Effects of mutations of conserved Lys-155 and Thr-156 residues in the phosphate-binding glycine-rich sequence of the F1-ATPase beta subunit of Escherichia coli.

beta Lys-155 in the glycine-rich sequence of the beta subunit of Escherichia coli F1-ATPase has been shown to be near the gamma-phosphate moiety of ATP by affinity labeling (Ida, K., Noumi, T., Maeda, M., Fukui, T., and Futai, M. (1991) J. Biol. Chem. 266, 5424-5429). For examination of the roles of beta Lys-155 and beta Thr-156, mutants (beta Lys-155-->Ala, Ser, or Thr; beta Thr-156-->Ala, Cys, Asp, or Ser; beta Lys-155/beta Thr-156-->beta Thr-155/beta Lys-156; and beta Thr-156/beta Val-157-->beta Ala-156/beta Thr-157) were constructed, and their properties were studied extensively. The beta Ser-156 mutant was active in ATP synthesis and had approximately 1.5-fold higher membrane ATPase activity than the wild type. Other mutants were defective in ATP synthesis, had < 0.1% of the membrane ATPase activity of the wild type, and showed no ATP-dependent formation of an electrochemical proton gradient. The mutants had essentially the same amounts of F1 in their membranes as the wild type. Purified mutant enzymes (beta Ala-155, beta Ser-155, beta Ala-156, and beta Cys-156) showed low rates of multisite (< 0.02% of the wild type) and unisite (< 1.5% of the wild type) catalyses. The k1 values of the mutant enzymes for unisite catalysis were lower than that of the wild type: not detectable with the beta Ala-156 and beta Cys-156 enzymes and 10(2)-fold lower with the beta Ala-155 and beta Ser-155 enzymes. The beta Thr-156-->Ala or Cys enzyme showed an altered response to Mg2+, suggesting that beta Thr-156 may be closely related to Mg2+ binding. These results suggest that beta Lys-155 and beta Thr-156 are essential for catalysis and are possibly located in the catalytic site, although beta Thr-156 could be replaced by a serine residue.


Casein kinase II and the tumor suppressor protein P53 associate in a molecular complex that is negatively regulated upon P53 phosphorylation.

Selective immunoisolation of P53 from Sf9 cells coexpressing wild-type P53 and casein kinase II yielded a preparation containing casein kinase II, thus suggesting that the two proteins may associate in a molecular complex in the intact cell. Such a complex could indeed be demonstrated in vitro between purified recombinant P53 and oligomeric casein kinase II and was shown to dissociate when P53 became phosphorylated by the kinase. This suggested that the P53 C-terminal domain, which contains the casein kinase II phosphorylation site was involved in the protein-protein interaction; this was confirmed by the fact that an anti-P53 monoclonal antibody directed to that domain inhibited the P53-casein kinase II association. Studies with isolated recombinant casein kinase II subunits disclosed that although the alpha (catalytic) subunit could phosphorylate P53, the formation of a stable P53-casein kinase II association required the presence of the beta subunit of the kinase. This was confirmed by immunoisolation of a P53-beta subunit complex from cells expressing both polypeptides. Although the biological significance of a reversible P53-casein kinase II molecular complex in the control of cell proliferation processes remains to be defined, these observations suggest the possibility of a novel mechanism regulating P53 and casein kinase II activities in the intact cell.


Identification of a cis-acting element that enhances the pigment cell-specific expression of the human tyrosinase gene.

To identify the cis-acting element that is responsible for the pigment cell-specific expression of the human tyrosinase gene, we analyzed the promoter activity of its 5'-flanking region by transient expression assays. The fusion genes were constructed by inserting the 5'-flanking region of the human tyrosinase gene upstream from the firefly luciferase gene and were introduced into human melanoma cells and HeLa cells. We thus found the element, located between 2.7 and 1.8 kilobase pairs upstream from the transcription initiation site, that enhances the transient expression of the luciferase reporter gene in melanoma cells, but not in HeLa cells, the tyrosinase gene expression of which is not detectable. Using the fusion genes containing putative enhancer elements under the control of the heterologous simian virus 40 promoter, we identified the pigment cell-specific enhancer of approximately 200 base pairs (bp) between -2.0 and -1.8 kilobase pairs and localized the core sequence to a 39-bp region. This 39-bp core element was then confirmed to direct the melanoma cell-specific expression of the reporter gene under the tyrosinase gene promoter. We thus propose that this core element is responsible for the pigment cell-specific expression of the human tyrosinase gene.


CDC43 and RAM2 encode the polypeptide subunits of a yeast type I protein geranylgeranyltransferase.

The question regarding the identity of the alpha and beta subunits of the yeast type I protein geranylgeranyltransferase was explored using prokaryotic expression of candidate genes. The Saccharomyces cerevisiae CDC43 and RAM2 genes were expressed in Escherichia coli and cell extracts examined for the ability to transfer [3H]geranylgeranyl diphosphate to an appropriate CaaX protein substrate. Individual expression of each gene yielded no activity; however, co-expression of the two genes resulted in high levels of [3H] geranylgeranyl incorporation into the substrate protein Ras-Cys-Val-Val-Leu. The activity was partially purified yielding approximately 12,600 units/liter. The partially purified enzyme geranylgeranylated the Ras-Cys-Val-Val-Leu, Ras-Cys-Ala-Ile-Leu, Ras-Cys-Ile-Ile-Leu, and Ras-Cys-Thr-Ile-Leu substrates but not the Ras-Cys-Val-Leu-Ser or Ras-Ser-Val-Leu-Ser substrates. The protein geranylgeranyltransferase was highly specific for geranylgeranyl diphosphate and poorly transferred farnesyl. The recombinant enzyme was indistinguishable from the native type I geranylgeranyltransferase in yeast extracts. As has been reported for the protein farnesyltransferase, the yeast type I protein geranylgeranyltransferase is also a magnesium-requiring, zinc metalloenzyme. Interestingly, the recombinant enzyme functioned with calcium as the only divalent cation, although addition of zinc increased calcium-dependent activity 2-fold.


Phosphatidylethanolamine:dolichol acyltransferase. Characterization and partial purification of a novel rat liver enzyme.

Incubation of rat or human post-heparin plasma with [3H]dolichol incorporated in liposomes consisting of dioleoyl phosphatidylcholine:dioleoyl phosphatidylethanolamine (3:1) resulted in the formation of radioactive dolichyl oleate. Non-heparinized plasma did not esterify dolichol, and, hence, the enzyme involved is probably associated with the cell surface and released into the blood by heparin. The major location of this activity was the liver, and, therefore, a partial purification of the enzyme from heparinized rat liver perfusates was performed using DEAE-Sephacel and heparin-Sepharose chromatography. The dolichol acyltransferase activity copurified with hepatic lipase activity in a lipid-protein complex of 350 kDa. Optimal acylation is achieved at pH 7.5 in the presence of 5% plasma and 20 mM Ca2+. Esterification can only be obtained when dolichol is present in a phospholipid bilayer, and the reaction is strongly stimulated by unsaturated phosphatidylethanolamine or phosphatidylserine. Radiolabeling experiments demonstrated that the primary acyl donor is phosphatidylethanolamine from which the fatty acid is transferred exclusively from position 1. Neither cholesterol nor retinol are esterified by the enzyme, and the reaction is not stimulated by acyl-CoA. Both the extracellular localization and the mechanism of transacylation clearly distinguish this new enzyme from the acyl-CoA:dolichol acyltransferase described earlier in microsomes.


Site-directed mutagenesis at the active site of Escherichia coli TEM-1 beta-lactamase. Suicide inhibitor-resistant mutants reveal the role of arginine 244 and methionine 69 in catalysis.

Arginine 244 is a highly conserved residue in Class A beta-lactamases, while methionine 69 is not. Informational suppression experiments show that replacement of M69 by a leucine, or that of R244 by most other amino acids lead to clavulanic acid-resistant phenotypes. The arginyl 244 side chain is tightly held in a network of interactions within the active site. Its replacement by a glutamine or a threonine perturbs the enzyme kinetics but to a smaller extent than would have been predicted if it were directly involved in substrate binding. Clavulanic acid and sulbactam still interact specifically with the mutant enzymes but are much less efficiently metabolized. Substitutions at position 244 also unveil interactions between the C6 substituent of substrates and the Asn132/Glu104 region of the active site. Methionine 69 is located in a region of strong structural constraints and presents an unusual conformation. Molecular dynamics simulation showed that its replacement by a leucine does not release the strain in this area and induces only minor structural changes. Accordingly, the kinetic behavior of the mutant is only marginally perturbed, except for suicide inhibitors. Both clavulanic acid and sulbactam are well degraded by the mutant enzyme, while irreversible inactivation is dramatically decreased. The contribution of both residues to catalysis is discussed in the light of the kinetic and structural data.


Isolation and partial characterization of lumican and decorin from adult chicken corneas. A keratan sulfate-containing isoform of decorin is developmentally regulated.

The proteoglycans extracted from adult chicken were initially purified by DEAE-chromatography. Digestion of these proteoglycans with chondroitinase ABC generated a single 40-kDa core protein while digestion with keratanase generated a single 52-kDa core protein. Digestion with both enzymes combined, however, increased the amount of 40-kDa core protein produced. This suggested that the 40-kDa core protein exists with chondroitin/dermatan sulfate (C/DS) side chains alone and with both C/DS and keratan sulfate (KS) side chains. The proteoglycan fraction was initially digested with chondroitinase ABC, and the M(r) = 40,000 core protein derived from proteoglycans containing C/DS side chains alone was isolated. Amino-terminal sequencing showed it to be the chick cognate of decorin. The remaining proteoglycans were then digested with keratanase, and both the 40-kDa core protein and the 52-kDa core proteins derived from KS-containing proteoglycans were purified. The M(r) = 40,000 core protein derived from proteoglycans containing both C/DS and KS side chains had the same amino-terminal sequence as decorin and cross-reacted with antibodies to decorin. Sequence from the 52-kDa core protein derived from KS-containing proteoglycans showed it to be lumican. The results of this study suggest that adult chick corneas contain two isoforms of decorin: one containing C/DS side chains and the other, a hybrid, containing both C/DS and KS side chains. Embryonic corneas did not contain the hybrid isoform of decorin. These results suggest that different post-translational modifications occur to the decorin gene product during corneal development and maturation.


Biochemical basis of the constitutive repressor cleavage activity of recA730 protein. A comparison to recA441 and recA803 proteins.

The recA730 mutation results in constitutive SOS and prophage induction. We examined biochemical properties of recA730 protein in an effort to explain the constitutive activity observed in recA730 strains. We find that recA730 protein is more proficient than the wild-type recA protein in the competition with single-stranded DNA binding protein (SSB protein) for single-stranded DNA (ssDNA) binding sites. Because an increased aptitude in the competition with SSB protein has been previously reported for recA441 protein and recA803 protein, we directly compared their in vitro activities with those of recA730 protein. At low magnesium ion concentration, both ATP hydrolysis and lexA protein cleavage experiments demonstrate that these recA proteins displace SSB protein from ssDNA in a manner consistent with their in vivo repressor cleavage activity, i.e. recA730 protein > recA441 protein > recA803 protein > recAwt protein. Additionally, a correlation exists between the proficiency of the recA proteins in SSB protein displacement and their rate of association with ssDNA. We propose that an increased rate of association with ssDNA allows recA730 protein to displace SSB protein from the ssDNA that occurs naturally in Escherichia coli and thereby to become activated for the repressor cleavage that leads to SOS induction. RecA441 protein is similarly activated for repressor cleavage; however, in this case, significant SSB protein displacement occurs only at elevated temperature. At physiological magnesium ion concentration, we argue that recA803 protein and wild-type recA protein do not displace sufficient SSB protein from ssDNA to constitutively induce the SOS response.


