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A V Gomes

Publications and source records attributed to A V Gomes.

12 recordsLinked to original sources
PubMed2001

A two-dimensional protein map of Caenorhabditis elegans.

A protein map of Caenorhabditis elegans was constructed by using two-dimensional gel electrophoresis followed by peptide mass fingerprinting. A whole worm extract of a mixed population was separated on immobilized pH gradient strips 4-7 L, 3-10 NL, 6-11 L and 12% sodium dodecyl sulfate-polyacrylamide gel eletrophoresis (SDS-PAGE) gels. Gels were stained with colloidal Coomassie blue and 286 spots representing 152 proteins were subsequently identified by matrix-assisted laser desorption/ionization-mass spectrometry after in-gel digestion with trypsin. Most of the identified proteins with known cellular function were enzymes related to carbohydrate and lipid metabolism, or structural proteins with subcellular locations in the cytoplasm, mitochondria or cytoskeleton.

Animals↗
PubMed2000

Familial hypertrophic cardiomyopathy mutations in the regulatory light chains of myosin affect their structure, Ca2+ binding, and phosphorylation.

The effect of the familial hypertrophic cardiomyopathy mutations, A13T, F18L, E22K, R58Q, and P95A, found in the regulatory light chains of human cardiac myosin has been investigated. The results demonstrate that E22K and R58Q, located in the immediate extension of the helices flanking the regulatory light chain Ca(2+) binding site, had dramatically altered Ca(2+) binding properties. The K(Ca) value for E22K was decreased by approximately 17-fold compared with the wild-type light chain, and the R58Q mutant did not bind Ca(2+). Interestingly, Ca(2+) binding to the R58Q mutant was restored upon phosphorylation, whereas the E22K mutant could not be phosphorylated. In addition, the alpha-helical content of phosphorylated R58Q greatly increased with Ca(2+) binding. The A13T mutation, located near the phosphorylation site (Ser-15) of the human cardiac regulatory light chain, had 3-fold lower K(Ca) than wild-type light chain, whereas phosphorylation of this mutant increased the Ca(2+) affinity 6-fold. Whereas phosphorylation of wild-type light chain decreased its Ca(2+) affinity, the opposite was true for A13T. The alpha-helical content of the A13T mutant returned to the level of wild-type light chain upon phosphorylation. The phosphorylation and Ca(2+) binding properties of the regulatory light chain of human cardiac myosin are important for physiological function, and alteration any of these could contribute to the development of hypertrophic cardiomyopathy.

Amino Acid Sequence↗
PubMed2000

Abnormal contractile function in transgenic mice expressing a familial hypertrophic cardiomyopathy-linked troponin T (I79N) mutation.

This study characterizes a transgenic animal model for the troponin T (TnT) mutation (I79N) associated with familial hypertrophic cardiomyopathy. To study the functional consequences of this mutation, we examined a wild type and two I79N-transgenic mouse lines of human cardiac TnT driven by a murine alpha-myosin heavy chain promoter. Extensive characterization of the transgenic I79N lines compared with wild type and/or nontransgenic mice demonstrated: 1) normal survival and no cardiac hypertrophy even with chronic exercise; 2) large increases in Ca(2+) sensitivity of ATPase activity and force in skinned fibers; 3) a substantial increase in the rate of force activation and an increase in the rate of force relaxation; 4) lower maximal force/cross-sectional area and ATPase activity; 5) loss of sensitivity to pH-induced shifts in the Ca(2+) dependence of force; and 6) computer simulations that reproduced experimental observations and suggested that the I79N mutation decreases the apparent off rate of Ca(2+) from troponin C and increases cross-bridge detachment rate g. Simulations for intact living fibers predict a higher basal contractility, a faster rate of force development, slower relaxation, and increased resting tension in transgenic I79N myocardium compared with transgenic wild type. These mechanisms may contribute to mortality in humans, especially in stimulated contractile states.

