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Biomedical subjects

S Clarke

Publications and source records attributed to S Clarke.

At least 289 records · Page 16Linked to original sources
PubMed1989

Specificity of endoproteinase Asp-N (Pseudomonas fragi): cleavage at glutamyl residues in two proteins.

Endoproteinase Asp-N, a metalloprotease from a mutant strain of Pseudomonas fragi, has been reported to specifically cleave on the N-terminal side of aspartyl and cysteic acid residues. We utilized this enzyme to generate fragments for determining the amino acid sequence of the D-aspartyl/L-isoaspartyl methyltransferase isozyme I from human erythrocytes. Surprisingly, we identified cleavage sites for this enzyme at the N-terminal side of several glutamyl residues in addition to the expected cleavage sites at aspartyl residues. The ability of this enzyme to cleave polypeptides at both glutamyl and aspartyl residues was confirmed by mapping additional sites on erythrocyte carbonic anhydrase I. These results indicate that a more appropriate name for this enzyme may be Endoproteinase Asp/Glu-N.

Amino Acid Sequence↗
PubMed1989

Enzymatic methylation of 23-29-kDa bovine retinal rod outer segment membrane proteins. Evidence for methyl ester formation at carboxyl-terminal cysteinyl residues.

A group of 23-29-kDa polypeptides in the membranes of bovine rod outer segments are substrates for S-adenosylmethionine-dependent methylation reactions. The bulk of the methyl group incorporation is in base-labile ester-like linkages, and does not appear to be due to the widespread D-aspartyl/L-isoaspartyl methyltransferase (EC 2.1.1.77). To determine the site(s) of methylation, 3H-methylated proteins separated by polyacrylamide gel electrophoresis in sodium dodecyl sulfate were eluted and digested with papain, leucine aminopeptidase-M, and prolidase. After performic acid oxidation of the digest, a base-labile radioactive material was recovered that coeluted with a synthetic standard of cysteic acid methyl ester upon cation exchange and G-15 gel filtration chromatography, as well as in two thin-layer electrophoresis and two thin-layer chromatography systems. These results provide direct evidence for the methylation of the alpha-carboxyl group of a carboxyl-terminal cysteinyl residue, a modification that has been proposed for the 21-kDa Ha-ras product and other cellular proteins (Clarke, S., Vogel, J. P., Deschenes, R. J., and Stock, J. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 4643-4647).

Amino Acid Sequence↗
PubMed1989

RAS2 protein of Saccharomyces cerevisiae is methyl-esterified at its carboxyl terminus.

Yeast and mammalian RAS gene products are GTP-binding proteins that are posttranslationally localized to the inner surface of the plasma membrane. This localization is accomplished by the addition of a lipid moiety to a conserved cysteine residue close to the carboxyl terminus. In a previous report we showed that the mammalia Ha-ras protein is also modified posttranslationally by methyl esterification. Here we show that the yeast RAS2 protein is posttranslationally modified by methyl esterification at or near the carboxyl terminus. We also present evidence indicating that the methyl ester is linked to the conserved cysteine residue, implying that RAS2 protein is cleaved to expose this cysteine as the carboxyl-terminal residue. This maturation pathway may be shared by a family of proteins that are initially synthesized as soluble proteins and must become membrane-localized to function.

Chromatography, Liquid↗
PubMed1989

Calcium affects the spontaneous degradation of aspartyl/asparaginyl residues in calmodulin.

We have previously shown that the D-aspartyl/L-isoaspartyl protein carboxyl methyltransferase recognizes two major sites in affinity-purified preparations of bovine brain calmodulin that arise from spontaneous degradation reactions. These sites are derived from aspartyl residues at positions 2 and 78, which are located in apparently flexible regions of calmodulin. We postulated that this flexibility was an important factor in the nonenzymatic formation and enzymatic recognition of D-aspartyl and/or L-isoaspartyl residues. Because removal of Ca2+ ions from this protein may also lead to increased flexibility in the four Ca2+ binding regions, we have now characterized the sites of methylation that occur when calmodulin is incubated in buffers with or without the calcium chelator ethylene glycol bis(beta-aminoethyl ether)-N,N,-N',N'-tetraacetic acid (EGTA). Calmodulin was treated at pH 7.4 for 13 days at 37 degrees C under these conditions and was then methylated with erythrocyte D-aspartyl/L-isoaspartyl methyltransferase isozyme I and S-adenosyl-L-[methyl-3H]methionine. The 3H-methylated calmodulin product was purified by reverse-phase HPLC and digested with various proteases including trypsin, chymotrypsin, endoproteinase Lys-C, clostripain, and Staphylococcus aureus V8 protease, and the resulting peptides were separated by reverse-phase HPLC. Peptides containing Asp-2 and Asp-78, as well as calcium binding sites II, III, and IV, were found to be associated with radiolabel under these conditions. When calmodulin was incubated under the same conditions in the presence of calcium, methylation at residues in the Ca2+ binding regions was not observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1989