Accumulation of a light-harvesting chlorophyll a/b protein in the chloroplast grana lamellae. The lateral migration of the membrane protein precursor is independent of its processing.

The events that follow the import of pLHCPIIb, the apoprotein precursor of the major light-harvesting complex of photosystem II, were studied in intact pea chloroplasts. The distribution of the events of insertion into the membrane, and processing, to yield the mature form (LHCP) between stromal and granal lamellae regions of the thylakoids were followed. pLHCP was preferentially inserted into stromal lamellae (SL) from which it migrated to granal lamellae (GL). Migration occurred before or after processing, suggesting that migration and processing are independent of each other. When migration was slowed down, LHCP accumulated in SL. Prolonged inhibition of migration induced degradation of LHCP that had accumulated in SL, whereas inhibition of processing did not affect the migration of pLHCP into GL. A small difference in electrophoretic mobility was noted between LHCP in SL and in GL. The predominant mature form in SL migrated more slowly than LHCP from GL. When thylakoids were subjected to trypsin, all of the LHCP embedded in SL underwent cleavage, whereas up to 60% of the radioactive LHCP in GL was resistant to the enzyme. The possible implications of the differences in size and in the sensitivity to trypsin of LHCP are discussed.


Degradation of secretory immunoglobulin M in B lymphocytes occurs in a postendoplasmic reticulum compartment and is mediated by a cysteine protease.

In 38C B lymphocytes, membrane IgM is expressed on the surface, whereas secretory IgM (sIgM) is rapidly degraded. Here, we localize this degradation and characterize the proteases involved in this process. Upon treatment with brefeldin A, degradation of sIgM in 38C cells was strongly inhibited, as was secretion from the sIgM-secreting D2 hybridoma. Moreover, the brefeldin A-induced Golgi resorption resulted in galactosylation of sIgM and partial resistance to endoglycosidase H. However, sIgM avoided degradation neither due to modified terminal glycosylation nor as a consequence of the brefeldin A-induced altered milieu of the endoplasmic reticulum. When these modifications were prevented by inhibiting retrograde transport with nocodazole or by abrogating terminal glycosylation with swainsonine, sIgM was still rescued from degradation. The unaffected breakdown in the presence of nocodazole also argued against recycling of sIgM to be degraded in the endoplasmic reticulum. Furthermore, upon removal of brefeldin A, degradation of galactosylated sIgM resumed in 38C cells, as did secretion from D2 cells. These results indicate that functional export of proteins from the endoplasmic reticulum is a prerequisite for sIgM degradation. Biochemical characterization of this novel postendoplasmic reticulum/pre-trans-Golgi proteolytic pathway included application of inhibitors to a broad spectrum of proteases. Among the compounds tested, only calpain inhibitor I exerted strong inhibition. The involvement of cysteine protease(s) in the degradation of sIgM was corroborated by the inhibitory effect of diamide. We conclude that B lymphocytes avoid secretion by active and selective targeting of sIgM to a developmentally regulated postendoplasmic reticulum degradation pathway in which degradation is mediated by a cysteine protease.


Purification and characterization of the periplasmic lysine-, arginine-, ornithine-binding protein (LAO) from Salmonella typhimurium.

The lysine-, arginine-, ornithine-binding protein (LAO) from Salmonella typhimurium has been purified to homogeneity and characterized. The dissociation constants (KD) were determined by equilibrium dialysis assay to be 14, 15, and 29 nM for L-arginine, L-lysine, and L-ornithine respectively. L-Histidine was found to be a relatively good ligand (KD, 500 nM). Methods have been developed for the separation of liganded from unliganded LAO, for the estimation of bound ligand, and for unliganding LAO. Liganded and unliganded LAO are shown to have distinct UV spectra. The UV spectrum also varies with the nature of the substrate. Inhibition studies with substrate analogs yielded information useful for understanding the nature of the ligand-binding pocket.


Influence of membrane potential changes on cytoplasmic Ca2+ concentration in an electrically excitable cell, the insulin-secreting pancreatic B-cell.

Glucose stimulation of insulin release involves metabolism of the sugar and elevation of cytoplasmic calcium (Ca2+i) in pancreatic B-cells. We compared the dynamic changes of metabolism (fluorescence of endogenous reduced pyridine nucleotides, NAD(P)H), membrane potential (intracellular microelectrodes), and Ca2+i (fura-2 technique), in intact mouse islets. Glucose (15 mM) sequentially triggered an increase in NAD(P)H fluorescence, a depolarization with electrical activity, and a rise in Ca2+i. The change in NAD(P)H was monophasic and regular, whereas the changes in membrane potential and Ca2+i were multiphasic, with steady-state regular oscillations of similar average frequencies (about 2.2/min). Digital image analysis revealed that Ca2+i oscillations were synchronous in all regions of the islets. Omission of extracellular Ca2+ abolished the rise in Ca2+i but not the increase in NAD(P)H. Both electrical and Ca2+i oscillations disappeared in low external Ca2+ (1 mM), and became larger but slower in high Ca2+ (10 mM). Sustained depolarization (by tolbutamide, arginine, or high K+) and hyperpolarization (by diazoxide) of B-cells caused sustained increases and decreases of Ca2+i, respectively. In conclusion, the changes in membrane potential induced by various secretagogues trigger synchronous changes in Ca2+i in all B-cells of the islets. The oscillatory pattern of the electrical and Ca2+i responses induced by glucose is not accompanied by and thus probably not due to similar oscillations of metabolism.


Angiotensin II stimulates glucose transport activity in cultured vascular smooth muscle cells.

The glucose transport system in cultured rat vascular smooth muscle cells has been examined by measuring the uptake of 2-deoxyglucose. Angiotensin II (Ang II) stimulated 2-deoxyglucose uptake in cells made quiescent by removing serum from the culture medium in a dose- and time-dependent manner that was shown to be receptor-mediated. Epidermal growth factor (EGF), fetal calf serum, thrombin, and arginine vasopressin also stimulated glucose transport. Cycloheximide did not affect the immediate-early (30 min) activation by either Ang II or EGF, but abolished any further increase. This suggested that, whereas the initial activation of glucose transport was independent of protein synthesis, the sustained increase required the synthesis of new glucose transporters. This was supported by 4-fold and 2-fold accumulations of GLUT-1 mRNA 4 h after exposure to Ang II and EGF, respectively. The induction of GLUT-1 mRNA was preceded by rapid and transient expression of c-fos and c-jun protooncogenes. In nuclear run-on assays, nuclei from Ang II-treated cells showed increased synthesis of GLUT-1 mRNA at 30 min and 1 h after hormone treatment. In contrast, in cells exposed to actinomycin D, pretreatment with Ang II had no effect on the turnover rates of GLUT-1 mRNa. These results are consistent with Ang II acting to stimulate the rate of transcription of the GLUT-1 gene leading to increased production of GLUT-1 protein and glucose transport.


Nitrilase from Rhodococcus rhodochrous J1. Sequencing and overexpression of the gene and identification of an essential cysteine residue.

The amino acid sequences of the NH2 terminus and internal peptide fragments of a Rhodococcus rhodochrous J1 nitrilase were determined to prepare synthetic oligonucleotides as primers for the polymerase chain reaction. A 750-base DNA fragment thus amplified was used as the probe to clone a 5.4-kilobase PstI fragment coding for the whole nitrilase. The nitrilase gene modified in the sequence upstream from the presumed ATG start codon was expressed to approximately 50% of the total soluble protein in Escherichia coli. The predicted amino acid sequence of the nitrilase gene showed similarity to that of the bromoxynil nitrilase from Klebsiella ozaenae. The 5,5'-dithiobis(2-nitrobenzoic acid) modification of the nitrilase from R. rhodochrous J1 resulted in inactivation with the loss of one sulfhydryl group/enzyme subunit. Of 4 cysteine residues in the Rhodococcus nitrilase, only Cys-165 is conserved in the Klebsiella nitrilase. Mutant enzymes containing Ala or Ser instead of Cys-165 did not exhibit nitrilase activity. These findings suggest that Cys-165 plays an essential role in the function of the active site.


Regulation of collagenase gene expression by serotonin and progesterone in rat uterine smooth muscle cells.

The regulation of collagenase gene expression by serotonin and progesterone was investigated in primary cultures of rat uterine smooth muscle cells. Northern blot analysis demonstrates that serotonin (5-hydroxytryptamine (5-HT)), when administered to cells in serotonin-depleted medium, causes 6-8-fold increases in levels of collagenase mRNA. Selective serotonin 5-HT2 receptor agonists were able to mimic the effect of the natural hormone, while the induction by serotonin could be blocked by 5-HT2 receptor antagonists. Addition of phorbol ester (PMA) to 5-HT-depleted cultures fully mimicked the effect of 5-HT on collagenase mRNA induction. Treatment with progesterone analogs caused a decrease in collagenase mRNA, even in the presence of saturating levels of serotonin or PMA. In all experiments, levels of secreted collagenase were observed to correspond to levels of collagenase mRNA. Experiments with cycloheximide demonstrate that serotonin- and PMA-induced increases in collagenase mRNA are dependent on protein synthesis. Furthermore, nuclear run-on analysis shows that mRNA increases are accompanied by increases in initiation of transcripts. These data indicate that transcription of collagenase mRNA in myometrial smooth muscle cells is stimulated by serotonin, possibly via activation of protein kinase C, but is in some way prevented by the negative influence of progesterone.


Possible salt bridges between transmembrane alpha-helices of the lactose carrier of Escherichia coli.

Although it is energetically extremely unfavorable to have charged amino acid residues of a polypeptide in the hydrophobic environment of the membrane phospholipid bilayer, a few such charged residues are found in membrane-spanning regions of membrane proteins. Ion pairs (salt bridges) would be much more stable in low dielectric media than single ionized residues. This paper provides indirect evidence for a salt bridge between Asp-240 and Lys-319 in the lactose carrier of Escherichia coli. When Asp-240 was changed to alanine by site-directed mutagenesis, there was a loss of the ability to accumulate methyl-beta-D-thiogalactopyranoside (TMG), melibiose, or lactose. Fast-growing revertants were isolated on melibiose minimal agar plates. Two second-site revertants were isolated: Asp-240-->Ala plus Gly-268-->Val and Asp-240-->Ala plus Lys-319-->Gln. These revertants showed extremely poor accumulation of TMG, melibiose, and lactose, but showed significant "downhill" lactose entry into beta-galactosidase-containing cells with sugar concentrations of 2 and 5 mM. It is concluded that there is some important interaction between Asp-240 and Lys-319, possibly a salt bridge.


Regulation of iron uptake in Saccharomyces cerevisiae. The ferrireductase and Fe(II) transporter are regulated independently.

