Calmodulin regulates fodrin susceptibility to cleavage by calcium-dependent protease I

The intracellular calcium-dependent proteolysis of fodrin has been postulated to be central to the regulation of plasticity of the cortical cytoskeleton of many eukaryotic cells. The close proximity of the sites of calmodulin (CaM) binding and calcium-dependent protease I (CDP-I) cleavage in mammalian alpha-fodrin suggested that their action may be linked. In hypotonic and isotonic buffers, CDP-I proteolysis of the beta subunit of fodrin was absolutely dependent upon the presence of active CaM. The stimulation by CaM was inhibited by CaM antagonists. The rate of CDP-I proteolysis of both subunits was enhanced by CaM, while the rate of fodrin proteolysis with other proteases was not influenced by CaM. The increase in the susceptibility of fodrin to CDP-I proteolysis was half-maximal at 80 nM CaM, and maximal at 200 nM CaM. The unusual and differential susceptibility of alpha- and beta-fodrin to proteolysis by CDP-I in the absence of CaM was exploited to investigate the quaternary structure of fodrin in which only the alpha subunit was cleaved. Cleavage of the alpha subunit alone did not destroy the tetrameric form of the molecule, whereas CDP-I cleavage of both subunits rendered the molecule incapable of reforming tetramers. These results provide structural and functional evidence that CaM and CDP-I act synergistically in the regulated proteolysis of fodrin.     

Isolation and characterization of postsynaptic densities from various brain regions: enrichment of different types of postsynaptic densities

Postsynaptic densities (PSDs) have been isolated from cerebral cortex, midbrain, cerebellum, and brain stem by the Triton X-100 method previously used in the isolation of cerebral PSDs (Cohen et al., 1977, J. Cell Biol. 74:181). These PSDs have been compared in protein composition, protein phosphorylation, and morphology. Thin-section electron microscopy revealed that cerebral cortex and midbrain PSDs were identical, being approximately 57 nm thick and composed of apparent aggregates 20-30 nm in diameter. Isolated cerebellar PSDs appeared thinner (33 nm) than cerebral cortex PSDs and lacked the apparent 20- to 30-nm aggregates, but had a latticelike structure. In unidirectional and rotary-shadowed replicas, the cerebrum and midbrain PSDs were circular in shape with a large central perforation or hole in the center of them. Cerebellum PSDs did not have a large perforation, but did have numerous smaller perforations in a lattice like structure. Filaments (6-9 nm) were observed connecting possible 20- to 30-nm aggregates in cerebrum PSDs and were also observed radiating from one side of the PSD. Both cerebral cortex and midbrain PSDs exhibited identical protein patterns on SDS gel electrophoresis. In comparison, cerebellar PSDs (a) lacked the major 51,000 Mr protein, (b) contained two times less calmodulin, and (c) contained a unique protein at 73,000 Mr. Calcium plus calmodulin stimulated the phosphorylation of the 51,000 and 62,000 Mr bands in both cerebral cortex and midbrain PSDs. In cerebellar PSDs, only the 58,000 and 62,000 Mr bands were phosphorylated. In the PSDs from all brain regions, cAMP stimulated the phosphorylation of Protein Ia (73,000 Mr), Protein Ib (68.000 Mr), and a 60,000 Mr protein, although cerebrum and midbrain PSDs contained very much higher levels of phosphorylated protein than did the cerebellum. On the basis of the morphological criteria, it is possible that PSDs isolated from cerebrum and midbrain were derived from the Gray type I, or asymmetric, synapses, whereas cerebellum PSDs were derived from the Gray type II, or symmetric, synapses. Since there is some evidence that the type I synapses are involved in excitatory mechanisms while the type II are involved in inhibitory mechanisms, the role of the PSD and of some of its proteins in these synaptic responses is discussed.     

