Biosynthesis of nonspecific lipid transfer proteins in germinating castor bean seeds

The biosynthesis of nonspecific lipid transfer proteins (ns-LTPs) in germinating castor bean (Ricinus communis L.) seeds were investigated. Lipid transfer activities of ns-LTPs in the cotyledons, axis, and endosperm increased with growth after germination. The activity increases were accompanied by increased amounts of ns-LTPs in each tissue, as measured by immunoblot using anti-ns-LTP serum. These results suggest that the ns-LTPs are synthesized de novo in each tissue after germination and not activated from inactive proteins synthesized before germination. Comparison of the immunoblot products in each tissue from 4-day-old seedlings indicate the occurrence of tissue-specific isoforms of ns-LTPs; 9 kilodaltons (major) and 7 kilodaltons (minor) in the cotyledons, and 7 kilodaltons (major) and 9 kilodaltons (minor) in the axis, whereas only the 8-kilodalton ns-LTP is present in the endosperm. In vitro translation from poly(A)⁺ RNAs from three tissues of castor bean seedlings and the detection of immunoprecipitated products indicate that translatable mRNAs for ns-LTPs exist in the three tissues a day before the synthesis of ns-LTPs; the translation products, which are 3.5 to 4.0 kilodaltons larger than ns-LTPs, were processed to the mature ns-LTPs. The production of mature ns-LTPs from translatable mRNAs without any delay suggests that gene expression of ns-LTPs in castor bean seedlings is controlled at a step before the formation of translatable mRNAs.     

Characterization of naturally elaborated blebs from serum-susceptible and serum-resistant strains of Neisseria gonorrhoeae

Outer-membrane blebs from two serum-susceptible and two serum-resistant strains of Neisseria gonorrhoeae were characterized. In general, bleb surfaces resembled cell surfaces, but there were qualitative and quantitative protein differences in blebs released by serum-susceptible and serum-resistant strains. Relative to blebs from serum-resistant strains, blebs from serum-susceptible strains expressed reduced amounts of major outer-membrane proteins I and III, and little if any 68,000 Dalton outer-membrane protein.     

Calmodulin-Binding Proteins in Chromaffin Cell Plasma Membranes

Abstract: Calmodulin-binding proteins present in chromaffin cell plasma membranes were isolated and directly compared with calmodulin-binding proteins present in chromaffin granule membranes. Chromaffin cell plasma membranes were prepared using Cytodex 1 microcarriers. Marker enzyme studies on this preparation showed a nine- to 10–fold plasma membrane enrichment over cell homogenates and a low contamination of these plasma membranes by subcellular organelles. Plasma membranes prepared in this manner were solubilized with Triton X-100 and applied to a calmodulin-affinity column in the presence of calcium. Several major calmodulin-binding proteins ( 240, 105 , and 65 kilodaltons) were eluted by an EGTA-containing buffer. ¹²⁵I-Calmodulin overlay experiments on nitrocellulose sheets containing both chromaffin plasma and granule membranes showed that these two membranes have several calmodulin-binding proteins in common ( 65, 60, 53 , and 50 kilodaltons), as well as unique calmodulin-binding proteins (34 kilodaltons in granule membranes and 240 and 160 kilodaltons in plasma membranes). The 65–kilodalton calmodulin-binding protein present in both membrane types was shown to consist of two isoforms (pI 6.0 and 6.2) by two-dimensional gel electrophoresis. Previous experiments from our laboratory, using two monoclonal antibodies (mAb 30 and mAb 48) specific for a rat brain synaptic vesicle membrane protein (p65), showed that the monoclonal antibodies reacted with a 65–kilodalton calmodulin-binding protein present in at least three neurosecretory vesicles (chromaffin granules, neurohypophyseal granules, and rat brain synaptic vesicles). When these monoclonal antibodies were tested on chromaffin cell plasma membranes and calmodulin-binding proteins isolated from these membranes, they recognized a 65–kilodalton protein. These results indicate that an immunologically identical calmodulin-binding protein is expressed in both chromaffin granule membranes (as well as other secretory vesicle membranes) and chromaffin cell plasma membranes, thus suggesting a possible role for this protein in granule/plasma membrane interaction.     

