Endoplasmic reticulum of rat liver contains two proteins closely related to skeletal sarcoplasmic reticulum Ca-ATPase and calsequestrin

Rat liver endoplasmic reticulum (ER) membranes were investigated for the presence of proteins having structural relationships with sarcoplasmic reticulum (SR) proteins. Western immunoblots of ER proteins probed with polyclonal antibodies raised against the 100-kDa SR Ca-ATPase of rabbit skeletal muscle identified a single reactive protein of 100 kDa. Also, the antibody inhibited up to 50% the Ca-ATPase activity of isolated ER membranes. Antisera raised against the major intraluminal calcium binding protein of rabbit skeletal muscle SR, calsequestrin (CS), cross-reacted with an ER peptide of about 63 kDa, by the blotting technique. Stains-All treatment of slab gels showed that the cross-reactive peptide stained metachromatically blue, similarly to SR CS. Two-dimensional electrophoresis (Michalak, M., Campbell, K. P., and MacLennan, D. H. (1980) J. Biol. Chem. 255, 1317-1326) of ER proteins showed that the CS-like component of liver ER, similarly to skeletal CS, fell off the diagonal line, as expected from the characteristic pH dependence of the rate of mobility of mammalian CS. In addition, the CS-like component of liver ER was released from the vesicles by alkaline treatment and was found to be able to bind calcium, by a 45Ca overlay technique. From these findings, we conclude that a 100-kDa membrane protein of liver ER is the Ca-ATPase, and that the peripheral protein in the 63-kDa range is closely structurally and functionally related to skeletal CS.     

Purification and ligand binding of a soluble class I major histocompatibility complex molecule consisting of the first three domains of H-2Kd fused to beta 2-microglobulin expressed in the baculovirus-insect cell system.

A recombinant baculovirus encoding a single-chain murine major histocompatibility complex class I molecule in which the first three domains of H-2Kd are fused to beta 2-microglobulin (beta 2-m) via a 15-amino acid linker has been isolated and used to infect lepidopteran cells. A soluble, 391-amino acid single-chain H-2Kd (SC-Kd) molecule of 48 kDa was synthesized and glycosylated in insect cells and could be purified in the absence of detergents by affinity chromatography using the anti-H-2Kd monoclonal antibody SF1.1.1.1. We tested the ability of SC-Kd to bind antigenic peptides using a direct binding assay based on photoaffinity labeling. The photoreactive derivative was prepared from the H-2Kd-restricted Plasmodium berghei circumsporozoite protein (P.b. CS) peptide 253-260 (YIPSAEKI), a probe that we had previously shown to be unable to bind to the H-2Kd heavy chain in infected cells in the absence of co-expressed beta 2-microglobulin. SC-Kd expressed in insect cells did not require additional mouse beta 2-m to bind the photoprobe, indicating that the covalently attached beta 2-m could substitute for the free molecule. Similarly, binding of the P.b. CS photoaffinity probe to the purified SC-Kd molecule was unaffected by the addition of exogenous beta 2-m. This is in contrast to H-2KdQ10, a soluble H-2Kd molecule in which beta 2-m is noncovalently bound to the soluble heavy chain, whose ability to bind the photoaffinity probe is greatly enhanced in the presence of an excess of exogenous beta 2-m. The binding of the probe to SC-Kd was allele-specific, since labeling was selectively inhibited only by antigenic peptides known to be presented by the H-2Kd molecule.     

