Showing papers on "Proteolytic enzymes published in 1980"

Mast cell heparin stimulates migration of capillary endothelial cells in vitro

Abstract: Migration of capillary endothelial cells is an important component of angiogenesis in vivo. Increased numbers of mast cells have been associated with several types of angiogenesis. We have used a quantitative assay in vitro to demonstrate that mast cells release a factor that significantly increases bovine capillary endothelial cell migration. The factor is present in medium conditioned by mast cells as well as lysates of mast cells. The stimulatory effect of mast cells on migration is specific for capillary endothelial cells. Furthermore, mast cells have no mitogenic activity for capillary endothelial cells. Of all the secretory products of mast cells tested, only heparin stimulated capillary endothelial cell migration in vitro. Heparin preparations from a variety of sources stimulated capillary endothelial cell migration to the same degree but did not stimulate migration of several other cell types. The migration activity of heparin and mast cell conditioned medium was blocked by specific antagonists of heparin (protamine and heparinase), but not by chondroitinase ABC. The migration activity of mast cell conditioned medium was resistant to heat (100 degrees C) and incubation with proteolytic enzymes. These results suggest that the role of mast cells in angiogenesis may be to enhance migration of the endothelial cells of growing capillaries.

Degradation of connective tissue matrices by macrophages. I. Proteolysis of elastin, glycoproteins, and collagen by proteinases isolated from macrophages.

Abstract: We have investigated the ability of neutral and lysosomal enzymes of mouse macrophages to degrade the insoluble extracellular matrices secreted by smooth muscle cells, endothelial cells, and fibroblasts. Matrices produced by smooth muscle cells contained glycoproteins, elastin, and collagens, but matrices of endothelial cells and fibroblasts contained no elastin. Sequential enzyme digestion of residual matrix revealed that plasmin, a product of macrophage plasminogen activation, degraded 50-70% of the glycoprotein in the matrices but did not degrade the elastin or the collagens. Purified macrophage elastase degraded glycoprotein and elastin components but had no effect on the collagens. The rate of elastin degradation by macrophage elastase was decreased in the presence of the glycoproteins. In contrast, human granulocyte elastase effectively degraded the matrix glycoproteins, elastin, and, to a lesser extent, collagens, Mammalian collagenase degraded only collagens. Conditioned medium from resident and inflammatory macrophages, containing mixtures of the secreted proteinases, degraded the glycoprotein and elastin components of the matrices. However, conditioned medium was less effective in degrading matrix than comparable amounts of purified macrophage elastase because > 90% of the elastase in the medium was in a latent form. Inclusion of plasminogen in the assays accelerated degradation. In the presence of plasminogen, glycoproteins were degraded readily by medium from P388D1, pyran copolymer-, thioglycollate-, and periodate-elicited macrophages and, to a lesser extent, by medium from endotoxin-elicited and resident macrophages; medium from P388D1, thioglycollate-, and periodate-elicited macrophages was most effective in elastin degradation, and resident, endotoxin-elicited and pyran copolymer-elicited macrophages degraded almost no elastin. The macrophage cathepsins D and B degraded all the matrix components at an optimum pH of 5.5 and acted with the secreted neutral proteinases to degrade the connective tissue macromolecules to amino acids and oligopeptides. These data indicate that macrophages at inflammatory sites contain and secrete proteolytic enzymes that could degrade the extracellular matrix.

Study of non-muscle cells of the adult mammalian heart: a fine structural analysis and distribution.

Abstract: The characterization and distribution of non-muscle cells of the adult rat heart were carried out by scanning and transmission electron microscopy. The non-muscle cells were examined during different phases of dissociation of the adult heart into single cell suspension. Non-muscle cells constituted approximately 65--70% of the ventricular cell suspension, while muscle cells constituted approximately 30--35%. Five types of non-muscle cells were observed: (1) endothelial cells, (2) fibroblasts, (3) pericytes, (4) smooth muscle cells, and (5) macrophages. The endothelial cells lining the internal wall of the ventricle possessed different surface morphology and shape than those lined with blood vessels. Fibroblasts were mostly scattered among the cardiac muscle cells and they contained highly developed rough-surfaced endoplasmic reticulum along with other structural features. Pericytes were characteristically observed on the periphery of the blood vessels and they showed structural similarities to the fibroblasts. Smooth muscle cells were relatively fewer than other non-muscle cells and generally served to line the wall of the medium calibre blood vessels of the heart. They were easily identified with their myofilaments. Although macrophages were observed in different regions of the heart, a small number of macrophage-like cells were strongly attached to the surfaces of the cardiac muscle cells. These cells were not released from the cardiac muscle cell surfaces even after prolonged treatment of proteolytic enzymes. Macrophages were mainly identified with their abundant filopodia, lysosomes and lysosomal degradation products. The functional implications of these non-muscle cells in relation to the heart was discussed.