Permeation of membranes by the neutral form of amino acids and peptides: Relevance to the origin of peptide translocation

The flux of amino acids and other nutrient solutes such as phosphate across lipid bilayers (liposomes) is 10⁵ slower than facilitated inward transport across biological membranes. This suggests that primitive cells lacking highly evolved transport systems would have difficulty transporting sufficient nutrients for cell growth to occur. There are two possible ways by which early life may have overcome this difficulty: (1) The membranes of the earliest cellular life-forms may have been intrinsically more permeable to solutes; or (2) some transport mechanism may have been available to facilitate transbilayer movement of solutes essential for cell survival and growth prior to the evolution of membrane transport proteins. Translocation of neutral species represents one such mechanism. The neutral forms of amino acids modified by methylation (creating protonated weak bases) permeate membranes up to 10¹⁰ times faster than charged forms. This increased permeability when coupled to a transmembrane pH gradient can result in significantly increased rates of net unidirectional transport. Such pH gradients can be generated in vesicles used to model protocells that preceded and were presumably ancestral to early forms of life. This transport mechanism may still play a role in some protein translocation processes (e.g., for certain signal sequences, toxins and thylakoid proteins) in vivo.Abbreviations LUV large unilamellar vesicle - pH transmembrane pH gradient - PAH polyaromatic hydrocarbon Correspondence to: A.C. Chakrabarti     

Cyclic AMP-Dependent Protein Phosphorylation in Isolated Neuronal Growth Cones from Developing Rat Forebrain

We have shown recently that neuronal growth cones isolated from developing rat forebrain possess an appreciable activity of adenylate cyclase, which produces cyclic AMP and can be stimulated by various neurotransmitter receptor agonists and by forskolin. To investigate cyclic AMP-mediated biochemical mechanisms in isolated growth cones, we have centered the present study on cyclic AMP-dependent protein phosphorylation. One-dimensional gel electrophoretic analysis showed that cyclic AMP analogs increased incorporation of 32P into several phosphoproteins in molecular mass ranges of 50-58 and 76-82 kilodaltons, including those of 82, 76, and 51 kilodaltons. Two-dimensional electrophoresis, using isoelectric focusing in the first dimension, resolved phosphorylated alpha- and beta-tubulin species, actin, a very acidic protein (isoelectric point 4.0) with a molecular mass of 93 kilodaltons, and two proteins (x and x') closely neighboring beta-tubulin. Two other phosphoproteins seen in the gels had molecular masses of 56 and 51 kilodaltons (respective isoelectric points, 4.5 and 4.4) and, along with the 93-kilodalton phosphoprotein, were highly enriched in the isolated growth cones. Only the tubulin and actin species were major proteins in the isolated growth cones. Cyclic AMP analogs enhanced incorporation of 32P into phosphoproteins x and x', and, as assessed by immunoprecipitation, into beta-tubulin. Peptide digest experiments suggested that phosphoproteins x and x' are unrelated to beta-tubulin. Nonequilibrium two-dimensional electrophoresis resolved many phosphoproteins, of which a 79- and 75-kilodalton doublet, a 74-kilodalton species, and a 58-kilodalton doublet showed enhanced incorporation of 32P in the presence of cyclic AMP.     

