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Biomedical subjects

A Ramaiah

Publications and source records attributed to A Ramaiah.

At least 19 recordsLinked to original sources
PubMed2000

Melanosomal proteins promote melanin polymerization.

In melanocytes, enzymes involved in the generation of melanin monomers are present and active in coated vesicles which are known to be acidic. Melanin polymerization however, occurs only in melanosomes. In vitro, it is not possible to generate melanin at the acidic pH of melanosomes using 3,4-dihydroxyphenylalanine (DOPA) and tyrosinase alone whereas melanin readily forms at higher pH with these reagents. Dimerization and elongation of the melanin polymer is known to require deprotonation. We have hypothesized that the amino acid side chains of melanosomal proteins act as proton acceptors to initiate polymerization and that the protonated basic groups serve to attract the negatively charged oligomers thus aiding polymerization and binding to proteins. We show that basic model proteins and basic premelanosomal proteins promote polymerization at an acidic pH and that positively charged surfaces allow binding of the growing melanin polymer. With progressive polymerization and exhaustion of the proton abstracting ability of melanosomal proteins, melanosomal pH drops further, which, we argue, is an additional controlling step that limits tyrosinase activity and melanin polymerization.

Animals↗
PubMed1999

Microphthalmia-associated transcription factor (MITF) locus lacks linkage to human vitiligo or osteopetrosis: an evaluation.

The microphthalmia-associated transcription factor (MITF) locus has been mapped to human chromosome 3p12-p14.1, and encodes a basic helix-loop-helix zipper (bHLH-ZIP) protein homologous to a number of transcription factors. Numerous mutations at the mouse microphthalmia (mi) locus have been described, and all have reduced or absent pigmentation of the eyes, ears, and/or pelage, with some genotypes exhibiting small or absent eyes and osteopetrosis. The mivit/vit mutation at the mouse mi locus produces a postnatal depigmentation that resembles human vitiligo. The mice homozygous for this mi allele show a progressive loss of cutaneous, hair and ocular pigmentation with age. Vitiligo, an acquired depigmentary disorder, is characterized by patchy depigmentation of skin that generally begins around puberty and tends to become more progressive over time. There is suggestive evidence that human vitiligo may be inherited; however, the mode of inheritance is still debated and the pathogenesis is not clearly delineated. The human disorder osteopetrosis is characterized by a generalized net accumulation of skeletal mass and results from reduced osteoclast function in the bone. This is an inherited disorder and has been associated with mi in a mutant mouse. Therefore, the possible involvement of the MITF locus in the pathogenesis of either familial vitiligo or osteopetrosis was investigated. Linkage analysis was performed using microsatellite polymorphic markers D3S2465, D3S1261, and D3S1766 on genomic DNA from 26 families with vitiligo/osteopetrosis. D3S1261 is physically located at or near the MITF locus, while D3S2465 and D3S1766 are flanking the locus at about 17.5 cM genetic distance each side. Evidence from LOD score analysis surprisingly indicated that none of the families with vitiligo or osteopetrosis are linked to these short tandem repeat polymorphisms (STRPs). Thus, the human homolog (MITF) of the mouse mi gene, a good candidate gene at the phenotypic level, may not be involved in the pathogenesis of familial human vitiligo or osteopetrosis.

Adolescent↗
PubMed1996

Lag kinetics of tyrosinase: its physiological implications.

The various theories put forward to explain the characteristic lag kinetics of oxidation of L-tyrosine by tyrosinase a rate regulatory step in the biosynthesis of melanin are reviewed Examination of the evidence in the literature and from experiments in the author's laboratory indicate that one of the hypotheses, that is, competition of tyrosine and dopa for met-tyrosinase and the formation of a dead-end complex of met-enzyme with tyrosine as explanation for lag kinetics is not consistent with available information. The alternative hypothesis that tyrosinase is an allosteric enzyme with tyrosine having negative effector site on the enzyme and dopa competing for it as an explanation for lag kinetics of tyrosinase is not yet disproved. Irrespective of the actual explanation for the lag kinetics of tyrosinase, it is suggested that the highly conserved lag kinetics may serve a physiological function. It is suggested that this function is to keep the enzyme essentially inactive during its transport to the specific organelle, namely the melanosome, in which an acidic environment exists. Only at acidic pH is the enzyme able to catalyze the biosynthesis of melanin.

Allosteric Site↗
PubMed1993

pH of melanosomes of B 16 murine melanoma is acidic: its physiological importance in the regulation of melanin biosynthesis.

