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V Guerriero

Publications and source records attributed to V Guerriero.

At least 19 recordsLinked to original sources
PubMed1991

Isolation of a human placenta cDNA coding for a protein related to the vascular permeability factor.

A human cDNA coding for a protein related to the vascular permeability factor (VPF) was isolated from a term placenta cDNA library; we therefore named its product placenta growth factor (PlGF). PlGF is a 149-amino-acid-long protein and is highly homologous (53% identity) to the platelet-derived growth factor-like region of human VPF. Computer analyses reveal a putative signal peptide and two probable N-glycosylation sites in the PlGF protein, one of which is also conserved in human VPF. By using N-glycosidase F, tunicamycin, and specific antibodies produced in both chicken and rabbit, we demonstrate that PlGF, derived from transfected COS-1 cells, is actually N-glycosylated and secreted into the medium. In addition, PlGF, like VPF, proves to be a dimeric protein. Finally, a conditioned medium from COS-1 cells containing PlGF is capable of stimulating specifically the growth of CPA, a line of endothelial cells, in vitro.

Amino Acid Sequence↗
PubMed1991

Quantitation of Hsp70 in tissues using a competitive enzyme-linked immunosorbent assay.

A competitive enzyme-linked immunosorbent assay has been developed to quantitate the Hsp70 levels in bovine tissues. Antibodies that show specificity to the low molecular weight form of Hsp70 (72 kDa) were developed in chickens, isolated from egg yolks, and characterized using Western blotting. Using this assay, we were able to verify quantitatively the previous observation that the low molecular weight form of Hsp70 exists at elevated levels in bovine skeletal muscle. Also, we report that the skeletal muscle contains the majority of the Hsp70 in the sarcoplasm, but a small amount of Hsp70 is localized in the sarcoplasmic reticulum. The amount of Hsp70 in bovine skeletal muscle is not at a maximal level because it can be increased by heat stress. The reliability, specificity, and use of enzyme-linked antibodies instead of radioactive materials make this assay preferable over previous quantitation techniques.

Animals↗
PubMed1991

Definition of the inhibitory domain of smooth muscle myosin light chain kinase by site-directed mutagenesis.

Site-directed mutagenesis of smooth muscle myosin light chain kinase was applied to define its autoinhibitory domain. Mutants were all initiated at Leu-447 but contained varying lengths of C-terminal sequence. Those containing the complete C-terminal sequence to Glu-972 possessed kinase activities that were calmodulin-dependent. Removal of the putative inhibitory domain by truncation to Thr-778 resulted in generation of a constitutively active (calmodulin-independent) species. Thus, the inhibitory domain lies to the C-terminal side of Thr-778. Truncation to Lys-793 and to Trp-800 also resulted in constitutively active mutants, although the specific activity of the latter was less than the other mutants. None of the truncated mutants bound calmodulin. For each mutant, the Km values with respect to ATP and to the 20,000-dalton light chain were similar to values obtained with the native enzyme. The presence of the inhibitory domain was detected by activation of kinase activity following limited proteolysis with trypsin. Using this procedure, it was determined that the inhibitory domain was manifest only in the mutant truncated to Trp-800 and was absent from that ending at Lys-793. These results indicate that a critical region of the inhibitory domain is contained within the sequence Tyr-794 to Trp-800. This region overlaps with the calmodulin-binding site for five residues. Our assignment of the inhibitory sequence is consistent with autoinhibition via a pseudosubstrate domain.

Amino Acid Sequence↗
PubMed1990

Synthesis of heat stress proteins in lymphocytes from livestock.

Cultured bovine, equine, ovine and chicken lymphocytes responded to heat stress by the increased synthesis of a specific set of proteins known as heat stress proteins (HSP). Proteins with molecular weights of 70 and 90 kDa were synthesized in all species. Additional proteins were found in bovine, ovine and chicken lymphocytes. A time course of induction showed an increased synthesis of some of these proteins with only 30 min of heat stress and of several proteins with 60 min of heat stress. A specific monoclonal antibody was used to identify HSP70 as one of the stress proteins in bovine lymphocytes. Cells from these animals are capable of responding to heat stress by the production of heat stress proteins. These may be of physiological importance.

Animals↗
PubMed1990

[Further characterization of the orange carotenoid protein extracted from the marine demosponge Axinella verrucosa].

