for proper sorting.

Structural basis of permeability in sequential segments of the microvasculature of the diaphragm: II. Pathways followed by microperoxidase across the endothelium

Abstract Bipolar microvascular fields of mouse diaphragm were used to investigate the existence of characteristic modulations in the structural basis of permeability along successive segments of the microcirculation. Using hemeundecapeptide (microperoxidase; MW, ∼1900; mol diam, ∼20 A) as a probe molecule, the observations indicate that the timing and pathways followed by this tracer across the endothelium were different in each microvascular segment. The first permeated by these molecules were the pericytic venules at the level of their endothelial junctions. Among the latter, ∼25 to 30% are normally open to a space of ∼30 to 60 A. The vesicular transport is particularly extensive in capillaries and pericytic venules, and the transition from phase I to phase III is faster in the venular segments of capillaries, probably due to their high frequency in transendothelial channels formed by single vesicles. In the arteriolar and middle segments of capillaries, the H11P passed via vesicular transport and transendothelial channels. Endothelial junctions in arterioles and along the entire length of capillaries appeared to be impermeable to molecules of ≥20 A in diameter.

Morphologic and biochemical evidence for a contractile cell network within the rat intestinal mucosa

Subepithelial and pericryptal fibroblastlike cells form a two-dimensional network immediately subjacent to the epithelial basal lamina in the small intestine and colon in several mammalian species. Stellate-shaped cells with similar, but not identical characteristics, form a three-dimensional network deep within the villar lamina propria. Electron microscopic studies indicate that these cells contain a putative contractile apparatus, are attached to each other and to apparently organized elements of the extracellular matrix by typical adhesive devices, and form gap junctions with each other. Comparative in situ immunoperoxidase localization studies document the presence in these cells of four contraction-associated proteins (smooth muscle isotropomyosin, cyclic guanosine monophosphate-dependent protein kinase, both nonmuscle and smooth muscle isomyosin, and actin) in amounts generally greater than those found in connective tissue fibroblasts, but less than in smooth muscle cells. Taken together, these results strongly suggest a smooth muscle-like, contractile function for these cells and indicate that this cellular network may provide a supportive tonus for the epithelium, as well as provide the force needed for active movement of the villus, expulsion of crypt secretion products, and propulsion of absorption products in the lamina propria, the microvasculature, and lacteals of the intestinal villus.

Structural basis of permeability in sequential segments of the microvasculature of the diaphragm: I. Bipolar microvascular fields

Abstract A new method is described for the reliable identification of successive segments in the microvasculature of skeletal muscle. The procedure relies on the existence in the postero-lateral regions of the mouse diaphragm of bipolar microvascular fields (BMFs) in which the supplying arteriole enters and the draining venules leave the capillary bed from opposite ends. In such fields the different segments of the microvasculature were initially recognized at the light microscope level in whole mounts of the muscle; their identity was confirmed by successive examination under the light and the electron microscope. In these BMFs, the average capillary path length is ∼580 μm, the average inner diameter increases progressively from ∼3 μm for the arteriolar, to ∼3.5 μm for the venular segments of capillaries; from this level, there is a sharp increase to the inner diameters of the postcapillary venules (∼11.5 μm). The new procedure has the advantage of allowing precise sampling in the muscle microvasculature and can be used to study regional differences in the structure and function of the small vessels of the peripheral vasculature.

Protein blotting in uniform or gradient electric fields.

Experimental data and computer mapping were used to analyze electric fields generated by a variety of electrode arrays in protein blotting apparatus. Asymmetric electrode arrays were found to generate nonuniform fields that effected uneven transfers of 125I-labeled albumin from gels to nitrocellulose membranes. Symmetric arrays with multiple (four), independent wire electrodes, supplied individually with electric current, generated the most uniform fields and effected the most even transfers of the test protein. With multiple independent electrodes, gradient electric fields can be generated in which differences in electrophoretic elution between large and small proteins can be eliminated. Transfer apparatus with either uniform or gradient electric fields are expected to improve qualitative results and make possible quantitation of protein blotting.

Partial chemical characterization of the anionic sites in the basal lamina of fenestrated capillaries

The distribution of anionic sites in the basal laminae of the blood capillaries of the murine pancreas was studied in specimens fixed in ruthenium red (RR)-glutaraldehyde mixtures. The sites appeared as discrete, small (6 to 18 nm) particles distributed throughout the three laminae but concentrated primarily in the lamina rara externa, in which--spaced 80-100 nm apart--they formed a planar, partially ordered lattice comparable to that revealed by cationized ferritin in previous studies (M. Simionescu, N. Simionescu, and G. E. Palade, 1982, J. Cell Biol. 95, 425-434). The chemical nature of the anionic sites was explored by incubating fresh tissue specimens in solutions of selected enzymes before fixation in RR-glutaraldehyde mixtures. Pronase P and papain removed completely the anionic sites and left behind an extensively degraded and disorganized basal lamina. Trypsin caused the removal of anionic sites only, did not degrade the rest of the basal lamina, but detached it completely from the endothelium. Chondroitinase ABC reduced slightly the size and the surface density of RR-stainable particles, and detached focally the rest of the basal lamina from the endothelium and pericytes. Crude heparinase caused a nearly complete removal of anionic sites, and pure heparitinase gave comparable but less extensive results. Similar effects were recorded on the basal laminae of smooth muscle fibers and pancreatic acini and ducts. The results indicate that the anionic sites of all basal laminae examined are contributed primarily by heparin sulfate proteoglycans and trace amounts of chondroitin sulfate proteoglycans.

