Electrostatic destabilization of the cytochrome b6f complex in the thylakoid membrane.

Three of the membrane-spanning polypeptides of the chloroplast cytochrome (cyt) b6f complex were sequentially released from the thylakoid membrane, in the order cyt b6, suIV and Rieske iron-sulfur protein, as the pH was increased from 10 to 12, a protocol usually employed to remove peripheral proteins from membranes. The fourth polypeptide of the cyt b6f complex, cyt f, which spans the membrane once, was apparently not released. The pH values for half-release at low ionic strength were approximately 10.7, 11.1 and 11.3 respectively. The separation of the polypeptides of the complex and the sequential release is readily seen at pH 11, where the loss from the membrane of cyt b6, suIV and Fe iron-sulfur center is approximately 90%, 50% and 20%, respectively. the release of cyt b6 from the membrane was reflected by the absence of its characteristic reduced minus oxidized absorbance signal. The pH values at which the release occurred increased as the ionic strength was raised, implying that the release of the b6f polypeptides arises from extrusion due to repulsive electrostatic interactions probably caused by deprotonation of tyrosine and lysine residues. The lipid content of the released polypeptides was very low, consistent with the observation of a non-membranous state. It is proposed that the pH-dependent extrusion requires two electrostatic effects at alkaline pH higher than approximately 10.5: (i) increased electrostatic repulsion between neighbouring polypeptides of the complex, arising from increased net negative charge in the peripheral segments of these polypeptides, which can cause separation of the polypeptides from the complex; and (ii) ionization of residues such as tyrosine in the membrane-spanning alpha-helices, and neutralization of residues such as lysine which can bind to the negative membrane surface.(ABSTRACT TRUNCATED AT 250 WORDS)     

Solubilization of proteins from bovine brain coated vesicles by protein perturbants and Triton X-100

To identify integral and peripheral membrane proteins, highly purified coated vesicles from bovine brain were exposed to solutions of various pH, ionic strength, and concentrations of the nonionic detergent Triton X-100. At pH 10.0 or above most major proteins were liberated, but four minor polypeptides sedimented with the vesicles. From quantitative analysis of phospholipids in the pellet and extract, we determined that at a pH of up to 12 all phospholipids could be recovered in the pellet. Electron microscopic examination of coated vesicles at pH 12.0 showed all vesicles devoid of coat structures. Treatment with high ionic strength solutions (0-1.0 M KCl) at pH 6.5-8.5 also liberated all major proteins, except tubulin, which remained sedimentable. The addition of Triton X-100 to coated vesicles or to stripped vesicles from which 90% of the clathrin had been removed resulted in the release of four distinct polypeptides of approximate Mr 38,000, 29,000, 24,000 and 10,000. The 38,000-D polypeptide (pK approximately 5.0), which represents approximately 50% of the protein liberated by Triton X-100, appears to be a glycoprotein on the basis of its reaction with periodic acid-Schiff reagent. Extraction of 90% of the clathrin followed by extraction of 90% of the phospholipids with Triton X-100 produced a protein residue that remained sedimentable and consisted of structures that appeared to be shrunken stripped vesicles. Together our data indicate that most of the major polypeptides of brain coated vesicles behave as peripheral membrane proteins and at least four polypeptides behave as integral membrane proteins. By use of a monoclonal antibody, we have identified one of these polypeptides (38,000 mol wt) as a marker for a subpopulation of calf brain coated vesicles.     