It was postulated by us that melanin biosynthesis cannot take place unless the melanosomal pH is acidic. That melanosomal pH may be acidic was suggested by other researchers as well. However, the actual pH was never determined. The current study was undertaken to estimate the intramelanosomal pH. Lightly melanized and heavily melanized melanosomes from B16 murine melanoma tissue were prepared by sucrose density gradient centrifugation and their internal pH was estimated. It was shown that quenching of fluorescence of 9-aminoacridine by melanosomes was to a large extent dependent on the pH difference between melanosomes and the medium in which they were suspended and it was used to determine the pH of lightly and heavily melanized melanosomes from B16 melanoma. The pH of lightly melanized melanosomes and heavily melanized melanosomes was found to be in the range of 4.2 to 4.6 and 3.0, respectively. These organelles were shown to hydrolyze fluorescein diacetate to fluorescein. The excitation spectrum of fluorescein generated in lightly melanized melanosomes and heavily melanized melanosomes also indicated that the pH in lightly melanized melanosomes was 4.6 and lower than 4.6 in heavily melanized melanosomes. The enzymes are active at such low pH. These organelles are also shown to contain proton-translocating ATPase, which may be involved in the acidification of lightly melanized melanosomes, possibly in addition to melanin biosynthesis. These results confirm our original postulate that melanin biosynthesis that occurs in melanosomes takes place under acidic conditions.

Ammonium Chloride↗
PubMed1989

Growth defects of melanocytes in culture from vitiligo subjects are spontaneously corrected in vivo in repigmenting subjects and can be partially corrected by the addition of fibroblast-derived growth factors in vitro.

Melanocytes cultured from uninvolved skin of untreated vitiligo subjects have decreased initial seeding capacities, manifest a lag period for the onset of the growth phase, and cannot be passaged. In contrast, melanocytes obtained from uninvolved and perilesional skin of vitiligo subjects actively repigmenting under 8-methoxy psoralen plus sunlight (PUVA) therapy have higher initial seeding capacities, grow faster without a lag period, and can be passaged to more than 12 passages. Extracts of a fetal lung fibroblast cell line (PMR-GF) that promote the growth rates and passage capacities of melanocytes from normal adult donors have been found also to promote the growth rates and passage capacities of melanocytes from the uninvolved skin of vitiligo subjects. Extracts of a fetal lung fibroblast cell line (PMR-GF), however, did not have any further stimulatory effect on the growth of melanocytes obtained from repigmenting vitiligo subjects. Melanocytes cultured from normal and untreated vitiligo subjects grew individually dispersed in the absence of PMR-GF, but tended to grow in clusters in its presence. Melanocytes from the repigmenting vitiligo subjects, however, tended to grow in clusters even in the absence of PMR-GF. These results indicate that the defective in vitro growth characteristics of melanocytes from vitiligo subjects may be related to the pathogenesis of this disease. It is possible that growth factors may be involved in the process of repigmentation in vitiligo subjects.

Adolescent↗
PubMed1989

Etiology of vitiligo. A new hypothesis.

Serum from actively repigmenting human vitiligo subjects had maximum mitogenic effect on the growth of melanocytes in culture, followed by the serum from normal donors, and from untreated vitiligo subjects in that order. Based on these findings, a new hypothesis is suggested for the etiology of vitiligo.

Adult↗
PubMed1988

pH-dependent interconversion of two forms of tyrosinase in human skin.

1. We have shown that the characteristic lag in cresolase activity of human skin tyrosinase at inhibitory concentration of tyrosine was absent at all pH values studied, i.e. pH 5.2, 5.7, 6.2 and 6.8, if the enzyme solubilized at low pH was used as the source of enzyme, but the same enzyme when dialysed against buffers of various pH values showed linear activity only at pH 5.2 and was not inhibited by excess tyrosine, whereas at higher pH values it exhibited a lag and inhibition by excess tyrosine. 2. However, the enzyme solubilized in buffer/detergent, pH 6.8, when dialysed against buffer of the same pH showed linear activity at pH 5.2 and non-linear activity at pH 6.8. 3. The water/detergent-solubilized enzyme from human skin melanosomes showed linear activity even at inhibitory concentrations of tyrosine at pH 5.2 and 6.8 up to 2 h, but acceleration of rate was observed after 2 h for the enzyme measured at pH 6.8. 4. After dialysis of the water/detergent-solubilized enzyme against double-glass-distilled water, it still exhibits linear activity at inhibitory concentration of tyrosines at pH 6.8 for the first 2 h, but the same enzyme when dialysed against 0.02 M-sodium phosphate buffer, pH 6.8, exhibits negligible activity up to 1/2 h, in contrast with considerable activity before dialysis during the same interval of time, but without any loss of activity at later intervals of incubation time. 5. On the basis of these results, it is concluded that the enzyme exists in at least two interconvertible forms, one without lag and inhibition by excess tyrosine and the other with lag and inhibition by excess tyrosine. These two forms are interconvertible only by gradual change in pH over a period of hours.

Catechol Oxidase↗
PubMed1987

pH-dependent interconvertible allosteric forms of murine melanoma tyrosinase. Physiological implications.