Shiverer and Normal Peripheral Myelin Compared: Basic Protein Localization, Membrane Interactions, and Lipid Composition

We have correlated membrane structure and interactions in shiverer sciatic nerve myelin with its biochemical composition. Analysis of x-ray diffraction data from shiverer myelin swollen in water substantiates our previous localization of an electron density deficit in the cytoplasmic half of the membrane. The density loss correlates with the absence of the major myelin basic proteins and indicates that in normal myelin, the basic protein is localized to the cytoplasmic apposition. As in normal peripheral myelin, hypotonic swelling in the shiverer membrane arrays occurs in the extracellular space between membranes; the cytoplasmic surfaces remain closely apposed notwithstanding the absence of basic protein from this region. Surprisingly, we found that the interaction at the extracellular apposition of shiverer membranes is altered. The extracellular space swells to a greater extent than normal when nerves are incubated in distilled water, treated at a reduced ionic strength of 0.06 in the range of pH 4-9, or treated at constant pH (4 or 7) in the range of ionic strengths 0.02-0.20. To examine the biochemical basis of this difference in swelling, we compared the lipid composition of shiverer and normal myelin. We find that sulfatides, hydroxycerebroside, and phosphatidylcholine are 20-30% higher than normal; nonhydroxycerebroside and sphingomyelin are 15-20% lower than normal; and ethanolamine phosphatides, phosphatidylserine, and cholesterol show little or no change. A higher concentration of negatively charged sulfatides at the extracellular surface likely contributes to an increased electrostatic repulsion and greater swelling in shiverer. The cytoplasmic surfaces of the apposed membranes of normal and shiverer myelins did not swell apart appreciably in the pH and ionic strength ranges expected to produce electrostatic repulsion. This stability, then, clearly does not depend on basic protein. We propose that P0 glycoprotein molecules form the stable link between apposed cytoplasmic membrane surfaces in peripheral myelin.     

Protein-catalyzed exchange of phosphatidylinositol between rat brain microsomes and myelin.

Exchange of phosphatidylinositol and phosphatidylcholine between microsomal and myelin membranes has been demonstrated. This exchange is reversible and catalyzed by soluble proteins from the brain homogenate precipitated at pH 5.1. The extent of exchange of phosphatidylinositol from microsomal membrane to myelin is dependent upon pH and temperature, with an optimum around pH 7 and at 50 degrees C. Maximum exchange was observed at approximately equal amounts of microsomal, myelin, and supernatant proteins. The extent of the catalyzed exchange increases 4- to 8-fold upon using sonicated or heat-treated myelin as an acceptor membrane. Heating of microsomal membranes results in no change. The extent of catalyzed exchange of phosphatidylcholine is less than that of the phosphatidylinositol. The exchange of other phospholipids and glycolipids between microsomal and myelin membranes cannot be demonstrated. The catalytic activity of the pH 5.1 supernatant proteins in rat brain for the exchange of phosphatidylinositol increases with age after birth and reaches a maximum around 21 days of age analogous to the process of myelination. The pH 5.1 supernatant proteins from quaking and jimpy mutant mice has normal catalytic activity.     

A Similar Calmodulin-Binding Protein Expressed in Chromaffin, Synaptic, and Neurohypophyseal Secretory Vesicles

The presence of calmodulin-binding proteins in three neurosecretory vesicles (bovine adrenal chromaffin granules, bovine posterior pituitary secretory granules, and rat brain synaptic vesicles) was investigated. When detergent-solubilized membrane proteins from each type of secretory organelle were applied to calmodulin-affinity columns in the presence of calcium, several calmodulin-binding proteins were retained and these were eluted by EGTA from the columns. In all three membranes, a 65-kilodalton (63 kilodaltons in rat brain synaptic vesicles) and a 53-kilodalton protein were found consistently in the EGTA eluate. 125I-Calmodulin overlay tests on nitrocellulose sheets containing transferred chromaffin and posterior pituitary secretory granule membrane proteins showed a similarity in the protein bands labeled with radioactive calmodulin. In the presence of 10(-4) M calcium, eight major protein bands (240, 180, 145, 125, 65, 60, 53, and 49 kilodaltons) were labeled with 125I-calmodulin. The presence of 10 microM trifluoperazine (a calmodulin antagonist) significantly reduced this labeling, while no labeling was seen in the presence of 1 mM EGTA. Two monoclonal antibodies (mAb 30, mAb 48), previously shown to react with a cholinergic synaptic vesicle membrane protein of approximate molecular mass of 65 kilodaltons, were tested on total membrane proteins from the three different secretory vesicles and on calmodulin-binding proteins isolated from these membranes using calmodulin-affinity chromatography. Both monoclonal antibodies reacted with a 65-kilodalton protein present in membranes from chromaffin and posterior pituitary secretory granules and with a 63-kilodalton protein present in rat brain synaptic vesicle membranes. When the immunoblotting was repeated on secretory vesicle membrane calmodulin-binding proteins isolated by calmodulin-affinity chromatography, an identical staining pattern was obtained. These results clearly indicate that an immunologically identical calmodulin-binding protein is expressed in at least three different neurosecretory vesicle types, thus suggesting a common role for this protein in secretory vesicle function.     