Iron is required for the growth of Saccharomyces cerevisiae. High concentrations of iron, however, are toxic, forcing this yeast to tightly regulate its concentration of intracellular free iron. We demonstrate that S. cerevisiae accumulates iron through the combined action of a plasma membrane ferrireductase and an Fe(II) transporter. This transporter is highly selective for Fe(II). Several other transition metals did not inhibit iron uptake when these metals were present at a concentration 100-fold higher than the Km (0.15 microM) for iron transport. Pt(II) inhibited ferrireductase activity but not the ability of cells to transport iron that was chemically reduced to Fe(II). Incubation of cells in a synthetic iron-limited media resulted in the induction of both ferrireductase and Fe(II) transporter activities. In complex media, Fe(II) transport activity was regulated in response to media iron concentration, while the activity of the ferrireductase was not. When stationary phase cells were inoculated into fresh media, ferrireductase activity increased independent of the iron content of the media; in contrast, transporter activity varied inversely with iron levels. These results demonstrate that the ferrireductase and Fe(II) transporter are separately regulated and that iron accumulation may be limited by changes in either activity.


Protein changes associated with reprotonation of the Schiff base in the photocycle of Asp96-->Asn bacteriorhodopsin. The MN intermediate with unprotonated Schiff base but N-like protein structure.

The difference Fourier transform infrared spectrum for the N intermediate in the photoreaction of the light-adapted form of bacteriorhodopsin can be recorded at pH 10 at 274 K (Pfefferlé, J.-M., Maeda, A., Sasaki, J., and Yoshizawa, T. (1991) Biochemistry 30, 6548-6556). Under these conditions, Asp96-->Asn bacteriorhodopsin gives a photoproduct which shows changes in protein structure similar to those observed in N of wild-type bacteriorhodopsin. However, decreased intensity of the chromophore bands and the single absorbance maximum at about 400 nm indicate that the Schiff base is unprotonated, as in the M intermediate. This photoproduct was named MN. At pH 7, where the supply of proton is not as restricted as at pH 10, Asp96-->Asn bacteriorhodopsin yields N with a protonated Schiff base. The Asn96 residue, which cannot deprotonate as Asp96 in wild-type bacteriorhodopsin, is perturbed upon formation of both MN at pH 10 and N at pH 7. We suggest that the reprotonation of the Schiff base is preceded by a large change in the protein structure including perturbation of the residue at position 96.


Stimulation and inhibition of human platelet adenylylcyclase by thiophosphorylated transducin beta gamma-subunits.

The effect of beta gamma-dimers isolated from the retinal guanine nucleotide-binding protein (G protein) transducin eluted from illuminated bovine rod outer segment membranes with GTP, guanosine 5'-O-(beta, gamma-imino)triphosphate (Gpp(NH)p), or guanosine 5'-O-(gamma-thio)triphosphate (GTP gamma S) on basal and forskolin-stimulated adenylylcyclase activities in membranes of human platelets was studied. beta gamma-Subunits isolated from transducin eluted with GTP gamma S (TD beta gamma GTP gamma S) had a concentration-dependent stimulatory effect on basal adenylylcyclase activity. The stimulatory agonist prostaglandin E1 increased the potency and the maximum extent of stimulation due to TD beta gamma GTP gamma S). With a similar concentration dependence, TD beta gamma GTP gamma S exerted an inhibitory influence on forskolin-stimulated adenylylcyclase activity. At the same concentrations, beta gamma-dimers isolated with either GTP or Gpp(NH)p did not alter enzyme activities. The observed effects of TD beta gamma GTP gamma S were similar to those of directly added GTP gamma S with regard to maximum levels, time dependence, and persistence; however, TD beta gamma GTP gamma S was approximately 10-fold more potent than GTP gamma S. Treatment of TD beta gamma GTP gamma S, but not of free GTP gamma S, with hydroxylamine caused a loss of adenylylcyclase regulation by TD beta gamma GTP gamma S. The data presented indicated that TD beta gamma GTP gamma S potently and efficiently activates the stimulatory and inhibitory G proteins of adenylylcyclase in human platelet membranes. Furthermore, evidence is provided suggesting that the observed effects of TD beta gamma GTP gamma S, which can be thiophosphorylated by GTP gamma S at the beta-subunit (Wieland, T., Ulibarri, I., Gierschik, P., and Jakobs, K. H. (1991) Eur. J. Biochem. 196, 707-716), are due to formation of GTP gamma S at the G proteins.


Cloning and characterization of the major promoter of the human protein kinase C beta gene. Regulation by phorbol esters.

The expression of the beta isoenzyme for protein kinase C is regulated developmentally and in response to inducers of cell differentiation (such as phorbol esters and 1 alpha,25-dihydroxyvitamin D3). The 5' segment of the gene for protein kinase C beta was cloned from a human leukocyte genomic library in EMBL3 bacteriophage. This segment of the gene (greater than 54 kilobases in length) encompassed the coding sequence for the amino-terminal regulatory domain of the enzyme, the 5'-untranslated region, and the 5'-flanking region. Initiation of transcription was identified by S1 nuclease analysis and confirmed by RNase protection analysis at 197 base pairs 5' of the initiator ATG. Sequence analysis of the 5'-flanking region revealed it to be extremely G+C-rich (> 80%) with many features of a CpG island. Comparison of sequence with known cis-regulatory motifs disclosed a number of potential regulatory elements including an octamer binding motif at -76, Sp1-binding sites at -94 and -63, E boxes at -110, -26, and +18, an AP-1 site at -442, and an AP-2 site at -330. To demonstrate promoter activity, a 630-base pair fragment extending from -587 to +43 was subcloned in front of a promoterless luciferase gene. This fragment was able to drive the expression of luciferase in transient transfections of human hematopoietic cells. Deletion analysis demonstrated that a fragment -111 to +43 was necessary and sufficient for promoter activity; this fragment did not contain TATA or CAAT motifs. The promoter was stimulated 8-20-fold by phorbol esters accounting for the previously observed transcriptional activation of protein kinase C beta. This phorbol ester responsiveness was conferred by the basal promoter (-111 to +43) and was independent of the AP-1 site. These results define a novel mechanism of protein kinase C autoregulation at a transcriptional level.


Role of essential sulfhydryl groups in drug interactions at the neuronal 5-HT transporter. Differences between amphetamines and 5-HT uptake inhibitors.

The sulfhydryl-selective alkylating agent, N-ethylmaleimide (NEM), has been used as a tool to discern whether different binding domains exist on the neuronal serotonin (5-HT) transporter for 5-HT and 5-HT uptake inhibitors (Reith, M. E. A., Allen, D. L., Sershen, H., and Lajtha, A. (1984) J. Neurochem. 43, 249-255; Graham, D., Esnaud, H., Habert, E., and Langer, S. Z. (1989) Biochem. Pharmacol. 38, 3819-3826). However, relatively high concentrations of NEM and long incubation times have been required for inactivation of the transporter-binding site which raises the possibility that NEM is reacting with other nucleophilic groups (Smyth, D. G., Blumenfeld, O. O., and Konigsberg, W. (1964) Biochem. J. 91, 589-595). In the present work, the reactivity and essential nature of sulfhydryl groups associated with substrate/inhibitor binding to the neuronal 5-HT transporter was assessed. [3H]Paroxetine, a potent and selective 5-HT uptake inhibitor, was used to label the 5-HT transporter. The effects of a relatively wide range of sulfhydryl reagents on [3H]paroxetine binding in digitoninsolubilized preparations of rat brain neuronal membranes and the relative abilities of different classes of drugs to protect against NEM-induced inactivation of [3H]paroxetine binding were studied. It was observed that digitonin-solubilized preparations were more sensitive than membrane preparations to the inactivating effects of NEM. The pKa of the reactive group was estimated to be 6.17, in the range expected for a reactive sulfhydryl. Sulfhydryls essential to ligand binding reacted preferentially with hydrophobic compounds (p-hydroxymercuribenzoate = dithiobisnitrobenzoate > methyl methanethiosulfonate > N-phenylmaleimide > N-ethylmaleimide) and were unreactive toward hydrophilic reagents such as iodoacetate and iodoacetamide. 5-HT, 5-HT uptake inhibitors and cocaine protected the digitonin-solubilized transporter from NEM-induced inactivation while the amphetamine-related releasing agents p-chloroamphetamine and fenfluramine were ineffective. The observation that the binding of some, but not all, ligands requires reduced sulfhydryl groups, suggests that differential mechanisms and/or different binding domains do exist for agents which interact at the neuronal 5-HT transporter.


F0F1-ATPase gamma subunit mutations perturb the coupling between catalysis and transport.

We introduced mutations to test the function of the conserved amino-terminal region of the gamma subunit from the Escherichia coli ATP synthase (F0F1-ATPase). Plasmid-borne mutant genes were expressed in an uncG strain which is deficient for the gamma subunit (gamma Gln-14-->end). Most of the changes, which were between gamma Ile-19 and gamma Lys-33, gamma Asp-83 and gamma Cys-87, or at gamma Asp-165, had little effect on growth by oxidative phosphorylation, membrane ATPase activity, or H+ pumping. Notable exceptions were gamma Met-23-->Arg or Lys mutations. Strains carrying these mutations grew only very slowly by oxidative phosphorylation. Membranes prepared from the strains had substantial levels of ATPase activity, 100% compared with wild type for gamma Arg-23 and 65% for gamma Lys-23, but formed only 32 and 17%, respectively, of the electrochemical gradient of protons. In contrast, other mutant enzymes with similar ATPase activities (including gamma Met-23-->Asp or Glu) formed H+ gradients like the wild type. Membranes from the gamma Arg-23 and gamma Lys-23 mutants were not passively leaky to protons and had functional F0 sectors. These results suggested that substitution by positively charged side chains at position 23 perturbed the energy coupling. The catalytic sites of the mutant enzymes were still regulated by the electrochemical H+ gradient but were inefficiently coupled to H+ translocation in both ATP-dependent H+ pumping and delta mu H+ driven ATP synthesis.


Intraluminal Ca2+ dependence of Ca2+ and ryanodine-mediated regulation of skeletal muscle sarcoplasmic reticulum Ca2+ release.

The action of ryanodine upon sarcoplasmic reticulum (SR) Ca2+ handling is controversial with evidence for both activation and inhibition of SR Ca2+ release. In this study, the role of the intraluminal SR Ca2+ load was probed as a potential regulator of ryanodine-mediated effects upon SR Ca2+ release. Through dual-wavelength spectroscopy of Ca2+:antipyrylazo III difference absorbance, the intraluminal Ca2+ dependence of ryanodine and Ca(2+)-induced Ca2+ release (CICR) from skeletal SR vesicles was examined. Ryanodine addition after initiation of Ca2+ uptake (a) increased the intraluminal Ca2+ sensitivity of CICR and (b) stimulated spontaneous Ca2+ release with a delayed onset. These ryanodine effects were inversely proportional to the intraluminal Ca2+ load. Ryanodine also inhibited subsequent CICR after reaccumulation of Ca2+ released from the initial CICR. These results provide evidence that ryanodine inhibits transitions between low and high affinity Ca2+ binding states of an intraluminal Ca2+ compartment, possibly calsequestrin. Conformational transitions of calsequestrin may be reciprocally coupled to transitions between open and closed states of the Ca2+ release channel.