Immunologically related multimeric forms of 30-40 kDa peptides associated with lung surfactant in various mammalian species

A comparative study of lung surfactant associated proteins was undertaken to determine which mammalian species would best serve as models for investigating alterations of the human lung surfactant system. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified surfactants in the presence of dithiothreitol revealed that surfactant invariably contains at least one peptide with molecular weight of 30 000-40 000. In the absence of disulfide reducing agents, the above peptides were in the form of high-molecular-weight proteins (greater than 400 kDa) in primates and cat, whereas in dog, rat and rabbit, the protein was a 72 kDa dimer. The 30-40 kDa peptide subunits were isolated from human, rat and dog surfactants and found to contain four or five residues of hydroxyproline. Antisera to either the human 34 kDa peptide or high-molecular-weight proteins reacted with the high-molecular-weight bands, the 34 kDa subunit and at least six intermediate disulfide-linked forms separated from purified human surfactant by electrophoresis under nonreducing conditions. Following electrophoresis in the presence of dithiothreitol, both antisera detected the 34 kDa peptide as well as other peptides ranging in molecular weight from 23 000 to 160 000. The isolated 34 kDa peptide readily reaggregated into disulfide-linked forms including 68 and 100 kDa complexes which were not reduced by 40 mM dithiothreitol. We conclude that the 34 kDa surfactant-associated peptide forms a complex system of monomeric and multimeric proteins, which varies among the species and could conceivably vary in distribution during lung development or disease.     

Identification of functional domains of the extrinsic 12 kDa protein in red algal PSII by limited rroteolysis and directed mutagenesis.

The extrinsic 12 kDa protein in red algal photosystem II (PSII) functions to minimize the chloride and calcium requirement of oxygen-evolving activity [Enami et al. (1998) Biochemistry 37: 2787]. In order to identify functional domains of the 12 kDa protein, we prepared the 12 kDa protein lacking N-terminal peptides or C-terminal peptides or both by limited proteolysis and directed mutagenesis. The resulting 12 kDa protein fragments were examined for their binding and functional properties by reconstitution experiments. (1) A peptide fragment from Gly-6 to C-terminus of the 12 kDa protein was prepared by V8 protease. This fragment rebound to PSII completely, and it reactivated oxygen evolution partially in the absence of Cl(-) and Ca(2+) ions but significantly in the presence of Cl(-) ion. (2) A peptide from Leu-10 to Phe-83 was obtained by chymotrypsin treatment. This peptide rebound to PSII effectively, but the rebinding did not restore oxygen evolution in both the absence and presence of Cl(-) and Ca(2+) ions. (3) Two mutant proteins, one lacking five residues and the other lacking nine residues of the N-terminus, were able to bind to PSII effectively. Recovery of oxygen evolution by their binding was almost the same as that reconstituted with the V8 protease-treated peptide. (4) Three mutant proteins lacking ten, seven or three residues of the C-terminus effectively rebound to PSII, but their binding did not result in recovery of the oxygen evolution. In contrast, reconstitution with a mutant protein lacking one residue of the C-terminus showed the same high restoration of oxygen evolution as reconstitution with the full-length 12 kDa protein. (5) These results indicate that two residues from lysine of the C-terminus of the 12 kDa protein constitute an important domain for minimizing the chloride and calcium requirement of oxygen evolution. In addition, the N-terminus of the protein, at least five residues, has a secondary function for the chloride requirement.     

Identification of the binding site of 55kDa tumor necrosis factor receptor by synthetic peptides.

We have synthesized a series of peptides, which cover almost the whole range of the N-terminal extracellular domain of human 55kDa TNF receptor (55kDa TNF-R). The peptides were examined for the binding activity to TNF by solid phase binding assay and for the inhibition of TNF cytotoxicity to mouse L-M cells. The peptide 159-178 exhibited remarkably higher binding activity to TNF than other peptides did. The specificity of the TNF binding to the peptides was confirmed by their inability to bind other cytokines. The peptide 159-178 also inhibited TNF cytotoxicity. These results indicate that the specific binding site of 55kDa TNF-R to TNF might reside within the peptide segment of amino acid numbers 159 to 178 in the N-terminal extracellular domain.     