Modification of Gs-Stimulated Adenylate Cyclase in Brain Membranes by Low pH Pretreatment: Correlation with Altered Guanine Nucleotide Exchange

Pretreatment of rat brain membranes at pH 4.5 before assay at pH 7.4 modifies the function of GTP-binding proteins (G-proteins) by eliminating Gs-stimulated adenylate cyclase activity while increasing opiate-inhibited adenylate cyclase activity. To help characterize the molecular nature of the low pH effect, we labeled Gs and Gi alpha-subunits in both control and low pH-pretreated membranes with the GTP photoaffinity analog [32P]P3 (4-azidoanilido)-P1-5'-GTP ([32P]AAGTP). When membranes were preincubated with unlabeled AAGTP, a persistent inhibitory state of adenylate cyclase was produced, which was overcome in untreated membranes with high (greater than 1 microM) concentrations of guanylyl-5'-imidodiphosphate [Gpp(NH)p]. In low pH-pretreated membranes, this inhibition could not be overcome, and stimulation by Gpp(NH)p was eliminated. Maximal inhibition of adenylate cyclase achieved by incubation with AAGTP was not altered by low pH pretreatment. Incorporation of [32P]AAGTP into Gs (42 kilodaltons) or Gi/o (40 kilodaltons) was unaffected by low pH pretreatment; however, transfer of 32P from Gi/o to Gs, which occurs with low (10 nM) concentrations of Gpp(NH)p in untreated membranes, was severely retarded in low pH-pretreated membranes. Both the potency and efficacy of Gpp(NH)p in producing exchange of [32P]AAGTP from Gi/o to Gs were markedly reduced by low pH pretreatment. These results correlate the loss of Gs-stimulated adenylate cyclase with a loss of transfer of nucleotide from Gi/o to Gs alpha-subunits and suggest that the nucleotide exchange participates in the modulation of neuronal adenylate cyclase.     

Studies on the persistence of differentiated functions in rat hepatocytes set into primary tissue culture. II. Production of specific exportable proteins and the effect of purine cyclic nucleotides: an immunofluorescent study.

Immunofluorescent studies showed that even after 15 days in vitro primary neonatal rat hepatocytes contained in their cytoplasm detectable amounts of different adult rat serum proteins, including fibrinogen and proalbumin. Estimation of the intensity of specific fluorescence revealed that in untreated cultures the hepatocytic content of the various exportable antigens progressively diminished between the 5th and 15th day in vitro. Treatment with cAMP (10(-5) M daily) alone increased in hepatocytic cytoplasm, with respect to parallel controls, the content of total exportable proteins and of proalbumin. Daily administration of an equimolar association (10(-5) M) of cAMP with cGMP increased the total protein, proalbumin and fibrinogen content of hepatocytes. Daily treatment with cGMP (10(-5) M) alone caused only light and transitory increases in the content of proalbumin and fibrinogen. Rocket immune electrophoresis showed that the hepatocytic secretion of specific proteins into the growth medium persisted up to the 15th day, although progressively diminishing in intensity. The secretion of total exportable proteins and of albumin, but not of fibrinogen, was stimulated by cGMP used alone or coupled with equimolar cAMP.     