Function of a calmodulin in postsynaptic densities. III. Calmodulin-binding proteins of the postsynaptic density

A method has been developed for binding calmodulin, radioiodinated by the lactoperoxidase method, to denaturing gels and has been used to attempt to identify the calmodulin-binding proteins of cerebral cortex postsynaptic densities (PSDs). Calmodulin primarily bound to the major 51,000 Mr protein in a saturatable manner; secondarily bound to the 60,000 Mr region, 140,000 Mr region, and 230,000 Mr protein; and bound in lesser amounts to a number of other proteins. The major 51,000 Mr calmodulin-binding protein is one of unknown identity. Binding of iodinated calmodulin to these proteins was blocked by EDTA, EGTA, chlorpromazine, and preincubation with unlabeled calmodulin. Calmodulin iodinated by the chloramine-T method, which inactivates calmodulin did not bind to the PSD but bound nonspecifically to histone. Calmodulin did not bind to proteins from a variety of sources for which calmodulin interactions have not been found. Except for three proteins, all of the proteins of synaptic membranes that bind calmodulin could be accounted for by proteins of the PSD which are a part of the synaptic membrane fraction. The major 51,000 M, protein and the corresponding iodinated calmodulin binding were greatly reduced in cerebellar PSDs and this difference between cerebral cortex and cerebellar PSDs is discussed in light of the possible function of calmodulin in synaptic excitatory responses.     

Fodrin CaM-binding domain cleavage by Pet from enteroaggregative Escherichia coli leads to actin cytoskeletal disruption

We have previously shown that the plasmid-encoded toxin (Pet) of enteroaggregative Escherichia coli produces cytotoxic and enterotoxic effects. Pet-intoxicated epithelial cells reveal contraction of the cytoskeleton and loss of actin stress fibres. Pet effects require its internalization into epithelial cells. We have also shown that Pet degrades erythroid spectrin. Pet delivery within the intestine suggests that Pet may degrade epithelial fodrin (non-erythroid spectrin). Here we demonstrate that Pet has affinity for alpha-fodrin (formally named alphaII spectrin) in vitro and in vivo and cleaves epithelial fodrin, causing its redistribution within the cells. When Pet has produced its cytoskeletal effects, fodrin is found in intracellular aggregates as membrane blebs. Pet cleaves recombinant GST-fodrin, generating two breakdown products of 37 and 72 kDa. Sequencing of the 37 kDa fragment demonstrated that the cleavage site occurred within fodrin's 11th repetitive unit between M1198 and V1199, in the calmodulin binding domain. Site-directed mutagenesis of these amino acids prevented fodrin degradation by Pet. Pet also cleaves epithelial fodrin from cultured Pet-treated cells. A mutant in the Pet serine protease motif was unable to cause fodrin redistribution or to cleave GST-fodrin. This is the first report showing cleavage of alpha-fodrin by a bacterial protease. Cleavage occurs in the middle of the calmodulin binding domain, which leads to cytoskeleton disruption.     

The calmodulin-binding site in alpha-fodrin is near the calcium-dependent protease-I cleavage site

Fodrin (brain spectrin) binds calmodulin and is susceptible to proteolysis by calcium-dependent protease I (CDP-I, calcium-activated neutral protease I, or calpain I). Both events involve the central region of the alpha-fodrin subunit, and calmodulin binding enhances the sensitivity of fodrin to CDP-I mediated proteolysis. Fragments of fodrin, generated chemically or proteolytically, which retain calmodulin binding activity have been identified and analyzed by two-dimensional peptide mapping and by direct protein sequencing. Both CDP-I and calmodulin interact with the terminal portion of the eleventh repetitive unit in fodrin, which is at the center of the molecule. CDP-I cleavage occurs between Tyr104 and Gly105 and preserves the calmodulin binding activity of the carboxyl-terminal fragment. In contrast, chymotryptic cleavage at Trp120 reduces the ability of this fragment to bind calmodulin, and tryptic cleavage beyond Trp120 completely eliminates calmodulin binding activity. It is concluded that Ser-Lys-Thr-Ala-Ser-Pro-Trp-Lys-Ser-Ala-Arg-Leu-Met-Val-His-Thr-Val-Ala- Thr- Phe-Asn-Ser-Ile-Lys, a 24-residue peptide which bridges repeats 11 and 12 of brain alpha spectrin contains the high affinity calmodulin binding domain.     