Populations of rat skeletal muscle mitochondria after exercise and immobilization

Abstract: We slightly modified an existing procedure (Palmer et al., J. Biol. Chem. 252: 8731-8739, 1977) to isolate two distinct populations of mitochondria from rat skeletal muscle; initial brief Polytron homogenization released the subsarcolemmal mitochondria, and brief exposure of the resultant intact myofibrils to the proteolytic enzyme, Nagarse, extracted the intermyofibrillar mitochondria. The intermyofibrillar mitochondria differed from the subsarcolemmal mitochondr. ia by higher state III respiration measurements and enzymatic activities. These two populations of mitochondria were then isolated from the gastrocnemius muscle that had been induced to perform different amounts of contractile activity. The endurance training program of daily running significantly increased state III respiration and respiratory control index in the subsarcolemmal mitochondria, but the program did not increase these measurements in the intermyofibrillar mitochondria. In addition, 2 days of hindlimb immobilization resulted in a significant decrease in state II respiration and the respiratory control index of the subsarcolemmal mitochondria; however, immobilization did not affect the intermyofibrillar mitochondria. These measurements suggest that the subsarcolemmal mitochondria adapt in response to chronic changes in the level of contractile activity.

Prorenin and Other Large Molecular Weight Forms of Renin

Abstract: PRORENIN is a completely inactive form of renin that circulates in human plasma in concentrations close to 10 times that of active renin. A similar inactive form of renin appears to be secreted from the kidney, but an extrarenal source can also contribute to the plasma level. Prorenin should not be confused with large molecular weight forms of renal renin that have intrinsic enzymatic activity. These appear to be, at least in part, the result of binding of renin to a renal cortical protein. Plasma prorenin can be activated by the techniques of cryoactivation and acid activation. It can also be converted to active renin by neutral serine proteases such as trypsin, glandular kallikrein, plasma kallikrein, and plasmin, and by acid proteases such as pepsin and cathepsin D. Both cryoactivation and acid activation appear to be mediated, in part, by endogenous plasma kallikrein which converts prorenin to renin in plasma in vitro after either cold inactivation or acid destruction of plasma kallikrein inhibitors. ...

Methods for microcarrier culture of bovine pulmonary artery endothelial cells avoiding the use of enzymes

Abstract: Enzymes situated along the luminal surface of pulmonary endothelial cells interact with circulating solutes, notably with vasoactive substances, to regulate the hormonal composition of systemic arterial blood. However, it is becoming clear that the range and complexity of reactions occurring at or near the surface of endothelial cells are greater than previously recognized. In addition, evidence indicates that the quality of cell cultures used to define specific endothelial functions must be carefully controlled, together with development of improved understanding of the effects of long-term culture on pulmonary endothelial cells. We have developed new techniques for the culture of pulmonary endothelial cells which avoid exposure to proteolytic enzymes at both the isolation step and during subculture. A combination of mechanical harvest and culture on microcarrier beads has provided a system for the long-term, large-scale culture of pulmonary endothelial cells, features which to a large extent determine the scope of biochemical studies which can be undertaken.

Release of somatomedin-like activity by cultured WI-38 human fibroblasts.