Structure and expression of two temperature-specific surface proteins in the ciliated protozoan Tetrahymena thermophila.

The presence of specific proteins (known as immobilization antigens) on the surface of the ciliated protozoan Tetrahymena thermophila is under environmental regulation. There are five different classes (serotypes) of surface proteins which appear on the cell surface when T. thermophila is cultured under different conditions of temperature or incubation medium; three of these are temperature dependent. The appearance of these proteins on the cell surface is mutually exclusive. We used polyclonal antibodies raised against 30 degrees C (designated SerH3)- and 40 degrees C (designated SerT)-specific surface antigens to study their structure and expression. We showed that these surface proteins contain at least one disulfide bridge. On sodium dodecyl sulfate-denaturing polyacrylamide gels, the nonreduced 30 degrees C- and 40 degrees C-specific surface proteins migrated with molecular sizes of 69 and 36 kilodaltons, respectively. The reduced forms of the proteins migrated with molecular sizes of 58 and 30 kilodaltons, respectively. The synthesis of the surface proteins responded rapidly and with a time course similar to that of the incubation temperature. The synthesis of each surface protein was greatly reduced within 1 h and undetectable by 2 h after a shift to the temperature at which the protein is not expressed. Surface protein synthesis resumed by the end of 1 h after a shift to the temperature at which the protein is expressed. The temperature-dependent induction of these surface proteins appears to be dependent on the synthesis of new mRNA, as indicated by a sensitivity to actinomycin D. Surface protein syntheses were mutually exclusive except at a transition temperature. At 35 degrees C both surface proteins were synthesized by a cell population. These data support the potential of this system as a model for the study of the effects of environmental factors on the genetic regulation of cell surface proteins.     

Size-variant pp60src proteins of recovered avian sarcoma viruses interact with adhesion plaques as peripheral membrane proteins: effects on cell transformation.

We have shown previously that the membrane association of the src proteins of recovered avian sarcoma viruses (rASVs) 1702 (56 kilodaltons) and 157 (62.5 kilodaltons), whose size variations occur within 8 kilodaltons of the amino terminus, is salt sensitive and that, in isotonic salt, these src proteins fractionate as soluble cytoplasmic proteins. In contrast, wild-type Rous sarcoma virus pp60src behaves as an integral plasma membrane protein in cellular fractionation studies and shows prominent membrane interaction by immunofluorescence microscopy. In this study we have examined the distribution of these size-variant src proteins between free and complexed forms, their subcellular localization by immunofluorescence microscopy, and their ability to effect several transformation-related cell properties. Glycerol gradient sedimentation of extracts from cells infected either with rASV 1702 or rASV 157 showed that soluble src proteins of these viruses were distributed between free and complexed forms as has been demonstrated for wild-type Rous sarcoma virus pp60src. Pulse-chase studies with rASV pp60src showed that, like wild-type Rous sarcoma virus pp60src, it was transiently found in a complexed form. Indirect immunofluorescence showed that size-variant pp60src proteins are localized in adhesion plaques and regions of cell-to-cell contact in rASV 1702- or 157-infected cells. This result is in contrast with the generalized localization of pp60src in plasma membranes of control rASV-infected cells which produce pp60src. Chicken embryo fibroblasts infected by rASVs 1702 and 157 display a partial-transformation phenotype with respect to (i) transformation-related morphology, (ii) cell surface membrane changes, and (iii) retained extracellular fibronectin. It is possible that the induction of a partial-transformation phenotype may be the result of the unique interaction of the src proteins encoded by these viruses with restricted areas of the plasma membrane.     