Murine melanoma melanosomal tyrosinase, solubilised at pH 6.8 and 1% Igepal, exhibits a lag in cresolase activity which increases with increasing concentration of tyrosine. The enzyme, solubilised at pH 5.0 and assayed at pH 5.0, does not exhibit lag even at inhibitory concentrations of tyrosine while the same enzyme when assayed at pH 6.8 exhibits characteristic lag. When the enzyme was solubilised from a melanosomal fraction with detergent/water without any buffer, significant linear activity for 2 h was seen at an inhibitory concentration of tyrosine, indicating for the first time the presence of a form of tyrosinase without lag and inhibition by excess tyrosine. Exposure of the enzyme solubilised in buffer/detergent at pH 6.8 to rapid decrease in pH to 5.0 or 4.7 makes the enzyme remain irreversibly in the form without characteristic lag, even at an inhibitory concentration of tyrosine and at pH 6.8. These results may be interpreted as follows. The enzyme at pH 6.8 exists in the E form with an allosteric site for tyrosine. Decrease of the pH of the enzyme solution from 6.8 to 5.0 or 4.7 by dialysis results in the reversible protonation of the enzyme, which no longer binds tyrosine at its allosteric site and consequently inhibition by excess tyrosine and lag were not observed at acidic pH. However, if the enzyme was rapidly brought to pH 5.0 from 6.8 it remains irreversibly in the protonated form even at pH 6.8. Ascorbic acid acts as an effective reductant for the hydroxylation of tyrosine by tyrosinase, while 3,4-dihydroxyphenylalanine is both an effective reductant and counteracts the inhibition by tyrosine at pH 6.8.

Allosteric Regulation↗
PubMed1987

In vitro growth characteristics of melanocytes obtained from adult normal and vitiligo subjects.

The in vitro growth characteristics of melanocytes obtained from uninvolved and perilesional skin of vitiligo vulgaris subjects have been investigated in comparison to those from healthy adult donors. Normal human melanocytes have been found to grow exponentially in the presence of 10(-11) M cholera toxin and 10 ng/ml of 12-O-tetradecanoylphorbol-13-acetate in routine tissue culture media. They could be trypsinized up to 3-4 passages. Melanocytes of the uninvolved skin of vitiligo subjects manifested a lag of 8-11 days for the onset of growth and they could not be passaged. Melanocytes obtained from both hypo- and hyper-pigmented perilesional skin failed to grow under these conditions. Only in a few cases where the perilesional skin was normally pigmented did the melanocytes manifest some growth after a lag of 15 days. The initial seeding capacity of the melanocytes from uninvolved and perilesional skin of vitiligo patients were, respectively, 50% and 25% of the normal individuals. Vitiligo lesions themselves gave rise to unidentified dendritic cells that survived for 10-15 days without manifesting any growth. Our results suggest that melanocytes of individuals with vitiligo are defective. This fact has to be taken into account in any theory on the etiology of vitiligo.

Adolescent↗
PubMed1983

Purification of phosphofructokinase from rabbit skeletal muscle based on its ability to aggregate in absence of its effectors.

Rabbit muscle phosphofructokinase was purified to homogeneity based on its property to form large aggregates with time at high concentration of its protein in absence of its effectors. The method involves no heat step or treatment with organic solvent or any ion-exchange columns. The enzyme thus prepared, however, exhibits the same kinetic properties as the enzyme purified by more drastic methods.

Adenosine Triphosphate↗
PubMed1982

Demonstration of tyrosinase in the vitiligo skin of human beings by a sensitive fluorometric method as well as by 14C(U)-L-tyrosine incorporation into melanin.

Tyrosinase activity (Monophenol, dihydroxyphenylalanine: oxygen oxidoreductase EC 1.14.18.1) in vitiligo and normal epidermal homogenates of skin from human beings was measured by estimating beta 3,4-dihydroxyphenylalanine (dopa) by a highly sensitive fluorometric method described in this paper. The tyrosine activity in the vitiligo skin was about 4 to 37% of corresponding normal skin. The activity of tyrosinase in normal human skin from different individuals and from different regions of the body was in the range of 4 to 140 picomoles of beta 3,4-dihydroxyphenylalanine formed per min/mg protein of epidermal homogenate. The enzyme from vitiligo and normal skin was severely inhibited by substance(s) of low molecular weight. The enzyme exhibits a lag of about 4 hr in the absence of added beta 3,4-dihydroxyphenylalanine and 1 hr in presence of 5 microM dopa. Tyrosinase from the normal and vitiligo skin was inhibited by excess concentration of tyrosine. The homogenates from vitiligo skin could synthesize melanin from C14(U)-L-Tyrosine. The rate of tyrosine incorporation into melanin by the epidermal homogenates is increased by 3,4-dihydroxyphenylalanine (dopa) disproportionate to its effect on tyrosinase activity. Based on the data presented in this paper it is concluded that melanocytes are present in the vitiligo skin. A tentative hypothesis is put forward to explain the lack of melanin synthesis by the vitiligo skin under in vivo conditions, although melanocytes are present.

Cadaver↗