Solubility properties and specific assembly pathways of the B-type lamin from Caenorhabditis elegans

Lamins are nucleus-specific intermediate filament (IF) proteins that together with a complex set of membrane proteins form a filamentous meshwork tightly adhering to the inner nuclear membrane and being associated with the nuclear pore complexes. This so-called nuclear lamina provides mechanical stability and, in addition, has been implicated in the spatial organization of the heterochromatin. While increasing knowledge on the biological function of lamins has been obtained in recent years, the assembly mechanism of lamin filaments at the molecular level has remained largely elusive. Therefore, we have now more systematically investigated lamin assembly in vitro. Using Caenorhabditis elegans lamin, which has been reported to assemble into 10-nm filaments under low ionic strength conditions, we investigated the assembly kinetics of this protein into filaments in more detail using both His-tagged and un-tagged recombinant proteins. In particular, we have characterized distinct intermediates in the filament assembly process by analytical ultracentrifugation, electron and atomic force microscopy. In contrast to the general view that lamins assemble only slowly into filaments, we show that in vitro association reactions are extremely fast, and depending on the ionic conditions employed, significant filamentous assemblies form within seconds.     

Identification of Tetrahymena ciliary surface proteins labeled with sulfosuccinimidyl 6-(biotinamido) hexanoate and Concanavalin A and fractionated with Triton X-114.

Tetrahymena thermophila cells were labeled with sulfosuccinimidyl 6-(biotinamido) hexanoate, a sensitive nonradioactive probe for cell surface proteins, and Western blots of axonemes and ciliary membrane vesicles were compared to cilia fractionated with Triton X-114 (TX-114) in order to study the orientation of ciliary membrane proteins. Greater than 40 ciliary surface polypeptides, from greater than 350 kDa to less than 20 kDa, were resolved. The major surface 50-60 kDa proteins are hydrophobic and partition into the TX-114 detergent phase. Two high molecular weight proteins, one of which is biotinylated, comigrate with the heavy chains of ciliary dynein, sediment at 14S in a sucrose gradient, and partition into the TX-114 aqueous phase. Fractions containing these high molecular weight proteins as well as fractions enriched in 88-kDa and 66-kDa polypeptides contain Mg(2+)-ATPase activities. Detergent-solubilized tubulins partition into the TX-114 aqueous phase, are not biotinylated, and must not be exposed to the ciliary surface. The detergent-insoluble axoneme and membrane fraction contains a 36-kDa polypeptide and a portion of the 50-kDa polypeptides that otherwise partition into the detergent phase. These polypeptides could not be solubilized by ATP or by NaCl extraction and appear to be associated with pieces of ciliary membrane tightly linked to the axoneme. The ciliary membrane polypeptides were also tested for Concanavalin A binding and at least sixteen Con A-binding polypeptides were resolved. Of the major Con A-binding polypeptides, three are hydrophobic and partition into the TX-114 detergent phase, three partition into the TX-114 aqueous phase, and four partition exclusively in the detergent-insoluble fraction, which contains axonemes and detergent-resistant membrane vesicles.     

Identification of Tetrahymena Ciliary Surface Proteins Labeled with Sulfosuccinimidyl 6-(Biotinamido) Hexanoate and Concanavalin A and Fractionated with Triton X-114

ABSTRACT. Tetrahymena thermophila cells were labeled with sulfosuccinimidyl 6-(biotinamido) hexanoate, a sensitive nonradioactive probe for cell surface proteins, and Western blots of axonemes and ciliary membrane vesicles were compared to cilia fractionated with Triton X-114 (TX-114) in order to study the orientation of ciliary membrane proteins. Greater than 40 ciliary surface polypeptides, from >350 kDa to <20 kDa, were resolved. The major surface 50–60 kDa proteins are hydrophobic and partition into the TX-114 detergent phase. Two high molecular weight proteins, one of which is biotinylated, comigrate with the heavy chains of ciliary dynein, sediment at 14S in a sucrose gradient, and partition into the TX-114 aqueous phase. Fractions containing these high molecular weight proteins as well as fractions enriched in 88-kDa and 66-kDa polypeptides contain Mg²⁺-ATPase activities. Detergent-solubilized tubulins partition into the TX-114 aqueous phase, are not biotinylated, and must not be exposed to the ciliary surface. The detergent-insoluble axoneme and membrane fraction contains a 36-kDa polypeptide and a portion of the 50-kDa polypeptides that otherwise partition into the detergent phase. These polypeptides could not be solubilized by ATP or by NaCl extraction and appear to be associated with pieces of ciliary membrane tightly linked to the axoneme. The ciliary membrane polypeptides were also tested for Concanavalin A binding and at least sixteen Con A-binding polypeptides were resolved. Of the major Con A-binding polypeptides, three are hydrophobic and partition into the TX-114 detergent phase, three partition into the TX-114 aqueous phase, and four partition exclusively in the detergent-insoluble fraction, which contains axonemes and detergent-resistant membrane vesicles.     