The orange coloration of the marine sponge A. verrucosa is provided by some carotenoids widespread in the ectosome and mesohyl of this sponge. These carotenoids are bound to a glyco(lipo)protein forming a non covalent complex. Six carotenoids are bound to the glyco(lipo)protein, but only alpha-carotene was identified by HPLC. The aminoacid composition is quite different from those previously reported in Porifera. The content of Ser and Gly and the total polar residues are high. The presence of Met and Pro was not evidenced. Some unusual aminoacids were detected, of which only Taurine was probably identified.

Amino Acids↗
PubMed1989

Identification in turkey gizzard of an acidic protein related to the C-terminal portion of smooth muscle myosin light chain kinase.

The isolation of an acidic protein, pI 4.5, that is abundant in turkey gizzard is described. Its apparent molecular weight measured by electrophoretic procedures is 24,000. This protein is phosphorylated by the catalytic subunit of the cAMP-dependent protein kinase and one phosphorylation site is indicated. From sequence determinations of tryptic peptides it is concluded that this protein is closely related to the C-terminal part of smooth muscle myosin light chain kinase. The initiation site for the protein is to the C-terminal side of the calmodulin-binding site. From the sequence data an estimated molecular weight is 18,000. This protein is expressed independently, as indicated by a blocked N terminus, and is probably the translation product of the 2.7-kilobase RNA detected previously in chicken gizzard (Guerriero, V., Jr., Russo, M. A., Olson, N. J., Putkey, J. A., and Means, A. R. (1986) Biochemistry 25, 8372-8381). Because of its putative origin as the C-terminal end of smooth muscle myosin light chain kinase, it is termed "telokin" (from a combination of kinase and the Greek telos, "end").

Amino Acid Sequence↗
PubMed1989

HSP70-related proteins in bovine skeletal muscle.

Constitutive expression of HSP70-related proteins was detected in a variety of bovine tissues using a specific antibody. All tissues contained a 73 kilodalton protein. A lower molecular weight form (72 kilodaltons) that co-migrated on two-dimensional gels with the stressed-induced HSP70 was present in high levels in bovine skeletal muscle, but absent from rat skeletal muscle. Two-dimensional gel analysis revealed several isoforms for both the 73 and 72 kilodalton forms. Purification of HSP70-related proteins from bovine skeletal muscle, thymus gland and rat skeletal muscle demonstrated that the antibody recognized all the forms present in the tissue homogenates. The two proteins are similar but distinct as detected by one-dimensional peptide mapping. The lower molecular form was not present in fetal tissue but was detectable in newborn animals, suggesting that the levels are regulated during development.

Animals↗
PubMed1989

[Caroteno-protein complexes in Axinella verrucosa (Porifera: Demospongae)].

Carotenoproteins are common to a wide variety of animals, most notably invertebrates although other animal groups have been shown to have these complexes as well. In a previous paper alpha-carotene, renieratene, isorenieratene and an unknown isomer of renieratene were isolated, but at present nothing is known about nature of the carotenoid-protein complexes in A. verrucosa. In this paper we report our results about a carotenoid-protein complex extracted by TRITON X-100 and purified by Sephadex G 200 Fine and DEAE-Sephacel chromatography. This carotenoprotein had a molecular weight of ca. 390,000 dalton (Gel electrophoresis). SDS PAGE plate showed only a single polypeptide chain of ca. 50,000 dalton. The proteic band separated by PAGE plate electrophoresis showed the presence of lipids and glycids when stained respectively by Sudan Black B and by Schiff's reagent. The extract obtained without TRITON X-100 showed the presence of a similar carotenoid-protein complex, but because of the low proteic concentration this result should be confirmed.

Animals↗
PubMed1987

The calmodulin binding domain of chicken smooth muscle myosin light chain kinase contains a pseudosubstrate sequence.