Animals↗
PubMed2000

Selective degradation of annexins by chaperone-mediated autophagy.

Annexins are a family of proteins that bind phospholipids in a calcium-dependent manner. Analysis of the sequences of the different members of the annexin family revealed the presence of a pentapeptide biochemically related to KFERQ in some annexins but not in others. Such sequences have been proposed to be a targeting sequence for chaperone-mediated autophagy, a lysosomal pathway of protein degradation that is activated in confluent cells in response to removal of serum growth factors. We demonstrate that annexins II and VI, which contain KFERQ-like sequences, are degraded more rapidly in response to serum withdrawal, while annexins V and XI, without such sequences, are degraded at the same rate in the presence and absence of serum. Using isolated lysosomes, only the annexins containing KFERQ-like sequences are degraded by chaperone mediated-autophagy. Annexins V and XI could associate with lysosomes but did not enter the lysosomes and were not proteolytic substrates. Furthermore, four annexins containing KFERQ-like sequences, annexins I, II, IV, and VI, are enriched in lysosomes with high chaperone-mediated autophagy activity as expected for substrate proteins. These results provide striking evidence for the importance of KFERQ motifs in substrates of chaperone-mediated autophagy.

Amino Acid Motifs↗
PubMed2000

Spectroscopic characterization of the interaction between calmodulin-dependent protein kinase I and calmodulin.

Calmodulin-dependent protein kinase I (CaM kinase I) is a member of the expanding class of protein kinases that are regulated by calmodulin (CaM). Its putative CaM-binding region is believed to occur within a 22-residue sequence (amino acids 299-320). This sequence was chemically synthesized and utilized for CaM interaction studies. Gel band shift assays and densitometry experiments with intact CaM kinase I and the CaM-binding domain peptide (CaMKIp) reveal that they bind in an analogous manner, giving rise to 1:1 complexes. Fluorescence analysis using dansyl-CaM showed that conformational changes in CaM on binding CaM kinase I or CaMKIp were nearly identical, suggesting that the peptide mimicked the CaM-binding ability of the intact protein. In the presence of CaM, the peptide displays an enhancement of its unique Trp fluorescence as well as a marked blue shift of the emission maximum, reflecting a transfer to a more rigid, less polar environment. Quenching studies, using acrylamide, confirmed that the Trp in the peptide on binding CaM is no longer freely exposed to solvent as is the case for the free peptide. Studies with a series of Met mutants of CaM showed that the Trp-containing N-terminal end of CaMKIp was bound to the C-terminal lobe of CaM. Near-UV CD spectra also indicate that the Trp of the peptide and Phe residues of the protein are involved in the binding. These results show that the CaM-binding domain of CaM kinase I binds to CaM in a manner analogous to that of myosin light chain kinase.

Acrylamide↗
PubMed2000

Fatty acid composition and possible health effects of coconut constituents.

The link between excessive consumption of dietary saturated fats and coronary heart disease (CHD) is now well established. Because of its high content of saturated fatty acids, the consumption of foods containing coconut oil may therefore be a risk factor for CHD. While the fatty acid composition of coconut oil is well established, relatively little is known about the other constituents of coconut: the milk, water, cream and meat fractions. In this study, we show that while the water fraction is low in lipid content, the milk contains about 24% of the fat content of oil and the cream and meat fractions about 34%. The other coconut constituents contain significant amounts of medium-chain triglycerides that are formed from fatty acids of chain length 8:0 to 14:0. It is these fatty acids, primarily 14:0, that are thought to be atherogenic. On the other hand, medium-chain triglycerides may be advantageous under some circumstances in that they are absorbed intact and do not undergo degradation and re-esterification processes. As a result, medium-chain triglycerides provide a ready source of energy and may be useful in baby foods or in diet therapy. Nevertheless, the possible negative effects of the saturated fatty acids and the absence of the essential fatty acid linolenic acid from all coconut constituents suggest that the coconut milk, oil and cream should not be used on a regular basis in adults.