Involvement of fodrin-binding proteins in the structure of the neuronal postsynaptic density and regulation by phosphorylation

Novel polypeptides with Mr values about 140,000 bind fodrin and spectrin and are enriched in the postsynaptic density (PSD) compared to other tissues or subcellular fractions. 125I-fodrin binding to these polypeptides is competed for by unlabeled spectrin. These polypeptides are distinct from ankyrin and its proteolytic fragments and from band 4.1 which also bind fodrin. Phosphorylation of PSDs by the endogenous calmodulin-dependent protein kinase markedly reduces 125I-fodrin binding to the transblotted preparation. Such an event may play a regulatory role in governing protein-protein interactions among elements of the PSD.     

Similarities between the Mr 245,000 calmodulin-binding protein of the dogfish erythrocyte cytoskeleton and alpha-fodrin

The Mr 245,000 calmodulin-binding protein of the dogfish erythrocyte cytoskeleton (D245) has been compared with human erythrocyte spectrin and mammalian brain fodrin [J. Levine and M. Willard (1981) J. Cell Biol. 90, 631-643]. Mammalian erythrocyte alpha-spectrin, brain alpha-fodrin, and D245 are all localized in the cell surface-associated cytoskeleton, and have similar molecular weights. Like mammalian erythrocyte spectrin, D245 was extracted from erythrocyte ghosts under low-ionic-strength conditions. However, D245 failed to bind an antibody which reacted strongly with both subunits of human erythrocyte spectrin. Unlike mammalian erythrocyte alpha- and beta-spectrin, D245 bound calmodulin in the absence of urea both in a "gel-binding" assay and in situ using azidocalmodulin [D.C. Bartelt, R.K. Carlin, G.A. Scheele, and W.D. Cohen (1982) J. Cell Biol. 95, 278-284]. Striking similarities were noted between D245 and alpha-fodrin in that both exhibited (a) comparable calcium-dependent calmodulin binding properties, (b) strong reactivity with two different anti-fodrin antibody preparations, (c) similar reactivity with antibody to brain CBP-I, now believed to be fodrin, (d) proteolytic degradation yielding an Mr 150,000 calmodulin-binding fragment, and (e) lack of reactivity with an anti-spectrin antibody. A protein with calmodulin-binding and anti-fodrin-binding properties similar to D245 was detected in cytoskeletal preparations of chicken erythrocytes. Moderate and consistent cross-reactivity of anti-fodrin with human erythrocyte alpha-spectrin was also observed. The data indicate that D245 is functionally and immunologically more closely related to alpha-fodrin than to alpha-spectrin of the mammalian erythrocyte.     

Function of calmodulin in postsynaptic densities. II. Presence of a calmodulin- activatable protein kinase activity

Because the calmodulin in postsynaptic densities (PSDs) activates a cyclic nucleotide phosphodiesterase, we decided to explore the possibility that the PSD also contains a calmodulin-activatable protein kinase activity. As seen by autoradiographic analysis of coomassie blue-stained SDS polyacrylamide gels, many proteins in a native PSD preparation were phosphorylated in the presence of [γ-(32)P]ATP and Mg(2+) alone. Addition of Ca(2+) alone to the native PSD preparation had little or no effect on phosphorylation. However, upon addition of exogenous calmodulin there was a general increase in background phosphorylation with a statistically significant increase in the phosphorylation of two protein regions: 51,000 and 62,000 M(r). Similar results were also obtained in sonicated or freeze thawed native PSD preparations by addition of Ca(2+) alone without exogenous calmodulin, indicating that the calmodulin in the PSD can activate the kinase present under certain conditions. The calmodulin dependency of the reaction was further strengthened by the observed inhibition of the calmodulin-activatable phosphorylation, but not of the Mg(2+)-dependent activity, by the Ca(2+) chelator, EGTA, which also removes the calmodulin from the structure (26), and by the binding to calmodulin of the antipsychotic drug chlorpromazine in the presence of Ca(2+). In addition, when a calmodulin-deficient PSD preparation was prepared (26), sonicated, and incubated with [γ-(32)P]ATP, Mg(2+) and Ca(2+), one could not induce a Ca(2+)-stimulation of protein kinase activity unless exogenous calmodulin was added back to the system, indicating a reconstitution of calmodulin into the PSD. We have also attempted to identify the two major phosphorylated proteins. Based on SDS polyacrylamide gel electrophoresis, it appears that the major 51,000 M(r) PSD protein is the one that is phosphorylated and not the 51,000 M(r) component of brain intermediate filaments, which is a known PSD contaminant. In addition, papain digestion of the 51,000 M(r) protein revealed multiple phosphorylation sites different from those phosphorylated by the Mg(2+)-dependent kinase(s). Finally, although the calmodulin-activatable protein kinase may phosphorylate proteins I(a) and I(b), the cyclic AMP-dependent protein kinase, which definitely does phosphorylate protein I(a) and I(b) and is present in the PSD, does not phosphorylate the 51,000 and 62,000 M(r) proteins, because specific inhibition of this kinase has no effect on the levels of the phosphorylation of these latter two proteins.     