Molecular characterization of fetal antigens on red blood cells of chickens,Japanese quail,and quail-chicken hybrids

The molecular nature of chicken fetal antigen (CFA) and quail fetal antigen (QFA) was studied on embryonic red blood cells (RBCs) of the chicken, the Japanese quail, and the quail-chicken hybrid. Specific immunoprecipitation of radiolabeled membrane proteins followed by electrophoretic separation and autoradiography were used to identify the protein molecules carrying these fetal antigens. CFA was found on molecules of 24, 50, 88, 99, 130, 170, and 220 kd (kilodaltons) in the chicken and hybrid and on molecules of 24, 50, 99, and 170 kd in the Japanese quail. Similarly, quail fetal antigen was associated with 24-, 50-, 99-, and 170-kd molecules in the quail and hybrid and was not detected in the chicken. Partial proteolytic digestion of the 50- and 170-kd molecules isolated from RBCs of all sources showed remarkably similar peptide patterns. Likewise, two-dimensional separation of the CFA-positive and QFA-positive 50-kd molecules from quail RBCs revealed a similar pattern of at least nine isomorphic variants. Sequential depletions of quail embryonic RBC extracts with either anti-CFA or anti-QFA followed by immune precipitation with the reciprocal antiserum suggested that most of the cell surface proteins carrying QFA also have CFA on the same molecules. It is suggested that specific glycosylations of a variety of distinct molecular weight proteins determines the antigenic phenotype characterized as "fetal antigens."     

Lateral diffusion of human histocompatibility antigens in isolated plasma membranes

We have prepared large (5–10 μm) plasma membrane fragments by lysis of VA-2, human, cells adherent to Sephadex beads. The membrane fragments may be removed from beads by sonication and stained with fluorescent antibodies to human histocompatibility antigens, HLA antigens. Lateral diffusion of labelled antigens is followed by the method of fluorescence photobleaching recovery (FPR). HLA antigens of isolated membranes diffuse at the same rate, approx. (2–4) · 10^?10 cm² · s^?1 as they do in intact cells. This rate may be modified by incubating membranes in a variety of media. Buffers of slightly acid pH (6.5 or less) enhance lateral diffusion, while the presence of divalent ions slightly reduces diffusion rates. Our major finding is that incubation of 37° in 0.10 M phosphate buffer increases lateral diffusion 3–5-fold.     

Composition of the outer membrane of Proteus mirabilis in relation to serum sensitivity in progressive stages of cell form defectiveness.

A serum-resistant strain of Proteus mirabilis was used to determine whether changes in the composition of surface components could be detected following induction of progressive stages of cell form defectiveness by beta-lactam antibiotics. The critical stage was the conversion from filaments to the spheroplast form, which was accompanied by increased susceptibility to the bactericidal action of human serum. Inner and outer membranes of the bacterium, its filament form and its spheroplast form were separated by sucrose density-gradient centrifugation after digestion of peptidoglycan, followed by osmotic lysis of the cells. Outer membranes of the bacterial and the filament forms sedimented at the same density, whilst the outer membrane fraction of the spheroplast form sedimented in a region of lesser density. In addition, the amounts of two major outer-membrane proteins as well as the O-polysaccharide content of the lipopolysaccharide were reduced in the spheroplast form. These results indicate a general disorganization in structure and assembly of components in regard to their interactions with one another in the outer membrane of the spheroplast form.     

Developmental changes in messenger RNAs and protein synthesis in Dictyostelium discoideum