The unraveling architecture of the junctional sarcoplasmic reticulum

The sarcoplasmic reticulum (SR) of skeletal muscle controls the contraction-relaxation cycle by raising and lowering the myoplasmic free-Ca²⁺ concentration. The coupling between excitation, i.e., depolarization of sarcolemma and transvers tubule (TT) and Ca²⁺ release from the terminal cisternae (TC) of SR takes place at the triad. The triad junction is formed by a specialized region of the TC, the junctional SR, and the TT. The molecular architecture and protein composition of the junctional SR are under active investigation. Since the junctional SR plays a central role in excitation-contraction coupling and Ca²⁺ release, some of its protein constituents are directly involved in these processes. The biochemical evidence supporting this contention is reviewed in this article.     

The effects of sphingosine on sarcoplasmic reticulum membrane calcium release.

In this study, we report that sphingosine is a potent inhibitor of sarcoplasmic reticulum (SR) calcium release. Evidence is presented demonstrating a direct effect of sphingosine on the SR ryanodine receptor. Calcium release from "skinned" rabbit skeletal muscle fibers and isolated junctional SR derived from the terminal cisternae (TC) was measured in response to caffeine, doxorubicin, 5'-adenylyl-beta,gamma-imidodiphosphate or calcium. Sphingosine inhibited caffeine-induced release in a dose-dependent manner with an IC50 of 0.1 microM for the single muscle fibers and 0.5 microM for the isolated TC vesicles. Near complete blockage of TC calcium release rate was observed with 3 microM sphingosine. Neither sphingomyelin nor sphingosylphosphorylcholine had any effect at the 3 microM level, suggesting that the sphingosine effect was specific. Doxorubicin-induced calcium release and spontaneous calcium release were also blocked by sphingosine. Sphingosine was also capable of stimulating calcium transport in the isolated TC vesicles without an effect on Ca-ATPase activity. Ruthenium red was not capable of substantial additional stimulation of calcium transport nor inhibition of calcium release beyond the action of sphingosine. Sphingosine's blockage of calcium release was not reversed by the protein kinase inhibitor, 1-(5-isoquinolinesulfonyl)-2- methylpiperazine dihydrochloride, suggesting that the action of sphingosine on calcium release was not dependent on ryanodine receptor phosphorylation. Sphingosine significantly increased (8-fold) the Kd for specific [3H]ryanodine binding to TC membranes and decreased the Bmax with a dose dependence similar to the inhibition of calcium release, but sphingosine did not affect the pCa tension relationship of skinned skeletal muscle fibers. These data are consistent with a direct effect of submicromolar sphingosine on the ryanodine receptor. Substantially higher concentrations of sphingosine (30-50 microM) or sphingosylphosphorylcholine (10-20 microM) were capable of inducing calcium release by themselves. Preliminary data indicate that the transverse tubule and not the SR contain substantial sphingomyelinase activity consistent with a transverse tubule source of sphingosine production. Considering that sphingosine is found in micromolar concentrations in some cells, our data indicate that sphingosine generated by the transverse tubule membranes may be a physiologically relevant mechanism for modulating SR calcium release.     

Species specificity of anti-acetylcholine receptor antibodies elicited by synthetic peptides

Two peptides corresponding to amino acid residues 351-368 of the alpha-subunits of Torpedo and human acetylcholine receptor (AChR) were synthesized. These peptides contain a segment (residues 355-364) which displays the greatest variability in amino acid sequence between the two species. Antibodies elicited against the two peptides cross-reacted with the respective native AChRs and were shown to be species specific by radioimmunoassay, immunoblotting, and immunofluorescence microscopy. Thus, antibodies against the Torpedo peptide cross-reacted with Torpedo AChR but did not bind to mammalian or chicken AChR. Antibodies against the human peptide proved to be specific probes for mammalian muscle AChR. They cross-reacted with mammalian AChR (human, calf, mouse, and rat) but not with Torpedo or chicken AChR. These antibodies were also shown to react preferentially with the extrajunctional form of muscle AChR, as compared to their reactivity with junctional muscle AChR. In immunofluorescence experiments, the anti-human peptide antibody stained AChR aggregates in sectioned or ethanol-permeabilized rat and mouse myotubes grown in culture but did not stain living myotubes. This indicates that the sequence 351-368 of the alpha-subunit of mammalian AChR is on the cytoplasmic face of muscle cell membranes, as predicted theoretically.     