Characterization of Na+-Independent GABA and Flunitrazepam Binding Sites in Preparations of Synaptic Membranes and Postsynaptic Densities Isolated from Canine Cerebral Cortex and Cerebellum

The binding of [3H]GABA and [3H]flunitrazepam was performed with synaptic membranes and post-synaptic densities (PSDs) isolated from canine cerebral cortex and cerebellum. Two GABA binding sites were found with cerebral cortex membranes but only one with cerebellar membranes. PSDs isolated from these showed only single binding sites, with cerebellar PSDs exhibiting lower KD values and a larger concentration of sites than did cerebral cortex PSDs. In the case of flunitrazepam, only one binding site was found for all four preparations, with cerebellar PSDs having twice the concentration of sites of cerebral PSDs. Photoaffinity labeling of the flunitrazepam receptor in PSDs resulted in the binding to a 51,000 Mr protein in both cases, with cerebellar PSDs again showing an increased concentration over that found in cerebral cortex PSDs. Based on this work, and on earlier work of ourselves and of others, we conclude that both populations of isolated PSDs contain inhibitory sites, but that the intact PSDs in both preparations are derived from Gray type I, probably excitatory, synapses, and that the inhibitory sites are found in the broken-up material in the PSD fractions which are derived from Gray type II, probably inhibitory, synapses.     

Identification and purification from bovine brain of a guanine-nucleotide-binding protein distinct from Gs, Gi and Go.

A guanine-nucleotide-binding protein (G-protein) was purified from cholate extracts of bovine brain membranes by sequential DEAE-Sephacel, Ultrogel AcA-34, heptylamine-Sepharose and Sephadex G-150 chromatography. Guanosine 5'-[gamma-[35S]thio]triphosphate (GTP[35S])-binding activity copurified with a 25,000 Da peptide and a 35,000-36,000 Da protein doublet. Neither pertussis toxin nor cholera toxin catalysed the ADP-ribosylation of a protein associated with the GTP[35S]-binding activity. Photoaffinity labelling of the purified protein with 8-azido[gamma-32P]GTP indicated that the GTP-binding site resides on the 25,000 Da protein. The 35,000-36,000 Da protein doublet was electrophoretically indistinguishable from the beta-subunits of other GTP-binding proteins, and the 36,000 Da protein was recognized by antiserum to oligomeric Gt. The purified protein specifically bound 17.2 nmol of GTP[35S]/mg of protein. The Kd of the binding site for radioligand was approx. 15 nM. The brain GTP-binding protein co-migrated during SDS/polyacrylamide-gel electrophoresis with a GTP-binding protein, named Gp, purified from human placenta [Evans, Brown, Fraser & Northup (1986) J. Biol. Chem. 261, 7052-7059], and cross-reacted with antiserum raised against the placental protein, but not with antiserum raised to brain Go. SDS/polyacrylamide-gel electrophoresis of the brain and placental GTP-binding proteins in the presence of Staphylococcus aureus V8 protease yielded identical peptide maps.     