Succinimide formation from aspartyl and asparaginyl peptides as a model for the spontaneous degradation of proteins.

Nonenzymatic intramolecular reactions can result in the deamidation, isomerization, and racemization of protein and peptide asparaginyl and aspartyl residues via succinimide intermediates. To understand the sequence dependence of these reactions, we measured the rate of succinimide formation in a series of synthetic peptides at pH 7.4. These peptides (Val-Tyr-Pro-X-Y-Ala) contained an internal aspartyl, asparaginyl, aspartyl beta-methyl ester, or aspartyl alpha-methyl ester residue (X) followed by a glycyl, seryl, or alanyl residue (Y). The rates of succinimide formation of the asparaginyl peptides were found to be 13.1-35.6 times faster than those of the aspartyl peptides. The rates of succinimide formation for the glycyl peptides were 6.5-17.6 times faster than those of the alanyl peptides, while the rates for the seryl peptides were 1.6-4.5 times faster than those of the alanyl peptides. The overall 232-fold range in these reaction rates for aspartyl and asparaginyl residues suggests that sequence can be an important determinant in their stability in flexible peptides. In proteins, there may be a much larger range in the rates of succinimide formation because specific conformations may greatly enhance or inhibit this reaction.

Amino Acid Sequence↗
PubMed1989

Forms and measures of adult and developing human corpus callosum: is there sexual dimorphism?

The sexual dimorphism of the human corpus callosum (CC) is currently controversial, possibly because of difficulties in morphometric analysis. We have reinvestigated the issue by using morphometric techniques specially designed to yield objective measurements of CC size and shape. The development of the CC was studied with similar techniques in order to investigate whether its final shape and size might be influenced by axonal elimination, as could be expected from previous animal studies. We have measured the CCs of 32 men and 26 women; 27 male and 19 female CCs were from brain tissue, the others were from magnetic resonance imaging graphs. Women tended to have 1) a smaller cross-sectional callosal area (CCA); 2) a larger fraction of CCA in the posterior fifth of the CC; 3) more slender CCs; and 4) more bulbous splenia. These differences could not be detected by simple inspection but were demonstrated by measurement and statistical analysis. However, CCA was correlated with the other sexually dimorphic parameters, and the sex-related differences in the latter became nonsignificant when variations in CCA were factored out or when male and female populations with similar CCA were compared. In addition, we analyzed CCs of 16 male and 16 female fetuses and of 13 male and 15 female infants and children. This sample ranged in age between 20 weeks of gestation and 14 years but covered in detail the period up to 14 months after birth. CCA increased throughout the latter period but decreased slightly between about 33 weeks of gestation and the beginning of the second postnatal mouth. This decrease coincided with thinning of the CC and a marked increase in bulbosity of the splenium. No sexual dimorphism could be demonstrated until the beginning of the postnatal period. In the age group between birth (at term) and the 14th month, CCA was, as in the adult, larger in males. Unlike in the adults, the CC was longer in males and the bulbosity index was the same in the two sexes. Axonal elimination may play a role in the perinatal pause in CCA growth and in the concomitant changes in callosal shape.

Adolescent↗
PubMed1989

Enzymatic methylation of L-isoaspartyl residues derived from aspartyl residues in affinity-purified calmodulin. The role of conformational flexibility in spontaneous isoaspartyl formation.