Isolation of terminal cisternae of frog skeletal muscle. Calcium storage and release properties

Sarcoplasmic reticulum (SR) terminal cisternae (TC) of frog (Rana esculenta) fast-twitch skeletal muscle have been purified by isopycnic sucrose density gradient centrifugation. Biochemical characteristics and Ca2+ release properties have been investigated and compared to those of the homologous fraction of rabbit skeletal muscle TC. The frog SR fraction obtained at the 38/45% sucrose interface appears to be derived from the terminal cisternae region as judged by: (a) thin section electron microscopy showing vesicles containing electron opaque material and squarelike (feet) projections at the outer surface; (b) protein composition (Ca2+-ATPase, calsequestrin, and high Mr proteins); (c) Ca2+ fluxes properties. The content of calsequestrin was higher in frog TC by 50% and the Ca2+ binding capacity (624 or 45 nmol of Ca2+/mg of TC protein, depending upon experimental conditions) was 3-4 times that of rabbit TC. Species-specific antigenic differences were found between junctional SR proteins of frog and rabbit TC. After active Ca2+ preloading in the presence of pyrophosphate (Palade, P. (1987) J. Biol. Chem. 262, 6135-6141), caffeine and doxorubicin elicited Ca2+ release from either TC fraction but with much faster rates in frog TC than in rabbit TC (14 versus 3 mumol of Ca2+/min/mg of protein). The present results provide new evidence for the existence of marked differences in Ca2+ release properties between TC of amphibian and mammalian fast-twitch muscle. Higher Ca2+ binding capacity and faster release rates in frog TC might compensate for the comparably greater diffusion distance being covered by the released Ca2+ from the Z-line to the actomyosin cross-bridges in the A-I overlap region.     

Identification of a novel recognition sequence for the integrin alpha 4 beta 1 in the COOH-terminal heparin-binding domain of fibronectin.

The type III connecting segment of fibronectin contains two cell binding sites, represented by the peptides CS1 and CS5, that are recognized by the integrin receptor alpha 4 beta 1. Using assays measuring the spreading of A375-SM human melanoma cells, we now report that the adhesion promoting activity of a 29 kDa protease fragment of fibronectin containing the COOH-terminal heparin-binding domain (HepII), but lacking CS1 and CS5, is completely sensitive to anti-alpha 4 and anti-beta 1 antibodies, suggesting that HepII contains a third alpha 4 beta 1-binding sequence. Examination of the primary structure of HepII revealed a sequence with homology to CS1. A 19mer peptide spanning this region (designated H1) was found to support cell spreading to the same level as the 29 kDa fragment. H1-dependent adhesion was completely sensitive to anti-alpha 4 and anti-beta 1 antibodies. When soluble peptides were tested for their ability to block cell spreading on the 29 kDa fragment, a 13mer peptide comprising the central core of H1 was found to be completely inhibitory. The active region of H1 was localized to the pentapeptide IDAPS, which is homologous to LDVPS from the active site of CS1. Taken together, these results identify a novel peptide sequence in the HepII region of fibronectin that supports alpha 4 beta 1-dependent cell adhesion.     