Isolation of insecticide resistance-related forms of cytochrome P-450 from Drosophila melanogaster.

Inositol 1,4,5-trisphosphate (InsP3) 3-kinase catalyses the ATP-dependent phosphorylation of InsP3 to inositol 1,3,4,5-tetrakisphosphate (InsP4). InsP3 3-kinase was purified from rat brain by Blue-Sepharose, phosphocellulose and calmodulin (CaM)-Sepharose affinity chromatography. The purified enzyme was stimulated by Ca2+/CaM by 3-6-fold as compared with the activity measured in the presence of EGTA. Rat brain InsP3 3-kinase activity was associated with two silver-stained bands of about equal activity which migrated with an apparent Mr of 50,000 on SDS/polyacrylamide gels. InsP3 3-kinase activity from rat brain could be immunoprecipitated by an antiserum against the SDS/PAGE-purified 50,000-Mr protein doublet. InsP3 kinase activity from bovine brain and the InsP3 5-phosphatase activity from rat brain were not immunoprecipitated. On Western blot, the human brain crude InsP3 3-kinase reacted specifically, but less strongly than the rat brain enzyme, with the antiserum.     

The cytoskeletal system of nucleated erythrocytes. II. presence of a high molecular weight calmodulin-binding protein

Calmodulin was detected in dogfish erythrocyte lysates by means of phosphodiesterase activation. Anucleate dogfish erythrocyte cytoskeletons bound calmodulin. Binding of calmodulin was calcium- dependent, concentration-dependent, and saturable. Cytoskeletons consisted of a marginal band of microtubules containing primarily tubulin, and trans-marginal band material containing actin and spectrinlike proteins. Dogfish erythrocyte ghosts and cytoskeletons were found to contain a calcium-dependent calmodulin-binding protein, CBP, by two independent techniques: (a) 125I-calmodulin binding to cytoskeletal proteins separated by SDS PAGE, and (b) in situ azidocalmodulin binding in whole anucleate ghosts and cytoskeletons. CBP, with an apparent molecular weight of 245,000, co-migrated with the upper band of human and dogfish erythrocyte spectrin. CBP was present in anucleate ghosts devoid of marginal bands and absent from isolated marginal bands. CBP therefore appears to be localized in the trans- marginal band material and not in the marginal band. Similarities between CBP and high molecular weight calmodulin-binding proteins from mammalian species are discussed.     

Purification of a novel calmodulin binding protein from bovine cerebral cortex membranes

A new calmodulin (CaM) binding protein, designated P-57, has been purified to apparent homogeneity from bovine cerebral cortex membranes. In contrast to other calmodulin binding proteins, P-57 has higher affinity for calmodulin in the absence of bound Ca2+ than in its presence. The protein was purified by DEAE-Sephacel chromatography and two CaM-Sepharose affinity column steps. The first CaM-Sepharose column was run in the presence of Ca2+; the second was run in the presence of chelator in excess of Ca2+. P-57 was adsorbed by CaM-Sepharose only in the absence of bound Ca2+ and was eluted from the second column by buffers containing Ca2+. Sodium dodecyl sulfate (SDS)-polyacrylamide gels of the purified protein showed only one band at Mr 57 000. The major form of the protein on Bio-Gel A-1.5m and native polyacrylamide gradient gel electrophoresis ran with an apparent Stokes radius of 41 A. Photoaffinity labeling of P-57 with azido[125I]calmodulin yielded one cross-linked product on SDS gels with an Mr of 70 000. This interaction occurred only when excess ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid was present and was inhibited by the presence of Ca2+ in excess of chelator. It appears that P-57 has novel binding properties for calmodulin distinct from all other calmodulin binding proteins described thus far.     