Calcium-activated neutral protease effects upon skeletal muscle sarcoplasmic reticulum protein structure and calcium release.

In this study, the effects of Ca(2+)-activated neutral protease (CANP) upon skeletal muscle heavy sarcoplasmic reticulum (HSR) structure and function were investigated. CANP was immunolocalized to the 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid detergent-insoluble fraction of purified HSR membranes. Ca2+ activation of the endogenous membrane-bound CANP produced a characteristic partial fragmentation of the HSR 565-kDa Ca2+ release channel. Similarly, the major substrate for both micromolar and millimolar Ca(2+)-sensitive isoforms of exogenous CANP was the Ca2+ release channel with proteolysis of a 88-kDa HSR protein also observed. Ca2+ release channel proteolysis was initiated at a single cleavage site with coincidental production of 410- and 150-kDa peptide fragments. Appearance of 160- and 137-kDa limiting peptides accompanied secondary proteolysis of the primary 410- and 150-kDa fragments, respectively. Despite extensive proteolysis of the Ca2+ release channel, CANP did not dramatically alter the Ca2+ handling and ryanodine binding properties of HSR membranes. The association of CANP with isolated HSR membranes suggests that, in vivo, this protease may modify an additional property of the Ca2+ release channel. This may be related to the CANP-susceptible structural association of the Ca2+ release channel with dihydropyridine receptors at T-tubule/sarcoplasmic reticulum junctions.


Cloning of human AMP deaminase isoform E cDNAs. Evidence for a third AMPD gene exhibiting alternatively spliced 5'-exons.

Higher eukaryotes express multiple isoforms of AMP deaminase (EC 3.5.4.6). In humans, four AMP deaminase variants, termed M (muscle), L (liver), E1, and E2 (erythrocyte) can be distinguished by a variety of biochemical and immunological criteria. Previous molecular studies have reported two genes, AMPD1 and AMPD2, that produce isoform M and L transcripts, respectively. This study identifies a third human AMP deaminase gene, AMPD3. Nucleotide sequence alignments between AMPD3 cDNAs isolated from several human libraries indicate three different extreme 5'-ends. Alternate forms of the AMPD3 cDNAs contain a common 2301-bp open reading frame (ORF) and 3'-untranslated region of 1245 bp. Two of the three forms, however, exhibit additional 5'-end nucleotide sequences that would extend their respective ORFs by 21 and 27 nucleotides. RNase protection analyses and the partial characterization of human AMPD3 genomic clones demonstrate alternative splicing of three different 5'-terminal exons. Western blot analyses detect anti-E-specific immunoreactivity in affinity-purified extracts derived from the bacterial expression of a truncated AMPD3 cDNA. These results are discussed in relation to AMP deaminase isoform diversity.


Differential irreversible insertion of protein kinase C into phospholipid vesicles by phorbol esters and related activators.

Incubation of protein kinase C (PKC) alpha with phorbol 12,13-dibutyrate and phospholipid vesicles promoted a time-dependent irreversible insertion of the enzyme into the vesicles and the generation of a calcium-independent kinase activity. Calcium neither caused insertion nor influenced the insertion induced by the phorbol ester. The effect was strongly dependent on the phosphatidylserine concentration in the vesicle and could also be supported by other anionic phospholipids. An analysis of the structure-activity relations of PKC activators for the calcium-independent kinase activity revealed marked relative differences in potencies for binding and for insertion. Compounds such as phorbol 13-myristate 12-acetate and mezerein were very efficient at inducing insertion. In contrast, 12-deoxyphorbol esters and diacylglycerol were relatively inefficient at inducing insertion, requiring higher concentrations than expected from their binding affinities. The insertion of PKC alpha depended substantially on the length of the aliphatic esters in the 12- and 13-positions of the phorbol derivatives, and once again, potencies for insertion and binding were not directly proportional. Our findings suggest two different sites for ligand interaction on the molecule of PKC alpha with different structure-activity requirements. We speculate that the differential ability of compounds to promote insertion could contribute to the documented marked differences in the biological behavior of PKC activators.


Central non-Pur.Pyr sequences in oligo(dG.dC) tracts and metal ions influence the formation of intramolecular DNA triplex isomers.

The effect of the central non-Pur.Pyr sequences in oligo(dG.dC) inserts on determining the type of intramolecular DNA triplex isomers formed in negatively supercoiled plasmids was investigated. Different triplex types (H-r3, H-r5, and H-y3), revealed by a combination of chemical probing and Maxam-Gilbert sequencing reactions, were adopted by the oligo(dG.dC) tracts depending on the length and composition of the central non-Pur.Pyr sequences (0, 3, or 5 base pairs) and the kind of metal ions. The H-r3 triplex conformer, one isomer of a Pur.Pur.Pyr structure, was formed in the (C)20 and (C)10GCG(C)10 inserts in plasmids in the presence of certain metal ions. Interestingly, H-r5, the other isomer of the Pur.Pur-Pyr triplex which had not been detected previously, was formed in a (C)9GAATT(C)9 insert in the presence of either Mg2+ or Ca2+. Alternatively, H-y3, one isomer of a Pyr.Pur.Pyr triplex, was formed in the (C)9GAATT(C)9 insert in the absence of metal ions. Thus, central non-Pur.Pyr sequences and metal ions play a role as determinants of the types of intramolecular triplexes formed; they also reduce the requirement of longer Pur.Pyr repeat sequences to form intramolecular triplexes. Furthermore, the effects of MgCl2 concentration and pH on the formation of triplex isomers were examined. The Pur.Pur.Pyr conformations (H-r3 and H-r5) may be the favored conformations in the cellular milieu, since they are stable at physiological pH and metal ion concentration.


Regulation of the actin-activated ATPase and in vitro motility activities of monomeric and filamentous Acanthamoeba myosin II.

The actin-activated Mg(2+)-ATPase activity of filamentous Acanthamoeba myosin II is inhibited by phosphorylation of 3 serine residues at the tip of the tail of each heavy chain. From previous studies, it had been concluded that the activity of each molecule in the filament was regulated by the global state of phosphorylation of the filament and was independent of its own phosphorylation state. The actin-activated Mg(2+)-ATPase activity of monomeric phosphorylated myosin II was not known because it polymerizes under the ionic conditions necessary for the expression of this activity. We have now found conditions to maintain myosin II monomeric and active during the enzyme assay. The actin-activated Mg(2+)-ATPase activities of monomeric dephosphorylated and phosphorylated myosin II were found to be the same as the activity of filamentous dephosphorylated myosin II. These results support the conclusion that phosphorylation regulates filamentous myosin II by affecting filament conformation. Consistent with their equivalent enzymatic activities, monomeric and filamentous dephosphorylated myosin II were equally active in an in vitro motility assay in which myosin adsorbed to a surface drives the movement of F-actin. In contrast to their very different enzymatic activities, however, filamentous and monomeric phosphorylated myosin II had similar activities in the in vitro motility assay; both were much less active than monomeric and filamentous dephosphorylated myosin II. One interpretation of these results is that the rate-limiting steps in the two assays are different and that, while the rate-limiting step for actin-activated Mg(2+)-ATPase activity is regulated only at the level of the filament, the rate-limiting step for motility can also be regulated at the level of the monomer.


Limited proteolysis reveals a structural difference in the globular head domains of dephosphorylated and phosphorylated Acanthamoeba myosin II.

Phosphorylation at three sites at the tip of the tail of myosin II from Acanthamoeba castellanii inactivates the actin-activated Mg(2+)-ATPase activity of filamentous myosin and the in vitro motility activity of both monomeric and filamentous myosin. To seek a structural explanation for these effects, we examined the susceptibilities of dephosphorylated and phosphorylated myosins II to endoproteinases. Endoproteinase Arg-C cleaved myosin II preferentially at two sites in the globular head, Lys-621 and Arg-638, producing an NH2-terminal fragment of about 67,000 Da and a COOH-terminal fragment of about 112,000 Da. Dephosphorylated monomers and filaments were cleaved about 3 times more rapidly than their phosphorylated counterparts principally because of a much greater rate of cleavage at Arg-638; the ratio of cleavage at Arg-638:Lys-621 was about 3 for dephosphorylated myosins and about 0.5 for phosphorylated myosins. These data demonstrate that phosphorylation at the tip of the tail of Acanthamoeba myosin II causes a conformational change in the globular head that contains the catalytic sites; therefore, this conformational change may be related to the different catalytic and motile activities of the dephosphorylated and phosphorylated enzymes.


Purification, structure, and characterization of caltrin proteins from seminal vesicle of the rat and mouse.

Caltrins, small basic proteins that inhibit calcium uptake by epididymal spermatozoa, have been purified from seminal vesicle content of the mouse and rat. Mouse caltrin (M(r) 8,476) contains 75 amino acid residues, 14 basic, 5 acidic, and 7 cysteines while rat caltrin (M(r) 6,217) has 56 residues, 10 basic, 5 acidic, and 6 cysteines; their pI values are 10.2 and 9.3, respectively. The proteins did not react with Ellman's reagent unless the cystine residues were previously reduced. The primary structures were determined by sequencing fragments generated by trypsin, clostripain, and endoproteinase Lys-C digestion. The sequences were ordered to give the total structural formula. The two molecules have no sequence similarity and are different from those of the bull and guinea pig previously reported. Only rat caltrin has a sequence of 13 residues nearly identical to that in guinea pig caltrin I. Both rat and mouse caltrin react with antibodies against bovine and guinea pig caltrins. Reduction and alkylation of cysteine residues suppressed the immunologic response of mouse caltrin; however, modified rat caltrin retained partially its immunoreactivity with the antiserum against guinea pig caltrin I. The same treatment abolished the calcium transport inhibitory activity of mouse caltrin and greatly reduced that of rat caltrin. It is likely that rat and mouse caltrins have the same physiological function as proposed for bovine caltrin; namely, to regulate the development of the Ca(2+)-dependent processes that "capacitate" sperm for fertilization.


A rho gene product in human blood platelets. II. Effects of the ADP-ribosylation by botulinum C3 ADP-ribosyltransferase on platelet aggregation.

In the accompanying paper (Nemoto, Y., Namba, T., Teru-uchi, T., Ushikubi, F., Morii, N., and Narumiya, S. (1992) J. Biol. Chem. 267, 20916-20920), we have identified rhoA protein as the sole substrate protein for botulinum C3 ADP-ribosyltransferase (C3 exoenzyme) in human blood platelets. Here we examined the role of rhoA protein in platelet functions. C3 exoenzyme added to washed platelets dose- and time-dependently ADP-ribosylated rhoA protein in situ in the cells. Concomitant with this modification, inhibition of thrombin-induced platelet aggregation was observed. This inhibition was not reversed by washing the treated platelets, but was not found when C3 exoenzyme was pretreated with mouse monoclonal anti-C3 exoenzyme antibody. C3 exoenzyme treatment did not affect thrombin-induced inositol 1,4,5-trisphosphate production. Secretion of preloaded [14C]serotonin was delayed by the enzyme treatment, but the extent of the secretion was not influenced. In addition, the enzyme treatment did not change the expression of the glycoprotein IIb-IIIa complex on the platelet surface. The enzyme treatment also suppressed platelet aggregation induced by phorbol myristate acetate. These results suggest that rhoA protein plays a role mainly in the aggregation process downstream from receptor-phospholipase C coupling. This, together with the previous finding that rhoA protein modulates stress fiber formation in cultured fibroblasts (Paterson, H. F., Self, A. J., Garrett, M. D., Just, I., Aktories, K., and Hall, A. (1990) J. Cell Biol. 111, 1001-1007), suggests that rhoA protein regulates the assembly of actin filaments and the avidity of the platelet integrin (glycoprotein IIb-IIIa) in the aggregation process.