Characterization of the actin polymerization-inhibiting protein from chicken gizzard smooth muscle

An actin polymerization-inhibiting protein, occurring in crude preparations of vinculin from chicken gizzard, has been found to be heterogeneous. The molecular masses of the polymerization-inhibiting peptides have been reported to range from 20 kDa to 80 kDa [Schr?er, E. & Wegner, A (1985) Eur. J. Biochem. 153, 515-520]. In this paper, a 21-kDa peptide was isolated from the bulk of the other peptides by gel chromatography. The 21-kDa peptide was identified as a polymerization-inhibiting peptide by its ability to retard nucleated actin polymerization and to bind polymeric actin when it was blotted onto nitrocellulose. Antiserum raised to the 21-kDa peptide was found to react with almost all peptides of the blotted heterogeneous polymerization-inhibiting protein. The same peptides which reacted with antiserum cosedimented with polymeric actin. The major peptides of the blotted polymerization-inhibiting protein bound polymeric actin. The largest peptide which reacted with antiserum and cosedimented with polymeric actin had a molecular mass of 85 kDa. The results suggest that the preparation of polymerization-inhibiting protein contains mainly polymerization-inhibiting peptides and only some contaminants, and that all the polymerization-inhibiting peptides are proteolytic fragments stemming from a common precursor.     

The 165-kDa peptide of the purified skeletal muscle dihydropyridine receptor contains the known regulatory sites of the calcium channel

The dihydropyridine receptor purified from rabbit skeletal muscle yields in the presence of dithiothreitol and sodium dodecyl sulfate on polyacrylamide gels bands of apparent molecular mass 165 +/- 5, 130 +/- 5, 55 +/- 3, 32 +/- 2 and 28 +/- 1 kDa (chi +/- SEM, n = 12). Under nonreducing conditions, the 130 kDa and 28-kDa peptides migrate as a single peptide of 165 kDa. These peptides were separated on a HPLC size-exclusion column. The specific absorption coefficients of the isolated peptides were determined. From these a stoichiometry of 1:1.7 +/- 0.2:1.4 +/- 0.3 (chi +/- SEM of 12 experiments with three different preparations) was calculated for the 165-kDa, 55-kDa and 32-kDa peptides. The relative amount of the 130/28-kDa peptide varied with different preparations. Tryptic, chymotryptic and V-8 protease peptides of the isolated proteins suggested that the 130/28-kDa peptide was not related to the 165-kDa peptide. The dihydropyridine photoaffinity analog (+/-)-azidopine was specifically incorporated only into the 165-kDa peptide with an efficiency of about 2.4%. The azido analog of desmethoxyverapamil, LU 49888, was specifically incorporated into the same peptide with an efficiency of 1.5%. These results suggest that only the 165-kDa peptide contains the regulatory sites detected so far in the voltage-operated L-type calcium channel. They suggest further that the 130/28-kDa peptide, which migrates as a 165-kDa peptide under nonreducing conditions, does not contain high-affinity binding sites for the calcium channel blockers.     

Properties of structure and interaction of the receptor for omega-conotoxin, a polypeptide active on Ca2+ channels

Binding properties of omega-conotoxin (GVIA) to avian and mammalian neuronal Ca2+ channels were investigated using a radioiodinated toxin derivative. An exceptionally high affinity of 0.6 to 2 pM was found both from equilibrium and kinetics measurements. Only one class of non-interacting binding sites was detected. In chick brain, dissucinimidyl suberate specifically cross-linked the toxin to 170 kDa component that comprises a 140 kDa peptide disulfide linked to a 30 kDa peptide, very similar to the heavily glycosylated component of the L-type DHP-sensitive Ca2+ channel. A large peptide of 210-220 kDa was labelled using the azidonitrobenzoyloxy derivative of omega-conotoxin as cross-linking reagent but not the 170/140+30 kDa component. The results suggest that the neuronal Ca2+ channel could be composed by the association of two distinct high molecular weight peptides of 220 kDa and 170/140+30 kDa.     