Herpesvirus glycoprotein synthesis and insertion into plasma membranes.

In the presence of the antibiotic tunicamycin (TM), glycosylation of herpes simplex virus glycoproteins is inhibited and non-glycosylated polypeptides analogous to the glycoproteins are synthesized (Pizer et al., J. Virol. 34:142-153, 1980). The synthesis of viral proteins and DNA occurs in TM-treated cells. By electron microscopy, nucleocapsids can be observed both in the nucleus and the cytoplasm of TM-treated cells; a small number of enveloped virions were observed on the cell surface. Analyses of the proteins in partially purified virus readily detects viral glycoproteins in the control cells, but neither glycoproteins nor nonglycosylated polypeptide analogs were observed in the virus prepared from TM-treated cells. By labeling the surface of infected cells with 125I, viral glycoproteins were detected as soon as 90 min after infection even when protein synthesis was inhibited with cycloheximide and glycosylation was blocked with TM. Labeling the proteins synthesized in infected cells with [35S]methionine showed that the surface glycoproteins detected in the cycloheximide- and TM-treated cells were not synthesized de novo after infection, but were placed on the cell surface by the infecting virus. Studies with metabolic inhibitors and a temperature-sensitive mutant blocked early in the infectious cycle showed that glycoproteins gA/gB and gD were synthesized soon after infection, but that the synthesis of gC was delayed. Under conditions of infection, in which gC and its precursor pgC are not produced, we have been able to observe the relationships between the glycosylated polypeptides that correspond to pgA/pgB and the nonglycosylated analog made in the presence of TM.     

Impaired intracellular migration and altered solubility of nonglycosylated glycoproteins of vesicular stomatitis virus and Sindbis virus.

Tunicamycin, an antibiotic which prevents the glycosylation of newly synthesized proteins, inhibits the replication of both vesicular stomatitis virus and Sindbis virus. In tunicamycin-treated infected cells, all of the viral proteins are synthesized but the glycoproteins are devoid of carbohydrate. The nonglycosylated glycoproteins could not be detected on the outside of the plasma membrane by lactoperoxidase labeling, indirect immunofluorescence staining, or chymotrypsin treatment of intact cells, whereas the glycosylated glycoproteins were readily detected by all three methods. These results indicate that the bulk of the nonglycosylated glycoproteins are unable to undergo the normal migration to the cell surface. In contrast to the normal glycosylated viral glycoproteins, the nonglycosylated glycoproteins were insoluble in nonionic detergents such as Triton X-100. The nonglycosylated glycoprotein of vesicular stomatitis virus could be solubilized using a combination of 6 M guanidine hydrochloride and 0.2% Triton X-100, but precipitated when the 6 M guanidine was removed by dialysis. These results suggest that the lack of carbohydrate alters the properties of the glycoproteins, which may explain their impaired mobility through the intracellular membranous system.     

Interactions of cytoskeletal elements with the plasma membrane of Sarcoma 180 ascites tumor cells