Regulation of Hsp70 function by a eukaryotic DnaJ homolog.

We report that a purified cytoplasmic Hsp70 homolog from Saccharomyces cerevisiae, Hsp70SSA1, exhibits a weak ATPase activity, which is stimulated by a purified eukaryotic dnaJp homolog (YDJ1p). Stable complex formation between Hsp70SSA1 and the permanently unfolded protein carboxymethylated alpha-lactalbumin (CMLA) was assayed by native gel electrophoresis. The affinity of Hsp70SSA1 for CMLA appeared to be regulated by YDJ1p. Significant reduction in both CMLA-Hsp70SSA1 complex formation and the release of CMLA pre-bound to Hsp70SSA1 was observed only in the presence of both YDJ1p and ATP. Thus, Hsp70SSA1 and YDJ1p interact functionally in the execution of Hsp70SSA1 chaperone activities in the eukaryotic cell.


p13suc1 suppresses the catalytic function of p34cdc2 kinase for intermediate filament proteins, in vitro.

The regulation of p34cdc2 kinase activity controls the entry into and exit from mitosis. Although genetic and biochemical evidence suggested close interactions between cyclins, p13suc1 and p34cdc2 kinase, the roles of p13suc1 on p34cdc2 kinase functions remain unclear. To examine the effects of p13suc1 on p34cdc2 kinase function we developed a simple purification procedure for p34cdc2 kinase, unassociated with p13suc1. The key to the purification procedures we used was buffer containing 0.5 M NaCl and 50% ethylene glycol, as a specific elutant of p34cdc2 kinase from p13suc1-Sepharose. This purified p34cdc2 kinase stoichiometrically phosphorylated vimentin and desmin. Exogenous p13suc1 suppressed the phosphorylation of these filament proteins by the kinase and prevented disassembly, although histone H1 phosphorylation was not affected. Peptide mapping analysis showed a similar extent of inhibition by p13suc1 for all five phosphorylation sites by p34cdc2 kinase of vimentin and desmin, hence these p13suc1-induced inhibitions are probably not site-specific. It thus appears that p13suc1 has a selective effect on the catalytic activity of p34cdc2 kinase for these filament proteins.


TATA-binding protein activates transcription when upstream of a GCN4-binding site in a novel yeast promoter.

In the gal-his3 hybrid promoter, his3-GG1, GCN4 stimulates transcription at the position normally occupied by a TATA element. This expression requires two elements within gal1-10 sequences, a REB1-binding site and a second element, Z, which resides 20 base pairs upstream of the GCN4-binding site. No obvious TATA element is present in this promoter. To characterize the function of Z, we replaced it with short random oligonucleotides and selected for expression in vivo. Fourteen elements were identified and classified into groups based upon sequence and phenotypic similarities. Group 1 elements contained functional TATA sequences that were essential for activity. TATA elements can thus function when positioned upstream of a GCN4-binding site. The Group 2 elements activated transcription poorly when used as conventional TATA elements; however, mutational analyses demonstrated that their activity required TATA-like sequences. These TATA-like sequences bound the yeast TATA-binding protein (TBP) poorly in vitro but function in vivo as TBP interaction sites based upon two criteria. First mutations that improved their TATA character correspondingly improved function and second their activity could be enhanced in the presence of an altered binding specificity mutant of TBP. Furthermore, the Group 2 elements enabled the identification of mutations outside of the TATA-like core that contribute to transcriptional activation without adversely affecting TBP binding. The finding that low affinity TBP-binding sites can be used at unconventional positions suggests that many "TATA-less" promoters contain a cryptic interaction site for TBP.


Human insulin-like growth factor I receptor 950tyrosine is required for somatotroph growth factor signal transduction.

Insulin-like growth factor I (IGF-I), a growth hormone (GH)-dependent growth factor exerts feedback regulation of GH by inhibiting GH gene expression. IGF-I inhibition of GH secretion is enhanced 3-5-fold in GC rat pituitary cells overexpressing the wild type 950Tyr human IGF-I receptor which autophosphorylates appropriately. To determine the critical amino acid sequence responsible for IGF-I signaling, insertion, deletion, and site-directed mutants were constructed to substitute for 950Tyr in exon 16 of the human IGF-I receptor beta-subunit transmembrane domain. All mutant transfectants bound IGF-I with a similar Kd to untransfected cells but had markedly increased (7-34-fold) IGF-I-binding sites. GH responsiveness to IGF-I was tested in mutant transfectants. Overexpressed site-directed and insertion mutant IGF-I receptors exhibited a modest suppressive effect on GH in response to the IGF-I ligand, similar to that observed in untransfected cells. Deletion mutant (IG-FIR delta 22) (amino acid 944-965) did not transduce the IGF-I signal to the GH gene. Site-directed and insertion mutants therefore did not enhance the IGF-I response of the endogenous rat receptor, unlike the 950Tyr wild type transfectants which enhanced the IGF-I signal. All mutant transfectants, except the deletion mutant, internalized radioactive ligand similarly to 950Tyr wild type transfectants. 950Tyr of the human IGF-I receptor is therefore required for IGF-I signal transduction in the pituitary somatotroph, but not for IGF-I-mediated internalization.


Catalytic effectiveness of human aldose reductase. Critical role of C-terminal domain.

Human aldose reductase and aldehyde reductase are members of the aldo-keto reductase superfamily that share three domains of homology and a nonhomologous COOH-terminal region. The two enzymes catalyze the NADPH-dependent reduction of a wide variety of carbonyl compounds. To probe the function of the domains and investigate the basis for substrate specificity, we interchanged cDNA fragments encoding the NH2-terminal domains of aldose and aldehyde reductase. A chimeric enzyme (CH1, 317 residues) was constructed in which the first 71 residues of aldose reductase were replaced with first 73 residues of aldehyde reductase. Catalytic effectiveness (kcat/Km) of CH1 for the reduction of various substrates remained virtually identical to wild-type aldose reductase, changing a maximal 4-fold. Deletion of the 13-residue COOH-terminal end of aldose reductase, yielded a mutant enzyme (AR delta 303-315) with markedly decreased catalytic effectiveness for uncharged substrates ranging from 80- to more than 600-fold (average 300-fold). The KmNADPH of CH1 and AR delta 303-315 were nearly identical to that of the wild-type enzyme indicating that cofactor binding is unaffected. The truncated AR delta 303-315 displayed a NADPH/D isotope effect in kcat and an increased D(kcat/Km) value for DL-glyceraldehyde, suggesting that hydride transfer has become partially rate-limiting for the overall reaction. We conclude that the COOH-terminal domain of aldose reductase is crucial to the proper orientation of substrates in the active site.


The Shaw-related potassium channel gene, Kv3.1, on human chromosome 11, encodes the type l K+ channel in T cells.

T lymphocytes exhibit three distinct types of voltage-gated K+ channels, n, n', and l, that are distributed in the T cell lineage according to subset, as well as the cells' activation and developmental status. Type l K+ channels are found sparingly in cytotoxic T cells from normal mice and abundantly in a specific T cell subset (CD4- CD8- Thy1+) from mice with autoimmune disease. Here, we show that the mouse Kv3.1 gene, when expressed in Xenopus oocytes, encodes a channel with properties remarkably similar to those of the l-type channel. Kv3.1 transcripts were found in T cells isolated from the lymph nodes of MRL-lpr mice with systemic lupus erythematosus and in a human lymphoma cell line that also expresses the l channel phenotype. By these criteria, we conclude that Kv3.1 encodes the voltage-gated type l K+ channel in lymphocytes. The Kv3.1 gene maps to human chromosome 11; the related Kv1.1 and Kv3.2 genes are localized on human chromosome 12, while the IsK gene maps to human chromosome 21.


Functional analysis of nucleosome assembly protein, NAP-1. The negatively charged COOH-terminal region is not necessary for the intrinsic assembly activity.

A nucleosome assembly protein (NAP-1) of Saccharomyces cerevisiae facilitates the association of histones with DNA to form nucleosomes in vitro at physiological ionic conditions. The cloned gene was expressed in Escherichia coli using a T7 expression system, and the protein (417 amino acid residues) was purified by Mono Q column chromatography. Various deletion fragments of NAP-1 protein were also produced, and their nucleosome assembly activity was examined by supercoiling assay. The internal fragment containing the residues 43-365 was necessary and sufficient for the activity, and a long stretch of negatively charged region near the carboxyl terminus was dispensable. This minimal size fragment could form the 12 S NAP-1-histone complex as the whole protein could, whereas deleted fragments on either side could bind with core histones only to form aggregates.


The muscle-derived lens of a squid bioluminescent organ is biochemically convergent with the ocular lens. Evidence for recruitment of aldehyde dehydrogenase as a predominant structural protein.

Many of the structural proteins of ocular lenses, commonly referred to as crystallins, are identical to specific enzymes or the result of a recent gene duplication (Piatigorsky, J., and Wistow, G. (1991) Science 252, 1078-1079). One such enzyme, aldehyde dehydrogenase (ALDH), has been recruited as a lens crystallin in certain mammals (Wistow, G., and Kim, H. (1991) J. Mol. Evol. 32, 262-269) and cephalopods (Tomarev, S., Zinovieva, R., and Piatigorsky, J. (1991) J. Biol. Chem. 266, 24226-24231). We report here that a transparent tissue, derived from muscle but functioning as a lens in the light-emitting organ of a squid, Euprymna scolopes, shows striking biochemical convergence with the epidermally derived ocular lenses of some mammals and cephalopods. In the light organ lens of E. scolopes, an ALDH-like protein is the predominant molecular component. The typical muscle-specific proteins are replaced as the dominant species by a protein composed of 54-kDa subunits. This protein, which we designate as L-crystallin, constitutes approximately 70% of the total soluble protein of the light organ lens. The amino acid sequences of three peptides of L-crystallin (approximately 9% of the total protein) showed 54.5% sequence identity with human cytosolic ALDH. Using polyclonal antiserum made against L-crystallin, we found that it is present in low abundance in other tissues of the squid, including muscle and the ocular lens. This polyclonal antiserum also cross-reacted with the ALDH-like crystallins found in the ocular lenses of certain mammals and cephalopods. L-Crystallin showed no ALDH activity, which is similar to several other enzyme/crystallins, including ALDH/eta-crystallin (Wistow, G., and Kim, H. (1991) J. Mol. Evol. 32, 262-269). The characteristics of this muscle-derived lens are evidence that a common biochemical basis underlies transparency and that certain proteins may possess properties that promote their selection as lens structural proteins.


Primary structure of Gal beta 1,3(4)GlcNAc alpha 2,3-sialyltransferase determined by mass spectrometry sequence analysis and molecular cloning. Evidence for a protein motif in the sialyltransferase gene family.