Abstract: Confluent cultures of normal diploid WI-38 human embryonic lung fibroblasts released somatomedin (SM)-like activity into their incubation medium during culture in serum-free medium. This postculture medium (conditioned medium) stimulated cell division in these same cultured WI-38 fibroblasts and 35SO4 uptake by hypophysectomized rat cartilage in vitro. The conditioned medium also contained immunoreactive SM (IRSM) activity which yielded parallel dose-response curves to human serum in a RIA for SM. The IRSM activity measured in conditioned medium was not the artifactual result of effects of possible SM-binding proteins or proteolytic enzymes in conditioned medium. These studies suggest that cultured WI-38 fibroblasts produce and release SM-like activity which has SM-like biological activity and is immunoreactive with a basic SM purified from human plasma Cohn fraction and having similarity with SM-C and insulin-like growth factor-I. Human GH appears to stimulate production and release of IRSM activity by these cells.

Human coagluation factor V purification and thrombin-catalyzed activation.

Abstract: Factor V was isolated from human plasma by barium citrate adsorption, polyethylene glycol fractionation, DEAE-Sepharose CL-6B chromatography, ammonium sulfate fractionation, and gel chromatography on Ultrogel 22. Degradation of Factor V during purification was largely prevented by ample use of inhibitors of proteolytic enzyme. The purified Factor V was a stable, single-chain molecule with an apparent molecular weight of 330,000. Activation of human Factor V by thrombin resulted in a 10- to 15-fold increase in activity. The activation pattern as monitored by sodium dodecyl sulfate polyacrylamide gel electrophoresis was compared with that of bovine Factor V. Differences in the patterns of thrombin activation were noticed between the two species, whereas the final products were similar. The products of human Factor V activation are two closely spaced doublets, one with an apparent molecular weight of approximately 110,000, and the other, approximately 72,000. An antibody was raised against the purified protein. Crossed immunoelectrophoresis showed that the antibody recognized Factor V both before and after activation with thrombin.

β-Endorphin and Adrenocorticotropin in Extrapituitary Sites: Gastrointestinal Tract

Abstract: β-Endorphin and ACTH immunoassays were employed to examine the concentrations, distributions, and character of those peptides in rat gastrointestinal tissues. Sections of the gastrointestinal tract were obtained from fasted and fed animals and were extracted in 5 N acetic acid containing proteolytic enzyme inhibitors. Aliquots immunoassayed for β-endorphin and ACTH revealed highest concentrations to be present in the small bowel, with stomach and colon containing little immunoreactivity. Tissues from fasted animals contained more immunoreactivity than did those from fed animals. Gel chromatography showed the presence of large molecular weight forms of β-endorphin and ACTH in gut extracts. Concanavalin A affinity chromatography revealed that approximately 5% of gut immunoreactivity contained carbohydrate. Therefore, β-endorphin and ACTH immunoreactivities are present in the gut. The demonstration of large molecular weight and glycosylated forms of immunoreactivity suggests the presence of biosynthetic prec...

Soybean diet lowers breast tumor incidence in irradiated rats

Abstract: This paper examines the relationship between feeding a diet rich in protease inhibitors and the reduction of mammary cancer induced by x-irradiation in Sprague-Dawley rats. Of a total of 145 irradiated animals, 44% of the 45 rats fed a raw soybean diet containing a high concentration of protease inhibitor developed mammary tumors as compared to 74% of 50 rats fed a casein diet containing no protease inhibitor. Animals fed Purina rat chow which contained low levels of protease inhibitor exhibited a 70% mammary tumor incidence. No spontaneous neoplasms were found in any of the non-irradiated animals on the raw soybean diet whereas about 10% of the animals on the protease-free diet developed tumors. Thus, soybeans which are rich in protease inhibitors reduced the induction of mammary cancer in x-irradiated rats. This work suggests that diets rich in protease inhibitors may contribute to reducing cancer incidence in man.

Chemical attraction of hermit crabs and other attendants to simulated gastropod predation sites