RECENT STUDIES ON VASCULAR ENDOTHELIUM

There is increasing evidence that processes that lead to vascular diseases, atherosclerosis included, are closely related to perturbations in endothelial permeability. Information has been accumulated that under various stress conditions, focal separations of endothelial junctions may occur preferentially in certain vascular segments. Such separations are involved in inflammatory reactions, hemorrhage, hypertension, thrombosis, and atheros~lerosis.~.~.’~.~~.~~.~~ A deeper knowledge of the normal structure and function of the endothelium is a prerequisite for a better understanding of its implication in vascular diseases. The vascular endothelium represents a special type of continuous, unistratified epithelium that behaves as a semipermeable partition across which, especially at the level of capillaries and venules, massive exchanges of small and large molecules occur between plasma and interstitial fluid. Like other cell membranes, the endothelial plasmalemma is assumed to be readily permeated by lipid-soluble molecules, but the endothelium as a whole has an unusually high permeability for water and solutes (much higher than that of other cells and of other epithelia). Moreover, unlike other epithelia, the endothelium is also permeable to large water-soluble molecules, which are assumed to pass through some water-filled channels or “pores” across the capillary W ~ I I . ~ . ’ ~ Physiologic experiments with graded molecules (dextrans) have indicated the presence of two categories of “pores”: small (diam., 90 A; frequency, 15-20/pm2; aggregate a r e a , 0.1%) and, large (diam., 500-700 A; frequency, 1/15-20 pm2). These figures have frequently been revised, the trend being toward smaller aggregate areas for the small-pore For the small pores, diffusion is restricted with increasing molecular size. Electron microscopic investigations of the vascular wallL.18.20 have revealed that blood vessels are provided with two basic types of endothelium: continuous endothehum, which is the most common type and is primarily encountered in all large vessels and in muscular capillaries, and fenestrated endothelium, which is found as several variants in visceral capillaries. Several peculiar features have been detected in the fine structure of the endothelial cells, the most characteristic being a high population of plasmalemmal vesicle^^^^^^ [outer diam., 600-700 A; frequency, 60-100/pm2 of endothelial surface; volume, 16% o f the endothelial cytoplasmic volume (in the peripheral zone)], about 60% of which are opened on one front o r the other of the C ~ I I S . ~ . ~ ~ I n addition, the visceral capillaries display transcellular round openings, the fenestrae (diam., 600-700 A; frequency, 15-25/pm2), usually closed by a thin single-layered diaphragm of unknown porosity and chemical n a t ~ r e . ~ . ’ ~ . ~ ~ * ~ * In thinsectioned specimens, the junctions of the endothelium appear to be of a relatively simple occludiag type with various degrees of membrane fusion, but the existence of open (20 40 A) junctions has been recorded.’” In fact, it is difficult to ascertain whether the endothelial tight junctions form complete sealing belts around each cell. However, none of the endothelial structures revealed by electron microscopy (vesicles, fenestrae, diaphragms, junctions) display the exact geometric dimensions and frequency postulated by the pore theory of capillary permeability.

Structural-functional correlates in the transendothelial exchange of water-soluble macromolecules

Abstract The transport of water-soluble macromolecules across the capillary wall is currently explained by assuming the existence of two pore systems: one of small pores with diameters of 90 A and relatively high frequency, and the other of large pores with diameter of up to 700 A and considerably lower frequency. There is at present general agreement concerning the structural equivalent of the two pore systems in the fenestrated capillaries, and the large pores in muscular capillaries. But the location of the small pores in the continuous endothelium of muscle capillaries is still an unsettled question: two hypothesis have been advanced, one favoring the intercellular junctions, another the plasmalemmal vesicles as structural equivalent for this system. Recent work with intravenously injected tracers of low molecular dimensions (Hemeundecapeptide, mol. wt. 1,900; mol. diam. 17–20 A and Hemeoctapeptide, mol. wt. 1,550) showed that such molecules cross the endothelium via vesicles, only. The latter can function either as isolated units shuttling, in revolvable phases, between the two endothelial fronts, or can fuse to form patent transendothelial channels made up by 1, or more connecting vesicles. Their size-limiting structures are represented either by strictures of 100 A diameter, or by diaphragms. The general geometry of the transendothelial channels could explain the physiological data, but their frequency remains to be established. Similar results were obtained by back-diffusion experiments in which the tracers were injected into the interstitial spaces. In both types of experiments, the endothelial junctions were not permeable to the tracers, but they may be permeated by molecules smaller than 20 A.