The Gal beta 1,3(4)GlcNAc alpha 2,3-sialyltransferase forms the NeuAc alpha 2,3Gal beta 1,3(4)GlcNAc sequences found in terminal carbohydrate groups of glycoproteins and glycolipids. High energy collision-induced dissociation analysis of tryptic peptides from only 300 pmol of the purified Gal beta 1,3(4)GlcNAc alpha 2,3-sialyltransferase provided 25% of the total amino acid sequence and led to the successful cloning of this enzyme. The peptide sequence information was used to design short degenerate primers for use in the polymerase chain reaction. A long specific cDNA fragment was amplified which was used to isolate a clone from a rat liver cDNA library. The cloned cDNA encodes a 374-amino acid protein containing an amino-terminal signal-anchor sequence characteristic of all cloned glycosyltransferases and produced sialyltransferase activity when transiently expressed in COS-1 cells. When compared with two other cloned sialyltransferases, the primary structure of Gal beta 1,3(4)GlcNAc alpha 2,3-sialyltransferase revealed a homologous region in all three enzymes consisting of a stretch of 55 amino acids located in their catalytic domains. This feature together with lack of homology in the remaining 85% of the sequence of the three sialyltransferases defines a pattern of sequence homology not found in cloned cDNAs of other glycosyltransferase families.


Preferential translation of heat shock mRNAs in HeLa cells deficient in protein synthesis initiation factors eIF-4E and eIF-4 gamma.

Expression of antisense RNA against eukaryotic translation initiation factor 4E (eIF-4E) in HeLa cells causes a reduction in the levels of both eIF-4E and eIF-4 gamma (p220) and a concomitant decrease in the rates of both cell growth and protein synthesis (De Benedetti, A., Joshi-Barve, S., Rinker-Schaffer, C., and Rhoads, R. E. (1991) Mol. Cell Biol. 11, 5435-5445). The synthesis of most proteins in the antisense RNA-expressing cells (AS cells) is decreased, but certain proteins continue to be synthesized. In the present study, we identified many of these as stress-inducible or heat shock proteins (HSPs). By mobilities on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by reactivity with monoclonal antibodies generated against human HSPs, four of these were shown to be HSP 90, HSP 70, HSP 65, and HSP 27. The steady-state levels of HSP 90, 70, and 27 were elevated in relation to total protein in AS cells. Pulse labeling and immunoprecipitation indicated that HSP 90 and HSP 70 were synthesized more rapidly in AS cells than in control cells. The accelerated synthesis of HSPs in the AS cells was not due, however, to increased mRNA levels; the levels of HSP 90 and 70 mRNAs either remained the same or decreased after induction of antisense RNA expression. Actin mRNA, a typical cellular mRNA, was found on high polysomes in control cells but shifted to smaller polysomes in AS cells, as expected from the general decrease in translational initiation caused by eIF-4E and eIF-4 gamma depletion. HSP 90 and 70 mRNAs showed the opposite behavior; they were associated with small polysomes in control cells but shifted to higher polysomes in AS cells. These results demonstrate that HSP mRNAs have little or no requirement in vivo for the cap-recognition machinery and suggest that these mRNAs may utilize an alternative, cap-independent mechanism of translational initiation.


Endo-xyloglucan transferase, a novel class of glycosyltransferase that catalyzes transfer of a segment of xyloglucan molecule to another xyloglucan molecule.

Xyloglucans are the major component of plant cell walls and bind tightly to the surface of individual cellulose microfibrils, thereby cross-linking them into a complex polysaccharide network of the cell wall. The cleavage and reconnection of xyloglucan cross-links are considered to play the leading role during chemical processes essential for wall expansion and, therefore, cell growth and differentiation. Although it is hypothesized that some transglycosylation is involved in these chemical processes, the enzyme responsible for the reaction was not identified. We have now purified a novel class of endo-type glycosyltransferase to apparent homogeneity from the extracellular space or the cell wall of the epicotyls of Vigna angularis, a bean plant. The enzyme is a glycoprotein with a molecular mass of about 33 kDa. The enzyme catalyzes both 1) endo-type splitting of a xyloglucan molecule and 2) linking of a newly generated reducing end of the xyloglucan to the nonreducing end of another xyloglucan molecule, thereby mediating the transfer of a large segment of the xyloglucan to another xyloglucan molecule. The transferase exhibits no glycosidase or glycanase activity. Substrate specificity of the enzyme was investigated using several polysaccharides with different glycosidic linkages as donor substrates and pyridylamino oligosaccharides as acceptor substrates, in which the reducing end of the carbohydrate was tagged with a fluorescent group. The enzyme required a basic xyloglucan structure, i.e. a beta-(1-->4)-glucosyl backbone with xylosyl side chains, for both acceptor and donor activity. Galactosyl or fucosyl side chains on the main chain were not required for the acceptor activity. The enzyme exhibited higher reaction rates when xyloglucans with higher M(r) were used as donor substrates. Xyloglucans smaller than 10 kDa were no longer the donor substrate. On the other hand, pyridylamino heptasaccharide acted as a good acceptor as did xyloglucan polymers. Based on these results we propose to designate this novel enzyme a xyloglucan: xyloglucano-transferase, to be abbreviated endo-xyloglucan transferase (EXT) or xyloglucan recombinase. This enzyme is the first enzyme identified that mediates the transfer of a high M(r) segment between polysaccharide molecules to generate chimeric polymers. We conclude that endo-xyloglucan transferase functions as a reconnecting enzyme for xyloglucans and is involved in the interweaving or reconstruction of cell wall matrix, which is responsible for chemical creepage that leads to morphological changes in the cell wall.


Molecular heterogeneity of the gamma-aminobutyric acid (GABA) transport system. Cloning of two novel high affinity GABA transporters from rat brain.

cDNA clones encoding two novel gamma-aminobutyric acid (GABA) transporters (designated GAT-2 and GAT-3) have been isolated from rat brain, and their functional properties have been examined in mammalian cells. The transporters display high affinity for GABA (Km approximately 10 microM) and exhibit pharmacological properties distinct from the previously cloned neuronal GABA transporter (GAT-1). Both transporters require sodium and chloride for transport activity. The nucleotide sequences of GAT-2 and GAT-3 predict proteins of 602 and 627 amino acids, respectively, which can be modeled with 12 transmembrane domains, similar to the topology proposed for other cloned neurotransmitter transporters. Localization studies indicate that both transporters are present in brain and retina, while GAT-2 is also present in peripheral tissues. The cloning of these transporter genes from rat brain reveals previously undescribed heterogeneity in GABA transporters.


Identification of N-acetylneuraminyl alpha 2-->3 poly-N-acetyllactosamine glycans as the receptors of sialic acid-binding Streptococcus suis strains.

Streptococcus suis is a common cause of sepsis, meningitis, and other serious infections in young piglets and also causes meningitis in humans. The cell-binding specificity of sialic acid-recognizing strains of Streptococcus suis was investigated. Treatment of human erythrocytes with sialidase or mild periodate abolished hemagglutination. Hemagglutination inhibition experiments with sialyl oligosaccharides indicated that the adhesin preferred the sequence NeuNAc alpha 2-3Gal beta 1-4Glc(NAc). Resialylation of desialylated erythrocytes with Gal beta 1-3(4)GlcNAc alpha 2-3-sialyltransferase induced a strong hemagglutination, whereas no or only weak hemagglutination was obtained with cells resialylated with two other sialyltransferases. Binding of radiolabeled bacteria to blots of erythrocyte membrane proteins revealed binding to the poly-N-acetyllactosamine-containing components Band 3, Band 4.5, and polyglycosyl ceramides and to glycophorin A. The involvement of glycophorin A as a major ligand was excluded by the strong hemagglutination of trypsin-treated erythrocytes and En(a-) erythrocytes defective in glycophorin A. Sensitivity of the hemagglutination toward endo-beta-galactosidase treatment of erythrocytes and inhibition by purified poly-N-acetyllactosaminyl glycopeptides indicated that the adhesin bound to glycans containing the following structure: NeuNAc alpha 2-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-.


Mapping single cysteine mutants of light chain 2 in chicken skeletal myosin.

The NH2- and COOH-terminal regions of the regulatory light chain (LC2) have been mapped to the head/rod junction by immunoelectron microscopy, using monoclonal and anti-fluorescyl antibodies as probes. In order to map the entire length of the light chain, we have engineered and purified mutants that contain a single cysteine residue at positions 2, 73, 94, 126, or 155. The single cysteine residues were labeled with either 5-iodoacetamido fluorescein or N-ethylmaleimide-biotin. We observed that the reactivity of Cys126 is far greater than that of Cys155, confirming that cysteine 126 is the fast-reacting thiol in wild-type light chain. The labeled light chains were exchanged into myosin stripped of its native LC2 by immunoaffinity chromatography, and the reconstituted myosin was reacted with anti-fluorescein antibody or avidin prior to rotary shadowing for electron microscopy. The position of the antibody or avidin was found to be near the head/rod junction for all mutants. These mapping studies, together with our finding that cysteines widely separated in the primary sequence can form multiple disulfide bonds (Saraswat, L.D., and Lowey, S. (1991) J. Biol. Chem. 226, 19777-19785), support a model for LC2 as a flexible, globular molecule localized mainly in the vicinity of the head/rod junction of myosin.


Expression and analysis of recombinant Amb a V and Amb t V allergens. Comparison with native proteins by immunological assays and NMR spectroscopy.

The Amb V allergens are small, highly disulfide-bonded ragweed pollen allergens that serve as useful models for understanding the molecular basis of the human immune response. We have produced recombinant Amb a V and Amb t V (from short and giant ragweed pollens, respectively) in Escherichia coli and have compared their structural and functional characteristics to those of the native proteins. Recombinant Amb t V was indistinguishable from native Amb t V as determined by NMR spectroscopy and antibody-binding studies. Whereas inhibition analysis showed that recombinant Amb a V possessed only approximately 50% of the antibody-binding activity of native Amb a V, the two proteins were similarly effective in stimulating Amb a V-specific T-cells. Our results demonstrate that even highly homologous proteins exhibit different abilities to fold into their native three-dimensional conformations and establish the potential and limits of expressing the recombinant Amb V allergens intracellularly in E. coli.


Conveyance of partial agonism/antagonism to bombesin/gastrin-releasing peptide analogues on Swiss 3T3 cells by a carboxyl-terminal leucine insertion.