Binding of brain spectrin to the 70-kDa neurofilament subunit protein

Brain spectrin, or fodrin, a major protein of the subaxolemmal cytoskeleton, associates specifically in in vitro assays with the 70-kDa neurofilament subunit (NF-L) and with glial filaments from pig spinal cord. As an initial approach to the identification of the fodrin-binding proteins, a crude preparation of neurofilaments was resolved by electrophoresis on SDS/polyacrylamide gels and then transferred to nitrocellulose paper, which was 'blotted' with 125I-fodrin. A significant binding of fodrin was observed on polypeptides of 70 kDa, 52 kDa and 20 kDa. These polypeptides were further purified and identified respectively as the NF-L subunit of neurofilaments, the glial fibrillary acidic protein (GFP) and the myelin basic protein. The binding of fodrin to NF-L was reversible and concentration-dependent. The ability of the pure NF-L and GFP to form filaments was used to quantify their association with fodrin. a) The binding of fodrin to reassembled NF-L was saturable with a stoichiometry of 1 mol fodrin bound/50 +/- 10 mol NF-L and an apparent dissociation constant Kd = 4.3 x 10(-7) M. b) The binding involved the N-terminal domain of the polypeptide chain derived from the [2-(2-nitrophenylsulfenyl)-3-methyl-3'-bromoindolenine] cleavage of NF-L. c) Binding occurred optimally at physiological pH (6.8-7.2) and salt concentrations (50 mM). d) Interestingly, calmodulin, a Ca2+-binding protein, which has been shown to bind to fodrin, was found to reinforce the binding of fodrin to the NF-L, at Ca2+ physiological concentrations. The binding of fodrin to pure neurofilaments was not affected by the presence of the 200-kDa (NF-H) and the 160-kDa (NF-M) subunits. The apparent dissociation constant for the binding of fodrin to NF-L in the pure NF was 1.0 x 10(-6) M with 1 mol fodrin bound/80 +/- 10 mol NF-L. Moreover, the binding of fodrin to GFP, demonstrated in blot assays, was confirmed by cosedimentation experiments. The apparent dissociation constant Kd for the fodrin binding was 2.8 x 10(-7) M and the maximum binding was 1 mol fodrin/55 +/- 10 mol GFP.     

Isolation and characterization of two 3-phosphatases that hydrolyze both phosphatidylinositol 3-phosphate and inositol 1,3-bisphosphate.

Inositol-polyphosphate 3-phosphatase catalyzes the hydrolysis of the 3-position phosphate bond of inositol 1,3-bisphosphate (Ins(1,3)P2) to form inositol 1-monophosphate and inorganic phosphate (Bansal, V.S., Inhorn, R.C., and Majerus, P.W. (1987) J. Biol. Chem. 262, 9444-9447). Phosphatidylinositol 3-phosphatase catalyzes the analogous reaction utilizing phosphatidylinositol 3-phosphate (PtdIns(3)P) as substrate to form phosphatidylinositol and inorganic phosphate (Lips, D.L., and Majerus, P.W. (1989) J. Biol. Chem. 264, 19911-19915). We now demonstrate that these enzyme activities are identical. Two forms of the enzyme, designated Type I and II 3-phosphatases, were isolated from rat brain. The Type I 3-phosphatase consisted of a protein doublet that migrated at a relative Mr of 65,000 upon sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The Mr of this isoform upon size-exclusion chromatography was 110,000, suggesting that the native enzyme is a dimer. The Type II enzyme consisted of equal amounts of an Mr = 65,000 doublet and an Mr = 78,000 band upon SDS-polyacrylamide gel electrophoresis. This isoform displayed an Mr upon size-exclusion chromatography of 147,000, indicating that it is a heterodimer. The Type II 3-phosphatase catalyzed the hydrolysis of Ins(1,3)P2 with a catalytic efficiency of one-nineteenth of that measured for the Type I enzyme, whereas PtdIns(3)P was hydrolyzed by the Type II 3-phosphatase at three times the rate measured for the Type I 3-phosphatase. The Mr = 65,000 subunits of the two forms of 3-phosphatase appear to be the same based on co-migration on SDS-polyacrylamide gels and peptide maps generated with Staphylococcus aureus protease V8 and trypsin. The peptide map of the Mr = 78,000 subunit was different from that of the Mr = 65,000 subunits. Thus, we propose that the differing relative specificities of the Type I and II 3-phosphatases for Ins(1,3)P2 and PtdIns(3)P are due to the presence of the Mr = 78,000 subunit of the Type II enzyme.     