The pattern of proteins synthesized at different stages of differentiation of the slime mold Dictyostelium discoideum was studied by two-dimensional polyacrylamide gel electrophoresis. Of the approximately 400 proteins detected during growth and/or development, synthesis of most continued throughout differentiation. Approximately 100 proteins show changes in their relative rates of synthesis. During the transition from growth to interphase, the major change observed is reduction in the relative rate of synthesis of about 8 proteins. Few further changes are noticeable until the stage of late cell aggregation, when production of about 40 new proteins begins and synthesis of about 10 is reduced considerably. Thereafter, there are few changes in the pattern of protein synthesis. Major changes in the relative rates of synthesis of a number of proteins are found during culmination, but few culmination-specific proteins are observed. In an attempt to understand the molecular basis for these changes, mRNA was isolated from different stages of differentiation and translated in an improved wheat germ cell-free system; the products were resolved on two-dimensional gels. The ratio of total translatable mRNA to total cellular RNA is constant throughout growth and differentiation. Messenger RNAs for many, but not all, developmentally regulated proteins can be identified by translation in cell-free systems. Actin is the major protein synthesized by vegetative cells and by early differentiating cells. The threefold increase in the relative rate of synthesis of actin during the first 2 hr of differentiation and the decrease which occurs thereafter can be accounted for by parallel changes in the amount of translatable actin mRNA. Most of the changes in the pattern of protein synthesis which occur during the late aggregation and culmination stages can also be accounted for by parallel increases or decreases in the amounts of translatable mRNAs encoding these proteins. It is concluded that mRNAs do not appear in a translatable form before synthesis of the homologous protein begins, and that regulation of protein synthesis during development is primarily at the levels of production or destruction of mRNA.     

Production of human c-myc protein in insect cells infected with a baculovirus expression vector.

A cDNA fragment coding for human c-myc was inserted into the genome of the baculovirus Autographa californica nuclear polyhedrosis virus adjacent to the strong polyhedrin promoter. Insect cells infected with the recombinant virus produced significant amounts of c-myc protein, which constituted the major phosphoprotein component in these cells. By immunoprecipitation and immunoblot analysis, two proteins of 61 and 64 kilodaltons were detected with c-myc-specific antisera. The insect-derived proteins were compared with recombinant human c-myc-encoded proteins synthesized in Escherichia coli and Saccharomyces cerevisiae cells. The c-myc gene product was found predominantly in the nucleus by subcellular fractionation of infected insect cells.     

Fish plasminogen activators: their identification and characterization

Immunoblots of proteins extracted from the skin of a small viviparous fish (Xiphophorus) showed that a monoclonal antibody against human urokinase recognizes multiple molecular weight species of antigens. The immunoaffinity-purified antigens had serine-protease activity for the hydrolysis of a chromogenic substrate and could convert human plasminogen to plasmin in a manner similar to that for human urokinase in vitro. Two antigens with apparent molecular weights of 55 and 50 kilodaltons that had been purified on a fibrin-Celite column were separable on SDS-polyacrylamide gels and were characterized as major plasminogen activators on fibrin-agar indicator plates. The 125I-tryptic peptide maps of both antigens were similar to that of human urokinase; therefore, the fish activators and human urokinase are structurally and functionally related.     

Effect of 3-Phenylpropanoic Acid on Capsule and Cellulases of Ruminococcus albus 8

The morphology and cellulases of Ruminococcus albus 8 were markedly affected by the inclusion of 3-phenylpropanoic acid (PPA) in a defined growth medium. PPA-grown bacteria produced substantial quantities of cell-bound cellulase, as well as a very high-molecular-weight extracellular enzyme and lesser amounts of two low-molecular-weight enzymes. PPA-deprived bacteria produced greater total amounts of cellulase, but all of it exists in soluble, low-molecular-weight forms. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the availability of PPA did not affect the kinds of proteins produced, but the distribution of two major proteins between cells and supernatant was PPA dependent. These two proteins (85 and 102 kilodaltons) were primarily associated with the cells of PPA-grown bacteria but were found chiefly in the supernatants of PPA-deprived cultures. Examination of thin sections of PPA-grown R. albus 8 by transmission electron microscopy showed a lobed ruthenium red-staining capsule surrounding the cell wall, as well as small vesicular structures (diameter, 0.05 to 0.06 μm) which appeared to aggregate into larger spherical units (diameter, 0.2 to 0.3 μm). In contrast, thin sections of PPA-deprived cells were devoid of vesicles and showed little or no capsule surrounding the cells.