Gastrin-releasing peptide (GRP) is a neuroendocrine hormone that may be involved in the pathophysiology of small cell lung carcinoma. We describe carboxylterminal peptide analogues of GRP and bombesin, a 14-residue amphibian homologue, that were modeled after the antagonist [Leu13-psi(CH2NH)-Leu14]bombesin and retained the psi bond. Three novel peptides contained a Leu insertion amino to the psi bond, i.e. ... Leu13Leu14 psi X (residues numbered after bombesin) where X = LeuNH2 or norleucine-NH2). The Leu-insertion analogues behaved as pure partial agonists/antagonists when examined for the ability to stimulate [3H]thymidine incorporation into quiescent Swiss 3T3 cells (agonist activity) and to diminish the agonist response of GRP (antagonist activity). A time course of [3H]thymidine incorporation into quiescent cells indicated maximal incorporation at 20-h post-peptide addition for bombesin and GRP and a Leu-insertion peptide, but the extent of the incorporation for the Leu-insertion peptide was half that of GRP and bombesin. The agonist dose responses of the Leu-insertion peptides (EC50 values of 1-10 nM) paralleled GRP and bombesin, but the maximal response of the Leu-insertion peptides, even at concentrations as high as 10(-4) M, was half the maximal value of GRP or bombesin. High concentrations of the Leu-insertion peptides antagonized 10 nM GRP (a concentration that produced a near-maximal GRP response) yielding a response that was half the maximal value of GRP and equivalent to the maximal response of the Leu-insertion peptides alone. Analogues of the form ... Leu13 psi X behaved as complete antagonists. The KD values of the Leu-insertion peptides for competitive binding versus 125I-GRP (2-50 nM) were as potent as parent ... Leu14 agonists. Stability studies indicated that peptide potencies for both agonist and antagonist activities diminished upon peptide incubation in medium or on cells. The results suggested that, for the Leu-insertion peptides, degradation into distinct products with different activities was not responsible for their partial agonist/antagonist behavior. Computer-generated molecular modeling studies indicated that the novel structures could adopt energy minimized conformations for either an agonist or an antagonist as proposed earlier (Coy, D.H., Heinz-Erian, P., Jiang, N.-Y., Sasaki, Y., Taylor, J., Moreau, J.-P., Wolfrey, W.T., Gardner, J.D., and Jensen, R. T. (1988) J. Biol. Chem. 263, 5056-5060).


Specific binding sites for the activator protein, ALCR, in the alcA promoter of the ethanol regulon of Aspergillus nidulans.

ALCR is the specific activator of the Aspergillus nidulans ethanol-utilization pathway, mediating the induction of its own transcription and that of the structural genes alcA and aldA, encoding respectively, alcohol dehydrogenase I and aldehyde dehydrogenase. ALCR is a DNA binding protein in which 6 cysteines are coordinated in a zinc binuclear cluster. This domain was fused to glutathione-S-transferase (GST) and isolated as a GST-ALCR(7-58*) fusion protein from Escherichia coli. Mobility shift assays showed that the ALCR fusion protein binds at sites upstream of the alcA promoter. DNaseI protection footprinting experiments revealed three specific binding sites, two that are direct repeats and one that is an inverted repeat with the same half-site 5'-CCGCA-3'. The half-sites are separated by a variable number of nucleotides in both types of target. The interaction of the ALCR fusion protein with direct and inverted repeats were examined by using interference and protection footprinting assays. In both binding sites, modification of the guanines in the half-sites interfered with the formation of the DNA complex, but the adjacent ones did not. Our results suggest that the ALCR protein makes contact in the major groove of the DNA helix of the half-sites. The functionality of two out of three binding sites of the GST-ALCR protein was demonstrated after their deletion. Therefore, the region encompassing these binding sites is a cis-acting element involved in the full induction of the alcA gene.


Transcriptional control of nuclear genes for the mitochondrial muscle ADP/ATP translocator and the ATP synthase beta subunit. Multiple factors interact with the OXBOX/REBOX promoter sequences.

The OXBOX promoter regions of the genes for the muscle-specific adenine nucleotide translocator (ANT1) and the beta subunit of the ATPsynthase (ATPsyn beta) have been implicated in the increased transcription of these nuclear-encoded oxidative phosphorylation (OXPHOS) genes in heart and skeletal muscle. DNA binding, electrophoretic mobility shift (gel-shift) assays now reveal that the OXBOX region has two unique but overlapping elements, the 13-base pair (bp) OXBOX and an 8-bp REBOX. The OXBOX binding factors are found only in myogenic cell lines, whereas the REBOX factors are ubiquitous. Methylation interference experiments have defined the boundaries of the OXBOX and REBOX elements, confirmed that the OXBOX factors are muscle-specific, and shown that the OXBOX and REBOX factors do not bind concurrently. The binding of the REBOX factors was found to be sensitive to NADH and thyroxine, suggesting that it may modulate OXPHOS gene expression in response to environmental and hormonal changes. Hence, the OXBOX/REBOX complex provides one mechanism by which mammalian energy metabolism can be adapted to developmental and environmental demands.


The 39-kDa receptor-associated protein interacts with two members of the low density lipoprotein receptor family, alpha 2-macroglobulin receptor and glycoprotein 330.

The 39-kDa receptor-associated protein (RAP) binds to the alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein (alpha 2MR/LRP) and inhibits binding of ligands to this receptor. The in vivo function of RAP may be to regulate ligand binding and/or assist in the correct biosynthetic processing or trafficking of the alpha 2MR/LRP. Here we show that RAP binds another putative receptor, the kidney glycoprotein 330 (gp330). Gp330 is a high molecular weight glycoprotein that is structurally similar to both the alpha 2MR/LRP and low density lipoprotein receptor. The ability of RAP to bind to gp330 was demonstrated by ligand blotting and solid phase binding assays, which showed that RAP binds to gp330 with high affinity (Kd = 8 nM). Exploiting the interaction of gp330 and RAP, we purified gp330 by affinity chromatography with a column of RAP coupled to Sepharose. Gp330 preparations obtained by this procedure were notably more homogeneous than those obtained by conventional methods. Immunocytochemical staining of human kidney sections localized RAP to the brush-border epithelium of proximal tubules. The fact that gp330 is also primarily expressed by proximal tubule epithelial cells strengthens the likelihood that the interaction between gp330 and RAP occurs in vivo. The functional significance of RAP binding to gp330 may be to antagonize ligand binding as has been demonstrated for the alpha 2MR/LRP or to assist in the biosynthetic processing and/or trafficking of this receptor.


Interactions of the eIF-4F subunits in the yeast Saccharomyces cerevisiae.

Recognition of the cap structure at the 5' end of mRNA is one of the first events in initiation of eukaryotic translation. This step is mediated by the translation initiation factor 4F (eIF-4F). In mammalian cells this factor is composed of the cap-binding protein eIF-4E, eIF-4A, and a 220-kDa polypeptide. In yeast Saccharomyces cerevisiae, eIF-4E is found associated with a 150-kDa protein (p150) and a 20-kDa protein (p20). The resulting protein complex is proposed to represent yeast eIF-4F. To study the functions of p150 and p20 and their interaction with eIF-4E, we disrupted the genes encoding p150 and p20 and analyzed the effects on protein complex formation and cell viability. Yeast cells with single and double disruptions of the genes encoding p150 and p20 are viable, but p150 single and p150/p20 double disruptions show a slow growth phenotype. Gel chromatography and immunoadsorption experiments with a monoclonal anti-eIF-4E antibody coupled to protein G-Sepharose show that both p150 and p20 bind independently of each other to eIF-4E.


FeMo cofactor synthesis by a nifH mutant with altered MgATP reactivity.

We have characterized a Nif- mutant of Azotobacter vinelandii, designated UW91 (Shah, V. K., Davis, L. C., Gordon, J. K., Orme-Johnson, W. H., and Brill, W. J. (1973) Biochim. Biophys. Acta 292, 246-255). The specific Fe protein mutation giving rise to the Nif- phenotype was shown by DNA sequencing and site-directed mutagenesis to be the substitution of a conserved alanine at position 157 by a serine. The UW91 Fe protein was purified and shown to have a normal [4Fe-4S] cluster and normal MgATP binding activity. The substitution of alanine 157 by serine, however, prevents the MgATP-induced conformational change that occurs for the wild-type Fe protein, prevents MgATP hydrolysis, and prevents productive electron transfer to the MoFe protein. The UW91 Fe protein does bind to the MoFe protein to give a normal cross-linking pattern; however, it does not compete very successfully with wild-type Fe protein in an activity assay. The UW91 MoFe protein was also purified and characterized and shown to be indistinguishable from the wild-type protein. Thus, the substitution of Fe protein residue alanine 157 by serine does not change the Fe protein's ability to function in FeMo cofactor biosynthesis or insertion. This demonstrates that these events do not require the MgATP-induced conformational change, MgATP hydrolysis, or productive electron transfer to the MoFe protein.


Recombinant von Willebrand factor Arg578-->Gln. A type IIB von Willebrand disease mutation affects binding to glycoprotein Ib but not to collagen or heparin.

von Willebrand factor (vWF) is a multimeric plasma glycoprotein that mediates platelet adhesion to the subendothelium via binding to platelet glycoprotein Ib (GPIb) and to components of the vessel wall. Recently, missense mutations that cause type IIB von Willebrand disease (vWD) were described, clustered within a disulfide loop in the A1 domain of vWF that has binding sites for GPIb, collagen, and heparin. In type IIB vWD, plasma vWF exhibits increased affinity for platelet GPIb, but decreased binding to collagen and heparin. The effect was studied of a type IIB vWD mutation, Arg578-->Gln, on the interaction of vWF with GPIb, collagen, and heparin. Recombinant wild type rvWF and mutant rvWF(R578Q) were expressed in COS-7 cells. Ristocetin-induced binding of rvWF(R578Q) to GPIb was markedly increased compared with rvWF, confirming that the Arg578-->Gln mutation causes the characteristic gain-of-function abnormality of type IIB vWD; botrocetin-induced binding was only slightly increased. Binding to collagen type III and heparin-agarose was compared for rvWF(R578Q) and plasma vWF from patients with four different type IIB mutations: Arg543-->Trp, Arg545-->Cys, Val553-->Met, Arg578-->Gln. For all of the plasma samples, binding to collagen and to heparin was reduced compared with normal plasma. In contrast, binding of rvWF(R578Q) to collagen and heparin was normal compared with wild type rvWF. Therefore, the Arg578-->Gln mutation increases the affinity of vWF for GPIb but does not directly impair vWF interaction with collagen or heparin. Arg578 may therefore be necessary to prevent normal vWF from interacting with GPIb. In type IIB vWD, the defective binding of plasma vWF to collagen and heparin may be secondary to post-synthetic modifications that occur in vivo, such as the loss of high molecular weight vWF multimers.


Structure, chromosomal assignment, and expression of the gene for proteinase-3. The Wegener's granulomatosis autoantigen.

Proteinase-3 (PR-3) is a neutral serine proteinase present in the azurophil granules of human polymorphonuclear leukocytes. It degrades a variety of extracellular matrix proteins including elastin in vitro and causes emphysema when administered by tracheal insufflation to hamsters. It is identical to the target autoantigen (c-ANCA) associated with Wegener's granulomatosis and to myeloblastin, a serine proteinase first identified in HL-60 leukemia cells. In this study, the gene encoding PR-3 was cloned and sequenced. The gene spans approximately 6.5 kilobase pairs and consists of five exons and four introns. The genomic organization of PR-3 is similar to that of the other serine proteinases expressed in hemopoietic cells. Each residue of the catalytic triad of PR-3 is located on a separate exon, and the positions of the residues within the exons are similar to those in human leukocyte elastase and cathepsin G. The phase and placement of the introns in the PR-3 gene are also similar to those in human leukocyte elastase and cathepsin G. The 400-base pair (bp) 5'-flanking sequence of the PR-3 gene contains a TATA box at position 379. There is no CAAT box promoter element. The 3'-untranslated region is 200 bp, extending from a TGA stop codon to the site of polyadenylation 10 bp after the canonical AATAAA signal. Amplification of PR-3 from a human/hamster hybrid cell line localizes the gene to human chromosome 19. Evidence from Northern analysis suggests that PR-3 expression is primarily confined to the promyelocytic/myelocytic stage of bone marrow development.