Smooth muscle myosin light chain kinase contains a 64 residue sequence that binds calmodulin in a Ca2+-dependent manner (Guerriero, V., Jr., Russo, M. A., and Means, A. R. (1987) Biochemistry, in press). Within this region is a sequence with homology to the corresponding sequence reported for the calmodulin binding region of skeletal muscle myosin light chain kinase (Blumenthal, D. K., Takio, K., Edelman, A. M., Charbonneau, H., Titani, L., Walsh, K. A., and Krebs, E. G. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 3187-3191). Inspection of these sequences reveals that they both share a similar number and spatial arrangement of basic residues with those present in the myosin light chain substrate. We have synthesized a 22-residue peptide corresponding to residues 480-501 (determined from the cDNA) of the smooth muscle myosin light chain kinase. This peptide, Ala-Lys-Lys-Leu-Ser-Lys-Asp-Arg-Met-Lys-Lys-Tyr-Met-Ala-Arg-Arg-Lys-Trp- Gln-Lys-Thr-Gly, inhibited calmodulin-dependent activation of the smooth muscle myosin light chain kinase with an IC50 of 46 nM. At saturating concentrations of calmodulin, the 22-residue peptide inhibited myosin light chain and synthetic peptide substrate phosphorylation competitively with IC50 values of 2.7 and 0.9 microM, respectively. An 11-residue synthetic peptide analog, corresponding to part of the calmodulin-binding sequence in skeletal muscle myosin light chain kinase, Lys-Arg-Arg-Trp-Lys-Lys-Asn-Phe-Ile-Ala-Val, also competitively inhibited synthetic peptide substrate phosphorylation with a Ki of 1 microM. The competitive inhibitory activity of the calmodulin binding regions is similar to the apparent Km of 2.7 microM for phosphorylation of the 23-residue peptide analog of the smooth muscle myosin light chain and raises the possibility that the calmodulin binding region of the myosin light chain kinase may act as a pseudosubstrate inhibitor of the enzyme.

Amino Acid Sequence↗
PubMed1987

Hormonal regulation of a chicken oviduct messenger ribonucleic acid that shares a common domain with gizzard myosin light chain kinase.

Changes in myosin light chain kinase (MLCK) and calmodulin (CaM) mRNAs have been evaluated during estrogen-mediated differentiation of the chicken oviduct. Also examined were acute changes that occur in oviduct RNA from animals stimulated with estrogen, withdrawn from hormone and then injected for 1, 2, and 4 days with synthetic estrogen [diethylstilbestrol (DES)], progesterone (P), or testosterone (T). Small changes were noted in both CaM and MLCK RNAs during primary stimulation when oviduct cells are actively dividing. On the other hand no significant changes were observed during secondary stimulation regardless of the steroid hormone injected. These data support the contention that CaM and MLCK are constitutively expressed but vary as a function of cell cycle. The MLCK mRNA is 5.5 kilobases (kb) but the MLCK cDNA also hybridizes to an oviduct RNA 2.7 kb long. This RNA species is acutely regulated by estrogen, P, and T but in a manner different from that of ovalbumin mRNA. The magnitude of stimulation of the 2.7 kb mRNA by diethylstilbestrol and T is greater than that of ovalbumin whereas changes in response to P are similar. The 12- to 16-fold increase of the 2.7 kb mRNA in response to T is the largest effect reported for this hormone acting on oviduct. The 2.7 kb mRNA encodes an unknown protein yet contains a 520 nucleotide segment that is highly homologous with the COOH-terminal coding portion of the MLCK mRNA. Since this homology does not include either catalytic or CaM-binding domains of MLCK, it is unlikely that the 2.7 kb mRNA encodes a CaM-dependent protein kinase.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1986

Domain organization of chicken gizzard myosin light chain kinase deduced from a cloned cDNA.

Myosin light chain kinases (MLCK) are the most studied of the calmodulin-activated enzymes; however, minimal sequence information is available for the smooth muscle form of the enzyme. The production of an antibody against the enzyme and the use of expression vectors for constructing cDNA libraries have facilitated the isolation of a cDNA for this kinase. The derived amino sequence was found to contain a region of high homology (54%) to the rabbit skeletal muscle enzyme and also very significant homology (35%) to the catalytic subunit of phosphorylase b kinase and cGMP-dependent protein kinase. All of these homologies were found in the known catalytic domains of these enzyme, thus enabling us to predict the location of the catalytic domain for the chicken gizzard myosin light chain kinase. Within the catalytic domain a consensus sequence for an ATP-binding site was located. Subcloning and expression of different regions of the cDNA defined a 192 base pair fragment coding for the calmodulin-binding domain of MLCK. Both of the cAMP-dependent protein kinase phosphorylation sites were identified by sequence homology. A linear model for MLCK is presented placing the various domains in relative position. Northern blot analysis and S1 protection and mapping experiments have revealed that the mRNA for MLCK is 5.5 kilobases in length, but there also exists a second mRNA of 2.7 kilobases that shares a high degree of homology with about 520 base pairs at the 3' end of the cDNA for MLCK.

Amino Acid Sequence↗
PubMed1985

Functional domains of chicken gizzard myosin light chain kinase.