Cocos↗
PubMed1997

Major proteins of yam bean tubers.

The tuberous roots of the Mexican yam bean, jicama, (Pachyrhizus erosus L. Urban) contained large quantities of two acidic glycoproteins which accounted for more than 70% of the total soluble proteins (about 3 g per 100 g of tuber on a dry weight basis). The two major proteins, tentatively named YBG1 and YBG2, had apparent M(r)s of 28,000 and 26,000, respectively, by SDS-PAGE. A third protein named YBP22 which accounted for 2-5% of the total soluble proteins had an M(r) of 22,000. YBG1 and YBG2 exhibited great similarity on the basis of their amino acid composition and had identical N-terminal amino acid sequences. The first 23 amino acids in the N-terminal region of YBG2 were DDLPDYVDWRDYGAVTRIKNQGQ which showed strong homology with the papain class of cysteine proteases. YBG1 and YBG2 were found to bind to a Concanavalin A-Sepharose column and were also stained positively by a sensitive glycoprotein stain. Both glycoproteins exhibited cysteine proteolytic activity. In contrast, YBP22 showed sequence homology with several known protease inhibitors, and a polyclonal antibody raised against this protein cross reacted with soybean trypsin inhibitor.

Amino Acid Sequence↗
PubMed1997

Protein phosphatases are pest containing proteins.

Protein phosphatases are required for removing phosphoryl groups in proteins involved in many physiological processes. Investigation of these enzymes for regions rich in proline (P), glutamic acid (E), serine (S) and threonine (T), called PEST regions showed that greater than 85% of the phosphatases investigated contained these regions. These regions are believed to be signals for degradation and could possibly serve as regulators of the intracellular localization and catalytic activity via limited proteolysis or as conditional signals for rapid degradation of these proteins by the ATP/ubiquitin-dependent and/or the ATP non-ubiquitin dependent proteolytic pathway. Many of these phosphatases were also found to contain a pentapeptide sequence biochemically related to the KFERQ motif which targets proteins for lysosomal degradation which suggest that several pathways may exist for the degradation of protein phosphatases.

Amino Acid Sequence↗
PubMed1995

Pest sequences in EF-hand calcium-binding proteins.

Calcium binding proteins are subdivided into two major families: the EF-family and the Annexin family. The EF-hand family is distinguished by the characteristic helix-loop-helix motif which consists of two alpha-helices separated by a loop. The EF-hand Ca2+(-)binding protein family contain subfamilies rich in proline, glutamic acid, serine and threonine residues (called PEST sequences) and non PEST-containing subfamilies. A few of the Ca2+(-)binding proteins contain KFERQ-like sequences which are thought to be possible signals for lysosomal degradation. Arginine pairs (RR) which have also been suggested to act as signals for proteolysis were found to be few or absent. However, most EF-hand proteins were found to possess lysine pairs (KK) which may also act as signals for proteolysis.

Amino Acid Sequence↗
PubMedDate not supplied

PEST sequences in calmodulin-binding proteins.

Many short-lived proteins which are devoid of proteolytic activity contain PEST sequences which are segments along the polypeptide chain that are rich in proline (P), glutamate (E), serine (S) and threonine (T). These designated PEST sequences are believed to be putative intramolecular signals for rapid proteolytic degradation. Calmodulin is a ubiquitous, 17 kDa, acidic Ca(2+)-binding protein which plays an important role in the regulation of many physiological processes through its interaction with a wide range of calmodulin-binding proteins. Several calmodulin-binding proteins are known to contain PEST sequences and are susceptible to proteolysis by endogenous neutral proteases such as calpain I and calpain II. In this report, we discuss the functions of PEST sequences in calmodulin-binding proteins and assess the correlation between calmodulin-binding proteins and PEST sequences.

Amino Acid Sequence↗