Abstract: Simulated gastropod predation sites were observed in the gulf intertidal near the Edward Ball Marine Laboratory, Sopchoppy, Florida,Fundulus similis, Callinectes sapidus, Melongena corona, Clibanarius vittatus, andPagurus longicarpus were attracted to the sites by small molecules released passively from the flesh of wounded or dead animals. Flesh consumers (F. similis, C. sapidus, andM. corona) were attracted to molecules released from the flesh of bivalves, gastropods, and crabs. Shell users (C. vittatus andP. longicarpus) were attracted only to small molecules from gastropod flesh, withP. longicarpus attendingP. duplicatas sites whileC. vittatus preferentially attendedM. corona, Busycon contrarium, andBusycon spiratum sites. Flesh consumers and shell users may be attracted to the sites by different sets of small molecules. The addition of proteolytic enzymes to the flesh increased the attendants at a site, indicating that the attractant molecules might be amino acids or small peptides. Flesh consumers were attracted to the sites primarily in the first 12 hr while the shell users were attracted from 2 hr to several days postinitiation. New shells were attractive to hermit crabs up to 12 hr after entry by a hermit crab. The shell species of the attendantC. vittatus were in different proportions than the generalC. vittatus population, and the shell fit of theP. longicarpus attendants was not as good as the general population ofP. longicarpus. Significantly moreC. vittatus attended than P. longicarpus, and it was speculated that there were moreC. vittatus in the area with a poor shell fit thanP. longicarpus.

Formation of the Semliki Forest virus membrane glycoprotein complexes in the infected cell.

Abstract: Summary In Semliki Forest virus (SFV)-infected cells, all structural proteins are translated from a 26S mRNA using a single initiation site. The capsid protein which is made first is released into the cytoplasm whereas the two membrane proteins, p62 (the precursor for E2 and E3) and E1, are inserted into the rough endoplasmic reticulum membrane. Based on gradient centrifugation and cross-linking studies, it can be seen that the p62 and E1 polypeptides form a complex immediately after synthesis and migrate to the plasma membrane in the form of a p62-E1 complex. The processing of p62 to E2 and E3 is first seen 25 to 30 min after a 10 min pulse of radioactive amino acids. This cleavage can be inhibited by addition of antisera specific for E1 and E3, thus supporting the view that, as in the case of the related Sindbis virus, this cleavage occurs on the external face of the plasma membrane. Proteolytic digestion of crude vesicle preparations derived from plasma membranes, combined with peptide mapping, indicate that the carboxy-terminal end of E2 spans the cell plasma membrane, there being a portion of mol. wt about 3000 located towards the cytosol.

Transmembrane Orientation of Proteins Present in Acetylcholine Receptor-Rich Membranes from Torpedo marmorata Studied by Selective Proteolysis

Abstract: The transmembrane orientation of the polypeptide chains present in acetylcholine receptor-rich membranes from Torpedo marmorata was studied by selective proteolysis. These membranes contain two major polypeptides of Mr 40000 and 43000 and two minor ones of Mr 50000 and 66000. With intact membranes all four proteins were found remarkably resistant to the action of the proteolytic enzymes pronase and trypsin. In contrast, when membranes were opened by sonication in the presence of trypsin, all four proteins were degraded. Stable degradation products of Mr 47000, 38000, 35000 and 32000 were found. Addition of protease after sonication was not effective; sonication alone did not remove the four major proteins from the membrane. It is concluded that: (a) these four chains were cut at sites normally exposed to the inside surface of the membrane and (b) the membrane fragments were sealed in vitro with the same orientation as in vivo Various treatments were found to render the intact receptor-rich membranes susceptible to proteolytic attack: incubation at pH 4 with the protease pepsin led to the selective degradation of the 40000-Mr, 50000-Mr and 66000-Mr chains; the same results were obtained when the membranes were heated at 62°C for 1 min and then digested with trypsin. The 43000-Mr chain remained resistant in both cases. No degradation fragments with Mr greater than 20000 were found. Furthermore, when these membranes were sonicated in the presence of pepsin, the 43000-Mr polypeptide was degraded. It is concluded that (a) the 43000-Mr polypeptide is entirely enclosed by the lipid bilayer, and (b) that the 40000-Mr, 50000-Mr and 66000-Mr polypeptides are transmembrane proteins. Trypsin digestion of the receptor-rich membranes opened by phospholipase A2 (crotoxin) or alkaline treatment, or solubilization by 1% Triton X-100 gave the same series of degradation products as the sonicated membranes. The 47000-Mr fragment was shown to be derived from the 50000-Mr chain; and the 35000-Mr and 32000-Mr fragments, which were labelled by the affinity reagent 4(N-maleimido)-phenyl-[3H]trimethylammonium, were derived from the 40000-Mr chain. After dodecylsulfate/polyacrylamide gel electrophoresis, fragments of Mr 47000, 38000, 35000 and 32000 were labelled by 25I-concanavalin A, like the native 40000-Mr, 50000-Mr and 66000-Mr chains. The NH2-terminal sequence of the 35000-Mr fragment was identical to that of the 40000-Mr chain.