Phosphorylation of caldesmon prevents its interaction with smooth muscle myosin

Caldesmon is known to bind to smooth muscle myosin. Ca2+/calmodulin-dependent phosphorylation of caldesmon completely blocks its interaction with myosin. Cleavage of caldesmon at its 2 cysteine residues by 2-nitro-5-thiocyanobenzoic acid (NTCB) occurs initially at one site to yield 108-kDa and 21.2-kDa peptides and subsequently at the second site within the 108-kDa peptide to yield 85-kDa and 23.5-kDa fragments. The 23.5-kDa peptide retains the ability to bind to myosin. The N-terminal (95 kDa) and C-terminal (42 kDa) chymotryptic peptides of caldesmon were isolated and digested with NTCB: the C-terminal actin- and calmodulin-binding peptide was not cleaved, indicating that it does not contain either of the cysteine residues, whereas the 95-kDa N-terminal peptide was cleaved at two sites to yield 56-kDa, 23.5-kDa, and 21.2-kDa fragments. The arrangement of NTCB fragments in caldesmon is, therefore: 21.2 kDa/23.5 kDa/85 kDa from N to C terminus. Digestion of phosphorylated caldesmon with NTCB suggested a single phosphorylation site in the 21.2-kDa peptide and three sites in the 23.5-kDa peptide. These results lead to the development of a model whereby caldesmon may cross-link actin to myosin and such cross-linking is blocked by phosphorylation of caldesmon. This mechanism may explain the formation of reversible "latch bridges" which permit force maintenance at low levels of myosin phosphorylation in intact smooth muscles.     

Immunologically related multimeric forms of 30–40 kDa peptides associated with lung surfactant in various mammalian species

A comparative study of lung surfactant associated proteins was undertaken to determine which mammalian species would best serve as models for investigating alterations of the human lung surfactant system. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified surfactants in the presence of dithiothreitol revealed that surfactant invariably contains at least one peptide with molecular weight of 30 000–40 000. In the absence of disulfide reducing agents, the above peptides were in the form of high molecular-weight proteins (> 400 kDa) in primates and cat, whereas in dog, rat and rabbit, the protein was a 72 kDa dimer. The 30–40 kDa peptide subunits were isolated from human, rat and dog surfactants and found to contain four or five residues of hydroxyproline. Antisera to either the human 34 kDa peptide or high-molecular-weight proteins reacted with the high-molecular-weight bands, the 34 kDa subunit and at least six intermediate disulfide-linked forms separated from purified human surfactant by electrophoresis under nonreducing conditions. Following electrophoresis in the presence of dithiothreitol, both antisera detected the 34 kDa peptide as well as other peptides ranging in molecular weight from 23 000 to 160 000. The isolated 34 kDa peptide readily reaggregated into disulfide-linked forms including 68 and 100 kDa complexes which were not reduced by 40 mM dithiothreitol. We conclude that the 34 kDa surfactant-associated peptide forms a complex system of monomeric and multimeric proteins, which varies among the species and could conceivably vary in distribution during lung development or disease.     

Detailed analysis of the region 9-30 of rat follicle stimulating hormone receptor: identification of peptide 20-30 as a potential hormone binding inhibitor

The extracellular domain (ECD) of the follicle stimulating hormone receptor (FSHR) has been shown to be a major determinant of hormone selectivity. The N-terminal 9-30 region, the sequence of which is unique to FSHR, has been extensively studied earlier and has been proposed to be an FSHR neutralizing epitope. In this study antipeptide antibodies specific to the peptide 9-30 were generated and used for identifying a specific immunodominant region within it. Overlapping peptides corresponding to the regions 9-19, 15-25 and 20-30 were synthesized. The ability of the antipeptide antibodies to 9-30 of FSHR to bind to different peptides was checked. The results indicated that the antibodies mainly recognized the peptide 20-30 and not the other two overlapping peptides. Further, the effect of the peptide 20-30 on the binding of radiolabeled FSH to its receptor was monitored. This peptide showed FSH-binding inhibitory activity with an IC(50) value of 0.598 x 10(-4)M and was more effective than the peptide 9-30 itself. Binding kinetics revealed that the observed effect of the peptide 20-30 is due to mixed type of inhibitory mechanism. This is the smallest peptide from the rat FSHR sequence having ability to inhibit FSH binding to its receptor by more than 90%.     