The association of cytoskeletal proteins with cell surface envelopes from Sarcoma 180 ascites cells has been studied by several techniques previously used successfully in studying the interaction of spectrin with erythrocyte membranes. By electron microscopy the envelopes exhibit irregular exterior surfaces and the presence of substantial amounts of “fuzz” at the interior surface. Extraction of the envelopes at low ionic strength and alkaline pH fragments the membranes and depletes them of the “fuzz” with concomitant elution of four major polypeptides of mol. wt >300 000 (Band E), 250 000, 100 000 and 43 000 D. The last three of these have been tentatively identified as actin-binding protein (ABP), α-actinin and actin. Membrane-associated myosin is not eluted under these conditions. Neither actin nor myosin is eluted under conditions commonly used to depolymerize them. However, myosin can be eluted at high salt concentrations if the envelopes have been previously extracted and fragmented with alkaline buffer as above. Extraction of the envelopes with Triton X-100 removes 60% of the membrane lipid and 70–80% of lactoperoxidase-iodinated cell surface proteins without removal of significant amounts of the cytoskeletal proteins. The Triton residues maintain the shape of the original envelopes but have lost the trilaminar membrane structure. Proteolysis of intact envelopes with trypsin or papain cleaves the high molecular weight polypeptides in the order E > ABP > myosin. Fragmentation occurs with cleavage of E or ABP, but does not appear to require cleavage of myosin. Actin and α-actinin are not appreciably cleaved when associated with the membrane. The results, combined with previous observations, suggest an extensive complex of cytoskeletal proteins attached to the membrane interior surface.     

Organization of lymphocyte plasma membrane. Surface protein-membrane matrix interactions in B-cell lines of different stages of differentiation

Composition of surface proteins and their interactions with cytoskeleton or membrane matrix were compared in tumor B-cell lines of different stages of B-lymphocyte maturation. All studied B-cell lines were found to share a similar set of cell surface proteins, which are tightly associated with the cytoskeleton. The increase in amount of detergent-unextractable cell surface proteins with B-cell maturation suggested that differentiation of B lymphocytes was accompanied by development of specific interactions between surface proteins and elements of the cytoskeleton or membrane matrix. Using a recently developed procedure for lymphocyte plasma membrane fractionation we demonstrate changes in distribution of cell surface proteins in membrane matrix-rich and membrane matrix-poor plasma membrane fractions during B-lymphocyte maturation. Thus, cell surface proteins of the mature B-cell line MOPC-315 were predominantly found in the plasma membrane vesicles of a high buoyant density. These vesicles mostly contained plasma membrane proteins tightly associated with elements of the membrane matrix. In immature B cells (line 70Z3) virtually all surface proteins were detected in both low and high buoyant density membrane vesicles. The tendency to increased associations between surface proteins and cytoskeleton/membrane matrix with maturation of B cells could not be explained by increased amounts of filamentous actin, since no correlation was found between the amount of globular or filamentous actin and the degree of surface protein-cytoskeleton (membrane matrix) interactions.     

Multiparticle computer simulation of plastocyanin diffusion and interaction with cytochrome <Emphasis Type="Italic">f</Emphasis> in the electrostatic field of the thylakoid membrane

A multiparticle computer model of plastocyanin-cytochrome f complex formation in the thylakoid lumen has been designed, which takes into account the electrostatic interactions of proteins and membrane. The Poisson-Boltzmann formalism was used to determine the electrostatic potentials of the electron carrier proteins and the thylakoid membrane at different ionic strengths. The membrane electrostatic field was shown to influence plastocyanin diffusion and interaction with cytochrome f. The rate constants for plastocyanin-cytochrome f complex formation were calculated as a function of ionic strength and membrane surface charge.     

Monoclonal antibodies identify a group of nuclear pore complex glycoproteins

Using monoclonal antibodies we identified a group of eight polypeptides of rat liver nuclear envelopes that have common epitopes. Most or all of these proteins are structurally distinct, as shown by tryptic peptide mapping and analysis with polyclonal antibodies. While these polypeptides are relatively tightly bound to nuclear membranes, only one is an integral membrane protein. The eight antigens cofractionate with the nuclear pore complex under various conditions of ionic strength and detergent. It can be seen by immunofluorescence microscopy that the monoclonal antibodies reacting with these antigens stain the nuclear surface of interphase cells in a finely punctate pattern. When the nuclear envelope is disassembled and subsequently reformed during mitosis, the proteins are reversibly dispersed throughout the cytoplasm in the form of minute foci. By EM immunogold localization on isolated nuclear envelopes, the monoclonal antibodies label exclusively the nuclear pore complex, at both its nucleoplasmic and cytoplasmic margins. Considered together, our biochemical and localization data indicate that the eight nuclear envelope polypeptides are pore complex components. As shown in the accompanying paper (Holt, G. D., C. M. Snow, A. Senior, R. S. Haltiwanger, L. Gerace, and G. W. Hart, J. Cell Biol., 104:1157-1164) these eight polypeptides contain a novel form of glycosylation, O-linked N-acetylglucosamine. The relative abundance and disposition of these O-linked glycoproteins in the pore complex are consistent with their having a role in nucleocytoplasmic transport.     