Nuclear protein binding to the 5' enhancer region of the intracisternal A particle long terminal repeat.

The interactions of nuclear proteins from embryonal carcinoma cells (PCC3) with the long terminal repeats (LTRs) of murine intracisternal A particle (IAP) genes were studied. Two protein-DNA complexes were detected between PCC3 nuclear extract and IAP LTRs in a gel mobility shift assay. An additional complex was observed when enriched fractions from a heparin-agarose column were used as the source of proteins. Two regions within the LTR of IAP 81 were identified as the sites of protein interaction by DNase I protection. One region encompasses 43 nucleotides within the U3 region at the 5' end of LTR 81. The other covers a 78 base pair region lying within 100 nucleotides upstream from the transcription initiation site. Studies using constructs containing intact or deleted versions of the LTR fused to the bacterial chloramphenicol acetyltransferase gene indicated that the absence of the 5' 47 base pairs reduced the level of chloramphenicol acetyltransferase transcription to 20% of that driven by the entire LTR. Southwestern analysis of PCC3 nuclear extracts and column fractions revealed that a 28,000- and a 46,000-dalton protein were the major species that interact with the 5' end of IAP LTR 81.


Transcription of IL-2 and IL-4 genes is not inhibited by cyclosporin A in competent T cells.

Cyclosporin A (CsA) inhibits T-cell proliferation primarily by blocking the transcription of several early activation genes, especially those of the important T-cell growth factors IL-2 and IL-4. This effect seems to be mediated through inhibition of the activity of the transcription factor NF-AT which is essential for IL-2 and probably for IL-4 gene transcription. However, once T cells are rendered "competent" to proliferate following a brief exposure to the phorbol ester, phorbol 12,13-dibutyrate (PDBu), and the calcium ionophore, ionomycin, CsA no longer inhibits cell cycle progression supported by the presence of PDBu alone. Here it is shown that transcription of the IL-2 and IL-4 genes occurs normally throughout this "progression" phase, even in the presence of CsA. However, further production of functional NF-AT, which began during the competence phase of the cell cycle, is inhibited. These data indicate that, although the primary initiation of transcription of IL-2 and IL-4 mRNA during induction of competence may be NF-AT-dependent and CsA-sensitive, the augmentation in the progression phase is both NF-AT-independent and CsA-resistant.


A novel form of fibroblast growth factor receptor 2. Alternative splicing of the third immunoglobulin-like domain confers ligand binding specificity.

The fibroblast growth factor receptor 2 (FGFR2) gene is expressed as alternatively spliced mRNAs that encode bacterially expressed kinase, the keratinocyte growth factor receptor, or K-sam. We have now isolated a novel FGFR2 cDNA that is identical with the previously cloned human bacterially expressed kinase, except in the third immunoglobulin-like domain. The ligand binding properties of FGFR2 were studied by expressing the protein in rat L6 muscle myoblasts. Unlike human bacterially expressed kinase which binds acidic and basic FGF with similar affinities, FGFR2 bound acidic FGF with approximately 1000-fold higher affinity than basic FGF. These results indicate that alternative splicing of the FGFR2 gene in the region encoding the carboxyl-terminal half of the third immunoglobulin domain determines the ligand specificity of this group of receptors.


Pulmonary surfactant protein D specifically binds to phosphatidylinositol.

Alveolar type II cells produce and secrete a complex mixture of lipids and proteins called pulmonary surfactant of which phospholipids are the major components. Surfactant proteins (SP) A, B, and C interact with phospholipids and are believed to play important roles in alveolar spaces. However, whether surfactant protein D (SP-D) interacts with phospholipids is unknown. In the present study, we examined whether SP-D binds to phospholipids and investigated phospholipid specificities of SP-D binding and the structural requirements of phospholipids for that binding using 125I-SP-D as a probe. 125I-SP-D bound exclusively to phosphatidylinositol (PI) in various phospholipids or a fraction containing phospholipids extracted from surfactant, which were developed on thin layer chromatography. 125I-SP-D also bound to PI coated on microtiter wells in a manner dependent upon the SP-D concentration. Unlabeled SP-D competed well with 125I-SP-D for PI binding and the antibody against SP-D abolished 125I-SP-D binding to PI. PI liposome also attenuated 125I-SP-D binding to the solid phase PI. Ca2+ is absolutely required for the binding of SP-D to PI. SP-D failed to bind to lyso-PI, fatty acids derived from PI digested with phospholipase A2, or diacylglycerol obtained after phospholipase C treatment of PI. SP-D bound to neither phosphatidylinositol 4-monophosphate nor phosphatidylinositol 4,5-diphosphate. We conclude that SP-D specifically binds to PI. This is the first report that demonstrates that SP-D interacts with surfactant phospholipids.


Inhibition of glycosylphosphatidylinositol anchor formation by mannosamine.

Many eucaryotic cell surface proteins are anchored to the plasma membrane via a glycosylphosphatidylinositol (GPI), of which the core region is highly conserved from protozoa to mammalian cells. Previous studies (Lisanti, M. P., Field, M. C., Caras, I. W., Menon, A. K., and Rodiguez-Boulan, E. (1991) EMBO J. 10, 1969-1977) showed that mannosamine blocked the expression of a recombinant GPI-anchored protein in Madin-Darby canine kidney cells and converted this protein to an unpolarized secretory product. In the present study, we examined the effect of mannosamine on the formation of the glycan portion of the GPI anchor precursors. This amino sugar inhibited the incorporation of mannose into the glycan portion, and the inhibition was dose-dependent. Mannosamine was shown to be incorporated into the glycan as mannosamine, probably mostly in the second mannose position and thereby to block the further addition of mannose and other anchor components. The products formed in the presence of this drug were characterized by gel filtration and high resolution TLC both before and after deamination with nitrous acid and dephosphorylation by HF. Galactosamine and trehalosamine were inactive in this system, whereas glucosamine also inhibited mannose incorporation into GPI intermediates.


Transforming growth factor beta 1 stimulates the production of basic fibroblast growth factor binding proteoglycans in Balb/c3T3 cells.

Basic fibroblast growth factor (bFGF) binds to cell surface receptors and to heparin sulfate proteoglycans. Heparan sulfate binding may limit bFGF degradation and be an obligatory step for bFGF cell interaction. Transforming growth factor-beta 1 (TGF-beta 1) is a potent regulator of proteoglycan production and composition. The possibility that TGF-beta 1 synergistically regulates bFGF activity by altering bFGF-proteoglycan interactions was investigated. TGF-beta 1 increased 125I-bFGF binding to the extracellular matrix (ECM) of Balb/c3T3 cells 2-4-fold by increasing the number of bFGF binding sites. Increased bFGF binding correlated with a 2-5-fold increase in the production of sulfated proteoglycans, including heparan sulfate proteoglycans. TGF-beta 1 selectively stimulated production of high molecular mass proteoglycans (190-300 kDa) in conditioned medium and stimulated all proteoglycans in ECM. 125I-bFGF bound to TGF-beta 1 induced proteoglycans immobilized onto cationic nylon filters. Furthermore, ECM isolated from TGF-beta 1-treated cells incorporated more mitogenically active bFGF than native ECM. The mitogenic potential of the ECM was significantly reduced by treatment with heparinase. These results suggest that the ability of TGF-beta 1 to stimulate binding of bFGF to ECM, increase ECM heparan sulfate proteoglycan, and potentiate the mitogenic activity of bFGF are linked. Thus one aspect of TGF-beta 1/bFGF synergy may involve modulation of the ECM.


Evolution of intestinal apolipoprotein B mRNA editing. Chicken apolipoprotein B mRNA is not edited, but chicken enterocytes contain in vitro editing enhancement factor(s).

Mammalian intestinal apolipoprotein B (apoB) messenger RNA (mRNA) undergoes posttranscriptional editing, changing codon 2153 from CAA in apoB100 mRNA to an in-frame translational stop codon (UAA) in apoB48 mRNA. By contrast, chicken intestinal apoB cDNA contains a CAA codon at the corresponding site and apoB mRNA from chicken enterocytes, kidney, and liver is unedited. The cDNA sequence of chicken apoB spanning the edited base is divergent from mammalian apoB cDNA sequence, with 70% homology over the conserved 29-nucleotide sequence (6662-6690) flanking codon 2153. Efficient in vitro editing of both human and rat, but not chicken, synthetic apoB RNA was achieved using rat enterocyte S-100 extracts. By contrast, chicken enterocyte S-100 extracts failed to edit chicken, rat, or human synthetic apoB RNA. Mixing experiments, however, revealed that chicken enterocyte S-100 extracts enhance the in vitro editing activity of rat, pig, and human enterocyte S-100 extracts upon homologous RNAs. The editing enhancement activity of chicken enterocyte S-100 extracts is tissue-specific, heat-sensitive, substrate-saturable, and sensitive to proteinase K, but resistant to micrococcal nuclease. The activity was partially purified by Q-Sepharose chromatography and has an average molecular mass of 49 kDa when analyzed by gel filtration chromatography. We conclude that the evolutionary adaptation of intestinal apoB mRNA editing requires both a requisite RNA motif and tissue-specific factors which mediate the site-specific modification.


Expression of the Duchenne muscular dystrophy gene products in embryonic stem cells and their differentiated derivatives.

Three protein products of the Duchenne muscular dystrophy (DMD) gene were identified so far. These include the two very similar muscle and brain type dystrophins, which are encoded by 14-kilobase (kb) mRNAs, and Dp71, which is much smaller. Dp71 is encoded by a 6.5-kb mRNA, which is transcribed from approximately 6% of the giant dystrophin gene. The present investigation shows that Dp71 is the first product of the DMD gene detectable during development. It is already expressed in the pluripotent embryonic stem cells. The two 14-kb mRNAs encoding the dystrophins are detectable only after differentiation of specialized cell types. The possible implication of these findings with regard to the ontogenetic activation and the evolution of the DMD gene are discussed.


Plant organelles contain distinct peptidylprolyl cis,trans-isomerases.

Peptidylprolyl cis,trans-isomerase (PPIase) activity was detected in the cytosol, mitochondria, and chloroplast of pea plants. Cyclosporin A inhibited the activity largely localized to the mitochondrial matrix while rapamycin inhibited the PPIase activity associated with the mitochondrial membranes. Differential inhibition by the two immunosuppressive drugs, the specific binding of these drugs to different mitochondrial fractions, and the immunological detection of a putative 25-kDa rapamycin-binding protein (RBP) in mitochondrial extracts attests to the presence in plant mitochondria of both cyclophilin and RBP classes of PPIases. Cyclosporin A-sensitive PPIase detected in the chloroplast was mostly localized to the thylakoids, which is suggestive of its function in the folding of membranal proteins. PPIase associated with the chloroplast stroma and the thylakoids was not inhibited by rapamycin nor was any cross-reactive RBP detected in chloroplast extracts. These results demonstrate the presence of distinct classes of PPIases in the mitochondria and the chloroplasts of plants.