Antimicrobial peptides from the skin of the Asian frog, Odorrana jingdongensis: De novo sequencing and analysis of tandem mass spectrometry data

Eight intact antimicrobial peptides were identified from the skin of Odorrana jingdongensis by de novo sequencing following low energy ESI CID Q-TOF MS/MS in positive-mode with the help of Edman degradation and structural similarity analysis. We devised exact mass measurements to discriminate the K/Q amino acid residue in the peptides between 2.0kDa to 3.8kDa. Moreover, the cleavage at the CS bond at the side chain of Met was observed in all the spectra of the peptides containing Met residue. And we found unusual cleavages within the intramolecular disulfide loop with high frequency. Our data revealed that the cleavage pathways are significantly different from those reported previously which are similar to the cycle peptide cleavage mode followed by the secondary cleavage at the CS bond on oxidized Cys. Thus, our results highly suggest that ion series generated from the cleavages within the intramolecular disulfide loop should be considered in both the top-down sequencing and the disulfide bridge location with the presence of a relatively high intensity of MH(+)-28 ion marker. Furthermore, our activity data implied that different AMPs may use different strategies to kill microbes.     

Specific binding sites for prohormone atrial natriuretic peptides 1-30, 31-67 and 99-126

Two peptides with vasodilatory properties consisting of amino acids 1-30 and 31-67 of the 98 a.a. N-terminal end of the prohormone of atrial natriuretic factor (proANF) which circulates in man were investigated to determine if they have specific binding sites on membranes isolated from DDT1 MF-2 smooth muscle cells. Smooth muscle is a known biologic target of these peptides. Competitive binding experiments revealed that proANFs (1-30), (31-67), and (99-126) (i.e., C-terminus; ANF) each had specific and separate binding sites. The dissociation constants for proANFs (1-30), (31-67), and (99-126) binding were 0.11 nM, 4 nM, and 7.3 nM, respectively. The binding site concentrations for proANFs (1-30), (31-67), and ANF were 2.57, 59.91 and 40 fmols/10(6) cells, respectively. The number of binding sites per cell were 1548, 36,087, and 24,090, respectively, for proANFs (1-30), (31-67), and (99-126) (ANF). Each peptide bound to DDT1 MF-2 membranes between 10(-8) to 10(-11) M but could only bind to the other peptides' receptors at concentrations of 10(-6) and 10(-7)M. These results suggest that proANF(1-30) and proANF(31-67) do not work through the ANF receptor but rather have their own separate and distinct receptors that mediate their biologic effects.     

Penicillin-binding proteins of Streptococcus pneumoniae: characterization of tryptic peptides containing the beta-lactam-binding site

Penicillin-binding proteins of Streptococcus pneumoniae were labeled with [3H] propionyl-ampicillin and treated with trypsin. The fragments were separated on sodium dodecyl sulfate/polyacrylamide gels, and peptides containing the beta-lactam-binding site visualized by fluorography. From native penicillin-binding proteins (PBP), either membrane-bound or solubilized with Triton X-100, relatively stable end products of proteolysis were obtained. The smallest radioactive peptides from PBP 1a (92 kDa), PBP 2b (77 kDa), and PBP 3 (43 kDa ) had sizes of 36.5 kDa, 26 kDa, and 29 kDa, respectively. When the PBP were trypsin treated prior to labeling with the radioactive beta-lactam, these small peptides were still able to bind the antibiotic. Under conditions of limited proteolysis, membrane-bound PBP 2b and PBP 3 were converted into soluble, hydrophilic derivatives after loss of a peptide of only 2 kDa and 1.5 kDa, respectively. These two PBP are therefore anchored in the membrane by a small terminal peptide. In contrast, PBP 1a could be digested to a Mr of 48000 without becoming water-soluble; the only hydrophilic tryptic peptide that could be found was the 36.5 kDa fragment. Therefore, large domains of this PBP seem to be embedded in the membrane.