The majority of cellular fatty acid acylated proteins are localized to the cytoplasmic surface of the plasma membrane

The BC3Hl muscle cell line was previously reported to contain a broad array of fatty acid acylated proteins [Olson, E. N., Towler, D. A., & Glaser, L. (1985) J. Biol. Chem. 260, 3784-3790]. Palmitate was shown to be attached to membrane proteins posttranslationally through thiol ester linkages, whereas myristate was attached cotranslationally, or within seconds thereafter, to soluble and membrane-bound proteins through amide linkages [Olson, E. N., & Spizz, G. (1986) J. Biol. Chem. 261, 2458-2466]. The temporal and subcellular differences between palmitate and myristate acylation suggested that these two classes of acyl proteins might follow different intracellular pathways to distinct subcellular membrane systems or organelles. In this study, we examined the subcellular localization of the major fatty acylated proteins in BC3Hl cells. Palmitate-containing proteins were localized to the plasma membrane, but only a subset of myristate-containing proteins was localized to this membrane fraction. The majority of acyl proteins were nonglycosylated and resistant to digestion with extracellular proteases, suggesting that they were not exposed to the external surface of the plasma membrane. Many proteins were, however, digested during incubation of isolated membranes with proteases, which indicates that these proteins face the cytoplasm. Two-dimensional gel electrophoresis of proteins labeled with [3H]palmitate and [3H]myristate revealed that individual proteins were modified by only one of the two fatty acids and did not undergo both N-linked myristylation and ester-linked palmitylation. Together, these results suggest that the majority of cellular acyl proteins are routed to the cytoplasmic surface of the plasma membrane, and they raise the possibility that fatty acid acylation may play a role in intracellular sorting of nontransmembranous, nonglycosylated membrane proteins.     

Protein filaments may initiate the assembly of the Bacillus subtilis spore coat.

The Bacillus subtilis spore coat consists of three morphological layers: a diffuse undercoat, a striated inner coat and a densely staining outer coat. These layers are comprised of at least 15 polypeptides and the absence of one in particular, CotE, had extensive pleiotropic effects. Only a partial inner coat was present on the spores which were lysozyme-sensitive. The initial rate of germination of these spores was the same as for the wild type but the overall optical density decrease was greater apparently due to the loss of the incomplete spore coat from germinated spores. Suppressors of the lysozyme-sensitive phenotype had some outer coat proteins restored as well as some novel minor polypeptides. These spores still lacked an undercoat and germinated as did those produced by the cotE deletion strain. The CotE protein was synthesized starting at stage II-III of sporulation, long before the appearance of the coat on spores at stage IV-V. Despite its apparent hydrophilic properties, this protein was present in the crude insoluble fraction from sporulating cells. CotE was not solubilized by high or low ionic strength buffers not by detergents used for the solubilization of membrane proteins. Either 8 M urea or 6 M guanidine HC1 was required and dialysis against a low ionic strength buffer resulted in aggregation into long, sticky filaments. Both the CotE and CotT spore coat proteins appeared to be necessary for the formation of these filaments. Each of these proteins contains sequences related to a bovine intermediate filament protein so their interaction could result in an analogous structure.(ABSTRACT TRUNCATED AT 250 WORDS)     

Properties of heparin binding to purified plasma membranes from bovine granulosa cells