We have investigated the formation of D-aspartyl and L-isoaspartyl (beta-aspartyl) residues and their subsequent methylation in bovine brain calmodulin by the type II protein carboxyl methyltransferase. Based on the results of studies with unstructured peptides and denatured proteins, it has been proposed that the major sites of carboxyl methylation in calmodulin are at L-isoaspartyl residues that originate from two Asn-Gly sequences. To test this hypothesis, we directly identified the sites of methylation in affinity-purified preparations of calmodulin by peptide mapping using the proteases trypsin, endoproteinase Lys-C, clostripain, chymotrypsin, and Staphylococcus aureus V8 protease. We found, however, that the major high-affinity sites of methylation originate from aspartyl residues at position 2 and at positions 78 and/or 80. The methylatable residue in the first case was shown to be L-isoaspartate by comparison of the properties of a synthetic peptide corresponding to the N-terminal 13 residues substituted with an L-iso-Asp residue at position 2. The second methylatable residue, probably derived from Asp78, also appears to be an L-isoaspartyl residue. These sites appear to be readily accessible to the methyltransferase and are present in relatively flexible regions of calmodulin that may allow the spontaneous degradation reactions to occur that generate L-isoaspartyl residues via succinimide intermediates. Interestingly, the four calcium binding regions, each containing 3-4 aspartyl and asparaginyl residues (including the two Asn-Gly sequences), do not appear to contribute to the high-affinity methyl acceptor sites, even when calcium is removed prior to the methylation reaction. We propose that methylatable residues do not form at these sites because of the inflexibility of these regions when calcium is bound.

Amino Acid Sequence↗
PubMed1989

The membrane binding domain of rod cGMP phosphodiesterase is posttranslationally modified by methyl esterification at a C-terminal cysteine.

Retinal rod cGMP phosphodiesterase (3',5'-cyclic-GMP phosphodiesterase; EC 3.1.4.35; PDE), a key regulatory enzyme involved in visual excitation, is one of several outer segment membrane proteins that are carboxyl methylated in the presence of the methyl donor S-adenosyl-L-[3H-methyl]methionine. By chromatographic analyses of the 3H-methyl amino acid generated by exhaustive proteolysis of purified PDE, followed by performic acid oxidation of the digest, we have shown that this modification occurs at a C-terminal cysteine residue of the alpha subunit of this enzyme. When PDE is subjected to limited proteolysis with trypsin, a 3H-methylated fragment of 1000 daltons or less is rapidly removed prior to the degradation of its inhibitory gamma subunit. This small fragment remains membrane bound, whereas the bulk of the enzyme is released, indicating that a domain responsible for anchoring PDE to the membrane is located near the C terminus. Based on the C-terminal amino acid sequence of Cys-Cys-Val-Gln predicted from the alpha cDNA sequence, we conclude that PDE undergoes posttranslational modifications, including the proteolytic removal of two or three terminal amino acids, and methyl esterification of the alpha-carboxyl group of the terminal cysteine residue. We speculate that the sulfhydryl group of the methylated cysteine is also lipidated to mediate membrane binding. These modifications may play an important role in delivering the nascent PDE chains to the membrane and in correctly positioning the PDE molecule in the rod disks for phototransduction.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1989

The Block Nurse Program.

In the Block Nurse Program, existing resources and agencies are used to help keep elderly persons in their own homes with case-management by public health nurses living in the community. In an external evaluation of the program it was documented that a neighborhood long-term care delivery model not only works, but keeps elderly persons in their homes at low cost and with high care standards.

Aged↗
PubMed1989

Combination therapy with gold and hydroxychloroquine in rheumatoid arthritis: a prospective, randomized, placebo-controlled study.

We studied combination therapy with two slow-acting antirheumatic drugs given concurrently in active rheumatoid arthritis (RA). A 12-month prospective randomized controlled trial compared gold and hydroxychloroquine in 52 patients to gold and placebo in 49. The patients continued to receive non-steroidal anti-inflammatory drugs and analgesics. They were selected from three rheumatology centres in the West Midlands. Combination therapy led to a greater number of withdrawals due to adverse reactions (18 cases compared to 10 receiving gold/placebo). Patients completing 12 months' therapy (27 taking gold/hydroxychloroquine and 32 on gold/placebo) were compared using five clinical, seven laboratory, and one radiological measure. All 13 variables favoured gold/hydroxychloroquine with an overall advantage of 20-25% for the combination. This only reached statistical significance (at the 1% level) for C-reactive protein. An overall disease activity index was better at 12 months (at the 5% level) and showed a more rapid response with gold/hydroxychloroquine. This is the first randomized prospective placebo-controlled trial to show a significant advantage from a combination of two slow-acting drugs. There are many different ways of giving such combinations and we consider these should be explored to maximize the effectiveness of treatment for RA.