The proteolytic susceptibility of chicken gizzard myosin light chain kinase, a calmodulin-dependent enzyme, has been utilized to define the relative location of the catalytic and regulatory domains of the enzyme. Myosin light chain kinase isolated from this source exhibits a Mr of 130,000 and is extremely sensitive to trypsin at 24 degrees C; however, the molecule is divided into susceptible and resistant domains such that proteolysis proceeds rapidly and at multiple sites in the sensitive regions even at 4 degrees C while the rest of the molecule remains relatively resistant to digestion. One of these sensitive areas is the calmodulin-binding domain. On the other hand, Staphylococcus aureus V8 protease digestion generates a calmodulin-binding fragment (Mr = 70,000) that retains Ca2+/calmodulin-dependent enzymatic activity and both of the phosphorylation sites recognized by cAMP-dependent protein kinase. In contrast, treatment with chymotrypsin produces a 95,000 Mr calmodulin-binding fragment that contains only the calmodulin-modulated phosphorylation site. Sequential proteolytic digestion studies demonstrated that the chymotryptic cleavage site responsible for the generation of this 95,000 Mr peptide is within 3,000 Mr of the V8 protease site which produces the 70,000 Mr fragment. Moreover, the non-calmodulin-modulated phosphorylation site must exist in this 3,000 Mr region. A calmodulin-Sepharose affinity adsorption protocol was developed for the digestion and used to isolate both the 70,000 and 95,000 Mr fragments for further study. Taken together, our results are compatible with a model for chicken gizzard myosin light chain kinase in which there is no overlap between the active site, the calmodulin-binding region, and the two sites phosphorylated by cAMP-dependent protein kinase with regard to their relative position in the primary sequence of the molecule.

Animals↗
PubMed1984

Estrogen stimulates the transient association of calmodulin and myosin light chain kinase with the chicken liver nuclear matrix.

Previous work has demonstrated that estrogen administration to immature chickens results in a rapid but transient increase in nuclear estrogen receptor content, a large portion of which is associated with the nuclear matrix. The present studies were undertaken to determine whether estrogen produced a more generalized change in the protein composition of the nuclear matrix. High-resolution two-dimensional gel analysis of the matrix revealed a very complex protein pattern, but several major qualitative differences were observed after estrogen treatment. To simplify the number of proteins evaluated, we examined the effects of estrogen on a subset of matrix proteins, namely, calmodulin and its binding proteins. Calmodulin was measured by radioimmunoassay and the binding proteins were detected by interaction of 125I-calmodulin with matrix proteins distributed on one-dimensional polyacrylamide gels. Calmodulin and two specific Ca2+-dependent calmodulin-binding proteins were found to be associated with matrix preparations. The two binding proteins exhibited apparent Mr of 200,000 and 130,000. The Mr 130,000 protein was identified as myosin light chain kinase on the basis of enzymatic activity and immunoreactivity with a specific antibody to this enzyme. Estrogen treatment of immature chickens did not alter the hepatic content of calmodulin. However, the steroid did result in an enrichment of the proportion of calmodulin and its two binding proteins associated with the nuclear matrix within 4 h after injection. The time course of these changes paralleled those previously documented for estrogen receptor. Taken together, these data are compatible with a role for calmodulin and myosin light chain kinase in the response of chicken liver cells to steroid hormones.

Animals↗
PubMed1982

Association of myosin light chain kinase with lymphocyte membrane-cytoskeleton complex.

A specific antibody against myosin light chain kinase (MLCK) was used to identify the presence of a Ca2+-calmodulin-activated MLCK in mouse 1-lymphoma cells. With a double immunofluorescence technique, MLCK was determined to be accumulated directly under Con A-capped structures in a manner similar to that of previously described accumulation of actomyosin. The lymphocyte MLCK was phosphorylated in the uncapped cell and, by immunoprecipitation with a specific MLCK antibody, was shown to possess a Mr of 130,000. The MLCK was also found to constitute a major fraction of the phosphoproteins present in the plasma membrane associated-cytoskeleton. Myosin light chain kinase catalyzed the phosphorylation of both endogenous lymphocyte myosin light chains and those from smooth and skeletal muscle. The enzyme activity was dependent on the presence of Ca2+-calmodulin and was inhibited by the calmodulin-binding drug, trifluoperazine. These data suggest that the membrane-cytoskeleton-associated MLCK activity may be important in regulation of the actinmyosin contraction which is believed to be required for the collection of surface receptors into capped structures.

Animals↗