Bovine granulosa cells were disrupted by nitrogen cavitation and the resulting membrane vesicles were isolated by centrifugation using a self-generating Percoll gradient. Transmission electron microscopy and marker enzyme assays revealed a highly enriched preparation of plasma membrane vesicles with little contamination from intracellular organelles. The membranes were examined for their ability to bind [3H]heparin under a variety of physical conditions. Binding was dependent largely on electrostatic interactions which were sensitive to alterations in the ionic strength and pH of the medium. Optimal binding was obtained in the absence of added salt and at pH 6.5 but reduced by 50% at 150 mM-NaCl or at pH values above 7.5. Heparin binding to the membranes was abolished by a 1-h pretreatment with chymotrypsin, plasmin, pronase or trypsin. Detergent treatment of the membranes had various effects, depending on the ionic characteristics of the detergents used. Sodium dodecyl sulphate-polyacrylamide gels of plasma membrane proteins revealed a complex pattern of polypeptides with Mr of 10,000-120,000. Autoradiographic analysis of plasma membrane proteins on Western blots labelled with 125I-labelled heparin revealed 3 major heparin-binding proteins with molecular weights of 14,000-16,000. These studies report a new method of rapidly obtaining purified membranes from a limited population of granulosa cells. The characterization of the binding domains as membrane-associated proteins provides opportunities for numerous additional studies. Detergent solubilization of the membranes without appreciable loss in binding activity should simplify attempts to purify the binding proteins. Further analysis of the interactions of these molecules with native follicular fluid GAGs at various stages of granulosa cell development should provide useful insights into the role of complex carbohydrates in follicular maturation.     

A single-amino-acid substitution eliminates the stringent carbohydrate requirement for intracellular transport of a viral glycoprotein.

In this report, we have investigated the contribution of primary sequence to the carbohydrate requirement for intracellular transport of two closely related glycoproteins, the G proteins of the San Juan and Orsay strains of vesicular stomatitis virus. We used site-directed mutagenesis of the coding sequence to eliminate the two consensus sites for glycosylation in the Orsay G protein. Whereas the nonglycosylated San Juan G protein required at least one of its two asparagine-linked oligosaccharides for transport to the plasma membrane at 37 degrees C, a fraction of the Orsay G protein was transported without carbohydrate. Of the 10 amino acid differences between these two proteins, residue 172 (tyrosine in San Juan, aspartic acid in Orsay) played the major role in determining the stringency for the carbohydrate requirement. The rates at which the glycosylated and nonglycosylated Orsay G proteins were transported to the cell surface were the same, although a smaller fraction of the nonglycosylated protein was transported. These results suggest that the carbohydrate does not promote intracellular transport directly but influences a polypeptide folding or oligomerization step which is critical for transport.     

Interaction of clathrin coat proteins with unilamellar and multilamellar vesicles of phosphatidylcholine

The binding of clathrin and accessory coat proteins to small unilamellar vesicles and to liposomes of uncharged phospholipids has been followed by chromatography, 31P-NMR, ESR and fluorescence anisotropy. At pH 6.5 and at an ionic strength value (0.1 M Mes) close to that used during the purification of clathrin-coated vesicles, the proteins do not restore the characteristic network found around the natural vesicles. Instead, a limited fusion leads to enlarged structures in which the perturbation of the dynamics of the phospholipids decreases gradually with the depth in the membrane. While the rate of motion of the outer polar heads is lowered, the order parameter of doxyl groups located either under or in the vicinity of the glycerol backbone is not affected by the proteins. In the inner core of the membrane, the main thermotropic transition of the hydrocarbon chains is unchanged. All the effects are the results of interactions limited to the membrane surface. The electrostatic nature of these interactions is evidenced when the embedded spin labels have a charge protruding at the membrane surface. An 'anchoring' effect appears which is due to the charged groups of the proteins. The lateral diffusion of the probes is reduced and, at low ionic strength, a cationic derivative no longer detects the thermotropic transition of the hydrocarbon chains. These results indicate that, although it is known that clathrin and accessory proteins bind to membranes by a series of protein-protein interactions, this system is not devoid of lipid-protein interactions, at least when it is not organized as in the natural system.     