Adult↗
PubMed1989

Experience in imaging medullary thyroid carcinoma using 99mTc (V) dimercaptosuccinic acid (DMSA).

99mTc (V) dimercaptosuccinic acid (DMSA) is a new tumor imaging agent that has been successfully used to image patients with medullary thyroid carcinoma (MTC). Since 1986, studies have been performed in 32 patients with histologically proven MTC at Guy's Hospital, London, England. Five patients with primary tumor were studied prior to surgery, four patients were studied after successful removal of the primary tumor, and 26 patients with biochemical evidence of recurrence were studied. Eight patients were studied serially to assess progression of disease, and four patients were studied before and after surgery. Twenty-one of the 26 patients with disease had positive scans with four false-negative scans and three true negative scans. One patient had a false-positive scan (sensitivity 80%, specificity 75%). Two of the false-negative scans were obtained in patients with moderate but stable elevations of calcitonin but no other evidence of recurrence. One false-negative scan was obtained in a patient who was discovered on screening to have an abnormal pentagastrin response, and a small 1 cm tumor was subsequently removed. Uptake in local neck recurrence was frequently intense, but uptake at sites of bone metastases was less marked. 99mTc (V) DMSA is an inexpensive radiopharmaceutical which produces good quality images and has been shown to have an acceptable sensitivity and specificity in the follow-up of patients with MTC and thereby contributes significantly to the management of these patients.

Adolescent↗
PubMed1988

Purification of homologous protein carboxyl methyltransferase isozymes from human and bovine erythrocytes.

We have purified the two major isozymes of the L-isoaspartyl/D-aspartyl protein methyltransferase from both human and bovine erythrocytes. These four enzymes all have polypeptide molecular weights of approximately 26,500 and appear to be monomers in solution. Each of these enzymes cross-reacts with antibodies directed against protein carboxyl methyltransferase I from bovine brain. Their structures also appear to be similar when analyzed by dodecyl sulfate gel electrophoresis for the large fragments produced by digestion with Staphylococcus aureus protease V8 or when analyzed by high-performance liquid chromatography (HPLC) for tryptic peptides. The structural relatedness of these enzymes was confirmed by sequence analysis of a total of 433 residues in 32 tryptic fragments of the human erythrocyte isozymes I and II and of the bovine erythrocyte isozyme II. We found sequence identify or probable identity in 111 out of 112 residues when we compared the human isozymes I and II and identities in 127 out of 134 residues when the human and bovine isozymes II were compared. These results suggest that the erythrocyte isozymes from both organisms may have nearly identical structures and confirm the similarities in the function of these methyltransferases that have been previously demonstrated.

Amino Acid Sequence↗
PubMed1988

Development of projections from auditory to visual areas in the cat.

In newborn kittens, cortical auditory areas (including AI and AII) send transitory projections to ipsi- and contralateral visual areas 17 and 18. These projections originate mainly from neurons in supragranular layers but also from a few in infragranular layers (Innocenti and Clarke: Dev. Brain Res. 14:143-148, '84; Clarke and Innocenti: J. Comp. Neurol. 251:1-22, '86). The postnatal development of these projections was studied with injections of anterograde tracers (wheat germ agglutinin-horseradish peroxidase [WGA-HRP]) in AI and AII and of retrograde tracers (WGA-HRP, fast blue, diamidino yellow, rhodamine-labeled latex beads) in areas 17 and 18. It was found that the projections are nearly completely eliminated in development, this, by the end of the first postnatal month. Until then, most of the transitory axons seem to remain confined to the white matter and the depth of layer VI; a few enter it further but do not appear to form terminal arbors. As for other transitory cortical projections the disappearance of the transitory axons seems not to involve death of their neurons of origin. In kittens older than 1 month and in normal adult cats, retrograde tracer injections restricted to, or including, areas 17 and 18 label only a few neurons in areas AI and AII. Unlike the situation in the kitten, nearly all of these are restricted to layers V and VI. A similar distribution of neurons projecting from auditory to visual areas is found in adult cats bilaterally enucleated at birth, which suggests that the postnatal elimination of the auditory-to-visual projection is independent of visual experience and more generally of information coming from the retina.

Aging↗