Demonstration of specific binding sites for human serum albumin in group C and G streptococci.

Abstract: A total of 297 bacterial strains belonging to 27 species was tested for quantitative uptake of radiolabeled human serum albumin. Specific binding sites with high affinity for human serum albumin were found exclusively in group C and G streptococci. The albumin binding was found to be a time-dependent, saturable, and displaceable process which obeyed simple kinetic equations. Scatchard analysis revealed that human serum albumin bound to a homogeneous population of receptors with an affinity in the order ot 10(7) liters/mol and that the average bacterial cell carried more than 80,000 binding sites. The albumin receptor is a heat-stable component susceptible to proteolytic digestion. It has a surface localization separate from the receptors for immunolgobulin G, fibrinogen, aggregated beta 2-microglobulin, and haptoglobin. In individual strains, albumin reactivity was also detected independently of these other types of interactions with human proteins.

Effect of Heat Treatment on Ruminal Degradation and Escape, and Intestinal Digestibility of Cottonseed Meal Protein

Abstract: The effect of heat treatment of ruminal protein degradation and escape was studied using in vitro incubations with autoclaved and commercial cottonseed meal (CSM) samples. Incubations using high ratios of protein to ruminal fluid appeared to overestimate ruminal degradation of CSM protein relative to casein. A biexponential model, assuming CSM contained two protein fractions degraded at two different rates, was used to interpret data from in vitro incubations conducted using ratios of protein to ruminal fluid similar to those expected in vivo. The first fraction was degraded at rates (0.68-1.19/hour) which were 2--3 1/2 times greater than that of casein (0.34/hour). Degradation rates of the second fraction were much slower (0.011-0.093/hour). The effect of heat treatment was to decrease the proportion of the rapidly degraded fraction and to both increase the proportion and to decrease the degradation rate of the more slowly degraded fraction. Estimated ruminal escape increased with each increment of heat treatment. Intestinal protein digestibility (ruminal escape times true digestibility) increased to maximum at 60 minutes autoclaving, and then declined. Estimates of intestinal protein digestibility averaged 30.6 and 50.3% for solvent-extracted and screw-press CSM, respectively. These and previous results suggest heat treatment decreases ruminal degradation partly by blocking reactive sites for microbial proteolytic enzymes and partly by reducing protein solubility.

Regulation of Glyoxysomal Enzymes during Germination of Cucumber: 3. IN VITRO TRANSLATION AND CHARACTERIZATION OF FOUR GLYOXYSOMAL ENZYMES

Abstract: Monospecific antibodies raised against four glyoxysomal enzymes (isocitrate lyase, catalase, malate synthase, and malate dehydrogenase) have been used to detect these proteins among the products of in vitro translation in a wheat germ system programmed with cotyledonary RNA from cucumber seedlings. In vitro immunoprecipitates were compared electrophoretically with the same enzymes labeled in vivo and also with the purified proteins. Isocitrate lyase yields two bands on sodium dodecyl sulfate-polyacrylamide gels, as synthesized both in vitro (61.5K and 60K products) and in vivo (63K and 61.5K polypeptides). Both the 63K and 61.5K subunits can also be demonstrated for the isolated enzyme. The two subunits are antigenically cross-reactive and yield similar electrophoretic profiles upon partial proteolytic digestion. A larger subunit is seen in vitro than in vivo for both malate dehydrogenase (38K versus 33K) and catalase (55K versus 54K); this suggests a need for processing which is often a characteristic of proteins that must be transported across or into membranes. Malate synthase has a molecular weight of 57K both in vitro and in vivo, but the isolated enzyme is a glycoprotein, containing N-acetyl glucosamine, mannose, and possibly also fucose and xylose. This indicates that the polypeptide portion of the isolated enzyme is smaller than the in vitro product and suggests processing of malate synthase also. None of the other three enzymes appears to be glycosylated. The implications of these size differences for the compartmentalization of matrix and membrane-bound glyoxysomal enzymes are discussed.