Secreted adenylate cyclase of Bordetella pertussis: calmodulin requirements and partial purification of two forms.

The extracellular adenylate cyclase of Bordetella pertussis was partially purified and found to contain high- and low-molecular-weight species. The high-molecular-weight form had a variable molecular weight with a peak at about 700,000. The smaller species had a molecular weight of 60 to 70,000 as determined by gel filtration. The low-molecular-weight form could be derived from the high-molecular-weight species. The high-molecular-weight complex purified from the cellular supernatant was highly stimulated by calmodulin, while the low-molecular-weight enzyme was much less stimulated. Active enzyme could be recovered from sodium dodecyl sulfate (SDS) gels at positions corresponding to molecular weights of about 50,000 and 65,000. Active low-molecular-weight enzyme recovered from SDS gels migrated with a molecular weight of about 50,000, which coincides with a coomassie blue-stained band. However, when both high- and low-molecular weight preparations were analyzed in 8 M urea isoelectrofocusing gels, the enzyme activity recovered did not comigrate with stained protein bands. The enzyme recovered from denaturing isoelectrofocusing or SDS gels was activated by calmodulin, indicating a direct interaction of calmodulin and enzyme. The high-molecular-weight form of the enzyme showed increasing activity with calmodulin concentrations ranging from 0.1 to 500 nM, while the low-molecular-weight form was fully activated by calmodulin at 20 nM. Adenylate cyclase on the surface of living cells was activated by calmodulin in a manner which resembled that found for the high-molecular-weight form.     

Crystallographic analysis of the three-dimensional structure of baboon alpha-lactalbumin at low resolution. Homology with lysozyme.

Previous investigations [Jones, Brown, von Glos & Gaunt (1985) Exp. Cell Res. 156, 31-44] have demonstrated the appearance of a new antigenic determinant (recognized by monoclonal antibody 2D6) on the plasma membrane of rat spermatozoa during post-testicular maturation in the epididymis. Identification of the 2D6 antigen on Western blots from one-dimensional SDS/polyacrylamide gels revealed that it co-migrated with a membrane protein (designated Mr 23,000 antigen) present on testicular and immature germ cells, suggesting that one antigen might be a modified version of the other. In the present work, however, we demonstrate that, although they have similar Mr and are present in soluble and membrane-bound forms, the 2D6 and Mr 23,000 antigens are biochemically and immunologically distinct molecules. The properties of the antigens are described and compared. The Mr 23,000 antigen is present on both testicular and cauda epididymidal spermatozoa, has a pI of 6.1, contains no detectable carbohydrate, is not tissue-specific and is degraded by V8 protease. By contrast, the 2D6 antigen is glycosylated, has a broad pI from 4.5 to 6.1, is tissue- and species-specific and is resistant to digestion with V8 protease. Its role in sperm-egg recognition is discussed.     

Polyclonal and monoclonal antibodies to human lysyl hydroxylase and studies on the molecular heterogeneity of the enzyme.

Human placental lysyl hydroxylase gave two bands in SDS/polyacrylamide-slab-gel electrophoresis: a broad, diffuse, major band corresponding to an apparent Mr of 80,000-85,000, and a sharp minor band with Mr 78,000. Mouse and chick-embryo lysyl hydroxylases gave only the broad, diffuse band, whereas the sharp band could not be detected. Polyclonal antibodies were prepared to the two bands of the human enzyme separately, and monoclonal antibodies were prepared to the whole purified enzyme preparation. Both types of polyclonal antibody inhibited and precipitated the enzyme activity, and both stained the two polypeptide bands in immunoblotting after SDS/polyacrylamide-gel electrophoresis. Only one out of five monoclonal antibodies inhibited the enzyme activity, whereas they all precipitated the activity when studied with antibody coupled to Sepharose. All five monoclonal antibodies stained the whole broad band in immunoblotting, and at least three of them also stained the sharp band. Peptide maps produced from the two polypeptide species by digestion with Staphylococcus aureus V8 protease were highly similar. Experiments with endoglycosidase H demonstrated that the Mr-80,000-85,000 polypeptide contains asparagine-linked carbohydrate units, which are required for maximal lysyl hydroxylase activity. The data suggest that the lysyl hydroxylase dimer consists of only one type of monomer, the heterogeneity of which is due to differences in glycosylation.     