Import of biopolymers into Escherichia coli: nucleotide sequences of the exbB and exbD genes are homologous to those of the tolQ and tolR genes, respectively.

Escherichia coli with mutations in the exb region are impaired in outer membrane receptor-dependent uptake processes. They are resistant to the antibiotic albomycin and exhibit reduced sensitivity to group B colicins. A 2.2-kilobase-pair DNA fragment of the exb locus was sequenced. It contained two open reading frames, designated exbB and exbD, which encoded polypeptides of 244 and 141 amino acids, respectively. Both proteins were found in the cytoplasmic membrane. They showed strong homologies to the TolQ and TolR proteins, respectively, which are involved in uptake of group A colicins and infection by filamentous bacteriophages. exbB and exbD were required to complement exb mutations. Osmotic shock treatment rendered exb mutants sensitive to colicin M, which was taken as evidence that the ExbB and ExbD proteins are involved in transport processes across the outer membrane. It is concluded that the exb- and tol-dependent systems originate from a common uptake system for biopolymers.     

Rat liver nuclear protein kinases NI and NII. Purification, subunit composition, substrate specificity, possible levels of regulation

Rat liver nuclear protein kinases NI and NII have been purified to homogeneity by an improved method. This method includes a casein-phosvitin-Sepharose column step, which separates the enzymes from the other chromosomal non-histone proteins, and a gel filtration at high ionic strength in the presence of a high concentration of protease inhibitors to separate the two enzymes from each other. NI has an apparent molecular mass of approximately 50 kDa and is composed of a single subunit. NII has an apparent molecular mass of 133 kDa and is composed of two subunits of identical molecular mass. The V and the Km of the two enzymes were determined for several substrates. Both enzymes phosphorylate chromosomal non-histone proteins with partly different specificities as shown by two-dimensional electrophoreses. When incubated in the absence of protease inhibitors, the enzymes were degraded into discrete polypeptides. Autophosphorylation of a polypeptide derived from NII was observed after incubation of the enzyme with ATP. This phosphorylation stimulated the enzyme activity. Several chromosomal proteins coeluted with NII from the casein-phosvitin-Sepharose column. They remained associated with the enzyme in sucrose gradients, during gel filtration performed at physiological ionic strength, and are dissociated at high ionic strength. These proteins were highly phosphorylated when the protein-NII complex was incubated with ATP.     

Specific and azurophilic granules from rabbit polymorphonuclear leukocytes. II. Cell surface localization of granule membrane and content proteins before and after degranulation

The compositional relationship between the cell surface of rabbit polymorphonuclear leukocytes (PMNs) and the membranes of PMN cytoplasmic granules has been investigated. Heterophilic PMNs obtained from peritoneal exudates contained 13 cell surface polypeptides ranging in molecular weight from 220,000 to 12,000 daltons as determined by lactoperoxidase-catalyzed protein iodination and gel electrophoresis. Of these, four polypeptides co-migrated with proteins identified as the major constituents of specific (SpG) and azurophilic (AzG) granule membranes. The most notable of these were cell surface proteins of 145,000 and 96,000 daltons that co-migrated with proteins identified as granule content proteins released from PMNs during exocytosis. Extensive washing did not remove these proteins from the cell surface. Iodination of PMNs after the release of SpG and AzG contents by calcium ionophore- induced exocytosis revealed that there was not a dramatic quantitative change in the proteins on the cell surface. Instead, there were large, quantitative increases in the relative amounts of (125)I that were incorporated into several pre-existing cell surface proteins; all of these cell surface proteins co-migrated as a set with those polypeptides identified as either granule membrane or content proteins. Although nearly all of the major polypeptides of SpG and AzG had counterparts on the cell surface of freshly isolated peritoneal exudates PMNs, there were several polypeptides that were unique to the cell surface. Thus, the PMN has at least three membrane compartments with strikingly different protein compositions.