Structural Chemistry of Outer Dense Fibers of Rat Sperm

Abstract: Rat sperm outer dense fibers (ODF) have been studied by several electron microscopic tech niques to determine fine structural features of the ODF cortex and medulla. Cross sections of Triton X-100 demembrananed flagella, fixed in the presence of ruthenium red, reveal a well defined ODF cortex which appears to be composed of a single lamina of 6—7nm diameter globular parti des, whereas the medulla appears electron dense with no obvious substructure. Surface replicas and positively or negatively stained whole mount preparations reveal a crossbanding pattern with a 40 nm major period repeat that extends obliquely over the outer dense fiber surface. These periodic ities appear to be confined no the outer dense fiber cortex. Sequential solubilization of spermatozoan organelles in sodium dodecyl sulfate or sodium lauroyl sacrosine results in the isolation of a flagellar subfraction containing only the ODF and attached connecting piece. Further solubilization of the complex in the ionic detergents results in an ODF subfraction which contains 4 polypeptide components. The 3 low molecular weight polypeptide bands have a high cysteine content (10—12%)while the high molecular weight com ponent (87,000 daltons) contains “? 3% cysneine. Brief nrypsin or chymotrypsin digestion of dc. membranated flagella gives an ODF subfracnion which by SDS polyacrylamide gel electrophoresis is shown to possess only the 3 low molecular weight polypeptide bands. These ODFs appear no possess an intact medulla but are lacking a cortex, thereby suggesting that the low molecular weight components are located in the medulla. 320 OLSON AND SAMMONS movement in mammalian spermatozoa results from sliding interactions of axonemal doublet microtubules (Lindemann and Gibbons, 1975; Afzelius et al., 1975; Pedersen and Rebbe, 1975) have led to recent suggestions that the ODFs function as passive elastic structures (for review see Fawcett, 1975). In view of the probable role of the ODFs in affecting the bending pattern of the mam malian sperm flagellum, this study was under taken to provide additional data on their structure and composition. Several electron microscopic techniques were employed to resolve new features of ODF substructure and to allow structural information on rat sperm ODF to be correlated with morphological data from other species. In addition, the principal structural polypeptides of the ODF were isolated on a preparative scale and their amino acid compositions are reported. Finally, selec tive proteolytic digestion of flagellar compo nents and correlated electron microscopic analysis were employed to localize specific polypeptides to the ODF medulla. MATERIALS AND METHODS Isolation of Spermatozoa Adult Sprague-Dawley rats were anesthetized with Nembutal. The cauda epididymides were removed, placed in a watch glass and covered with PBS (phos phate buffered saline composed of 0.145 M NaC1 and 0.01 M NaH2PO4/Na2HPO4, pH 7.0). The epididy mides were minced with a razor blade; the spermato zoa released into the PBS were collected with a Pasteur pipette. Spermatozoa were pelleted by centrif ugation at 500 g for 10 mm and used in the following protocols. Isolation ofSperm Tails Sperm flagella were isolated according to a modifi cation of the procedure reported by Calvin (1976). Spermatozoa from each animal were suspended in 7—8ml ice cold PBS and sonicated with ten, 10 sec bursts at 100 W with a Braun sonicator to detach the heads and tails. One aliquot of the sperm suspension was mixed with 6 parts of 75% sucrose (w/v), 10 mM sodium phosphate (pH 7.0). Aliquots (3 ml) of the sperm mixture were layered over sucrose step gradi ents containing equal volume steps of 65%, 70% and 75% sucrose (w/v), each containing 20 mM sodium phosphate (pH 7.0). The gradients were centrifuged for 1 h at 100,000 X g in a SW 41 rotor (12 ml gra dients) or a SW 25.1 rotor (32 ml gradients) and the tails that layered at the 65—70% interface were removed with a Pasteur pipette, diluted with PBS and pelleted at 50,000 X g for 15 mm. Triton X-100 Extraction Pellets of intact spermatozoa or isolated flagella were extracted in 2 changes of 15 mm each with at least 20 volumes of 1% Triton X-100, 2 mM dithio threitol (DTT), 100 mM NaCI and 25 mM Tris HC1 (pH 9.0). After extraction, the spermatozoa were washed twice in 100 mM NaCl, 25 mM Tris HC1 (pH 7.5) by centrifugation. The final pellets were immediately prepared for electron microscopy or subjected to enzymatic digestion or used for SDS polyacrylamide gel electrophoresis. Enzymatic Digestion ofSperm Tails Pellets of Triton-DTT extracted sperm tails were suspended at room temperature in 20 volumes of PBS containing 1 mg/mI trypsin or chymotrypsin (Sigma Chemical Co.). At 15 mm intervals for periods up to 1 h, duplicate aliquots of the suspension were removed and the nondigested material was pelleted by centrif ugation at 10,000 X g for 10 mm. The pellets were washed twice in PBS by resuspension and centrif ugation and the final pellets were used for SDS poly acrylamide gel electrophoresis and electron micro scopy. isolation ofthe Connecting Piece-Outer Dense Fiber Complex Two procedures were employed to isolate connect ing piece-outer dense fiber complexes. In the first, intact spermatozoa suspended in PBS at a concentra tion of 4—6 x 10' cells/mI were mixed with an equal volume of 2% sodium dodecyl sulfate (SDS), 4 mM DTT and 50 mM Tris HC1 (pH 9.0). In some experi ments 2% sodium lauroyl sarcosine (SLS) was substi tuted for the sodium dodecyl sulfate. The suspension was monitored microscopically until the sperm heads detached from the tails and the tails separated into individual fibers joined together at the connecting piece (usually 10—15 mm). Aliquots (1 ml) of the sperm extract were layered on top of 40 ml, 30—60% linear sucrose gradients containing 0.1% SDS. The tubes were centrifuged at room temperature for 15 mm at 1500 rpm in a Sorval GLC-1 tabletop centri fuge equipped with a swinging bucket head. Two bands were visible after centrifugation, the upper containing the flagellar subfraction and the lower the sperm heads. The bands were removed from the tubes with an ISCO density gradient fractionator. Fractions in which tail components accounted for at least 95% of the particles were pelleted by centrifugation at 50,000 X g for 15 mm and the pellets used for bio chemical and ultrastructural analysis. In the second procedure, pellets of isolated sperm tails were cx tracted in 20 volumes of 1% SDS, 2 mM DTT, 25 mM Tris HC1 (pH 9.0) or 1% SLS, 2 mM DTT, 25 mM Tris HO (pH 9.0). After various extraction times, aliquots of the suspension were removed and the nonsolubilized components were pelleted by centrif ugation at 10,000 X g for 10 mm and the pellets used for electron microscopy and SDS polyacrylamide gel electrophoresis. SPERM DENSE FIBERS 321