Brain postsynaptic densities: the relationship to glial and neuronal filaments

Preparations of isolated brain postsynaptic densities (PSDs) contain a characteristic set of proteins among which the most prominent has a molecular weight of approximately 50,000. Following the suggestion that this major PSD protein might be related to a similarly sized component of neurofilaments (F. Blomberg et al., 1977, J. Cell Biol., 74:214- 225), we searched for evidence of neurofilament proteins among the PSD polypeptides. This was done with a novel technique for detecting protein antigens in SDS-polyacrylamide gels (immunoblotting) and an antiserum that was selective for neurofilaments in immunohistochemical tests. As a control, an antiserum against glial filament protein (GFAP) was used because antisera against GFAP stain only glial cells in immunohistochemical tests. They would, therefore, not be expected to react with PSDs that occur only in neurons. The results of these experiments suggested that PSDs contain both neuronal and also glial filament proteins at higher concentrations than either synaptic plasma membranes, myelin, or myelinated axons. However, immunoperoxidase staining of histological sections with the same two antisera gave contradictory results, indicating that PSDs in intact brain tissue contain neither neuronal or glial filament proteins. This suggested that the intermediate filament proteins present in isolated PSD preparations were contaminants. To test this possibility, the proteins of isolated brain intermediate filaments were labeled with 125I and added to brain tissue at the start of a subcellular fractionation schedule. The results of this experiment confirmed that both neuronal and glial filament proteins stick selectively to PSDs during the isolation procedure. The stickiness of PSDs for brain cytoplasmic proteins indicates that biochemical analysis of subcellular fractions is insufficient to establish a given protein as a synaptic junctional component. An immunohistochemical localization of PSDs in intact tissue, which has now been achieved for tubulin, phosphoprotein I, and calmodulin, appears to be an essential accessory item of evidence. Our findings also corroborate recent evidence which suggests that isolated preparations of brain intermediate filaments contain both neuronal and glial filaments.     

Calmodulin-binding proteins and calmodulin-regulated enzymes in dog pancreas.

Calmodulin was isolated and purified to homogeneity from dog pancreas. Highly purified subcellular fractions were prepared from dog pancreas by zonal sucrose-density ultracentrifugation and assayed for their ability to bind 125I-calmodulin in vitro. Proteins contained in these fractions were also examined for binding of 125I-calmodulin after their separation by polyacrylamide-gel electrophoresis in SDS. Calmodulin-binding proteins were detected in all subcellular fractions except the zymogen granule and zymogen-granule membrane fractions. One calmodulin-binding protein (Mr 240,000), observed in a washed smooth-microsomal fraction, has properties similar to those of alpha-fodrin. The postribosomal-supernatant fraction contained three prominent calmodulin-binding proteins, with apparent Mr values of 62,000, 50,000 and 40,000. Calmodulin-binding proteins, prepared from a postmicrosomal-supernatant fraction by Ca2+-dependent affinity chromatography on immobilized calmodulin, exhibited calmodulin-dependent phosphodiesterase, protein phosphatase and protein kinase activities. In the presence of Ca2+ and calmodulin, phosphorylation of smooth-muscle myosin light chain and brain synapsin and autophosphorylation of a Mr-50,000 protein were observed. Analysis of the protein composition of the preparation by SDS/polyacrylamide-gel electrophoresis revealed a major protein of Mr 50,000 which bound 125I-calmodulin. This protein shares characteristics with the calmodulin-dependent multifunctional protein kinase (kinase II) recently observed to have a widespread distribution. The possible role of calmodulin-binding proteins and calmodulin-regulated enzymes in the regulation of exocrine pancreatic protein synthesis and secretion is discussed.