Molecular Evolution of Biologically Active Polypeptides

Abstract: Any biological function is at least bimolecular and involves primarily a specific recognition between the shapes (conformations) of the reacting molecules. The selective pressure of evolution therefore acted on the interaction so that coordinated changes probably occurred in two lines of molecules. Because the structure of the specific partner (receptor, macromolecular substrate, naturally occurring inhibitor, antigen, etc.) is rarely known, evolutionary speculations are often arbitrarily limited to the active polypeptide. During the life of a polypeptide chain, its conformation can be modified by ligands, by `conformers' or by morphogenic cleavages. Inactive preprohormones and prohormones (e.g. preproparathyrin, proopiocortin) are successively split by specific proteolytic enzymes. Several modulator- or activator-binding sites can be distinguished in addition to the active site, so that the chain can be regarded as the result of a multiple evolution. The conformation of an active polypeptide chain on the one hand displays a variable degree of flexibility, and on the other may include a hierarchy of organized substructures: secondary ($\alpha $-helix, $\beta $-pleated sheet, $\beta $-bend), super-secondary and domain. The amino acid sequence appears to program largely the organized substructures and the potential adaptability. However, general architectural rules on which selective pressure could primarily act remain unknown. Duplication seems to be a fundamental mechanism for increasing both the size and the number of polypeptide chains. Duplication without fusion may lead to parallel lines of peptides which differentiate functionally by subsequent mutations (e.g. neurohypophysical hormones and neurophysins). Duplication with fusion may give single-chain proteins with internal homology between two or several domains (e.g. somatotrophin). Repetitive duplication could involve fusion in the first steps and not in the last step so that several lines of homologous proteins, each with internal homology, could arise (e.g. somatotrophin, prolactin, choriomammotrophin). The assembly of polypeptide chains, whether covalent or not, is likely to represent a higher level of evolution. Each chain or subunit may have a distinct function, as in dimeric hormones (e.g. lutrophin, follitrophin, thyrotrophin, choriogonadotrophin), or the association may determine new cooperative properties (allostery). The integration of molecular evolution at the organelle, cellular and organismal levels raises the problem of the evolution of regulatory mechanisms.

Technical aspects of lymphoma immunohistology.

Abstract: Immunohistological techniques (i.e., based on tissue sections) for the study of human lymphoma have been developed in recent years as alternatives to immunocytochemical methods (based on cell suspensions). These techniques not only allow the important architectural features of the lymphoma sample to be preserved, but are also more convenient to use in that tissues can be processed rapidly and studied at leisure. The relative advantages and disadvantages of frozen versus fixed embedded tissue, and of immunofluorescence versus immunoenzymatic methods are reviewed, and technical aspects of proteolytic digestion (as a technique for enhancing immunohistochemical labeling) are discussed. This review concludes with a consideration of the problems encountered in the interpretation of immunohistochemical labeling for lymphoma cell immunoglobulin in paraffin sections.

Secretion of Alpha-2-macroglobulin by human alveolar macrophages.

Abstract: Cultured human alveolar macrophages were shown to secrete alpha-2-macroglobulin (α2M), a potent inhibitor of bacterial and mammalian proteases. Secretion could be blocked by 2.0 µg/ml cycloheximide, an inhibitor of protein synthesis. Detection of the inhibitor was achieved by radioimmunoelectrophoresis of concentrated culture medium obtained from cells cultured in the presence of35S-methionine. In addition, concentrated culture medium was treated with antihumanα2M antiserum and antigen: antibody complexes were precipitated with protein A-bearing staphylococci. When the precipitate thus formed was dissolved in a reducing buffer and subjected to SDS electrophoresis, a single radioactive peak of about 185,000 daltons was obtained. This molecular weight corresponds closely to the molecular weight of theα2M subunit. Secretion ofα2M by alveolar macrophages may facilitate the internalization and disposal of proteolytic enzymes in the alveolar microenvironment, and thus play an important role in the defense of the lung.

Identification of proteolytically resistant domains of human erythrocyte spectrin

Abstract: Digestion of purified human erthrocyte spectrin with proteolytic enzymes at 0 degrees C results in the production of intermediate-size peptides that resist further cleavage at 0 degrees C. By two-dimensional peptide analysis of these intermediate peptides it has been determined that five unique peptides are produced by tryptic cleavage of the alpha subunit of spectrin (band 1); these have apparent molecular weights of 80,000, 46,000, 46,000, 41,000, and 30,000 and account for 97% of the alpha subunit. Similarly, four unique peptides having apparent molecular weights of 74,000, 65,000, 33,000, and 38,000 account for 90% of the beta subunit (band 2). By examining larger peptide fragments, the linear alignment of the unique peptides along each of the spectrin subunits has been established. These results indicate that spectrin is composed of two nonidentical subunits, each containing multiple proteolytically resistant domains. These domains, which may be largely alpha-helical, seem to be connected by small protease-sensitive segments. The proteolytic resistance of these domains is not influenced by the multimeric state of the spectrin molecule.

Proteolytic digestion studies of chromatin core-histone structure. Identification of a limit peptide of histone H2A.

Abstract: Tryptic digestion of chicken erythrocyte nuclei, to a level at which no intact histone remained, resulted in a set of resistant peptides. These were partially separated by exclusion chromatography. One of the peptides was shown to represent the central sequence 12--118 of histone H2A. This was established by amino acid analysis and by Edman degradations. Comparison of the sequence of histone H2A from a wide range of cell types shows that the tryptic cleavage points correspond closely to the limits of the highly conserved central sequence and not to the limits of the strongly basic regions. It is proposed that the 11 N-terminal and 10 C-terminal residues cleaved by trypsin are exposed in chromatin and play a structural and functional role different from the central 107 residues. The exposed position of the 118--119 bond accords with the known linkage point of ubiquitin to residue 119 of histone H2A in the semi-histone A24.