Biomedical subjects

M Saleemuddin

Publications and source records attributed to M Saleemuddin.

At least 37 records · Page 2Linked to original sources

PubMed2001

A study of plasma alpha-2-macroglobulin levels in type 2 diabetic subjects with microalbuminuria.

BACKGROUND: Alpha-2 macroglobulin (Alpha-2-M) is a major plasma protease inhibitor that also regulates the activity of a variety of bioactive peptides including interleukins and exerts a range of immunomodulatory effects. OBJECTIVE: We conducted the present study with the objective to study the alpha-2-M levels in type 2 diabetic subjects with microalbuminuria in an attempt to establish alpha-2-M as a predictor of microvascular complications in diabetes. MATERIAL AND METHODS: Plasma Alpha-2-M levels were assayed in 100 (53 males and 47 females) randomly selected type 2 diabetic subjects with microalbuminuria. Diabetes was diagnosed according to the expert committee report of 1998. Patients with any acute metabolic complication like hypoglycemia, ketoacidosis, cerebrovascular accident or any acute infection were not included in the study group. RESULTS: Majority of patients belonged to 40-60 years age group. In our study alpha-2-M levels indicated a clear increase in diabetic subjects with the increasing age of subjects confirmed by multiple logistical analysis. Alpha-2-M levels were not found to be significantly different between males and females (55.6 +/- 11.3 vs. 53.7 +/- 10.5). Duration of diabetes was found to be an important confounding variable showing a direct positive correlation with alpha-2-M levels and also a significant correlation was found between alpha-2-M levels with different levels of microalbuminuria on multiple logistical analysis. No significant relation of alpha-2-M levels with either fasting blood sugar or HbA1 was observed. CONCLUSION: The increase in plasma alpha-2 macroglobulin levels in diabetes may be a correlative measure to encounter the potential proteolytic challenge associated with diabetic microangiopathy, even very early in the course of the disease. Alph-2 macroglobulin may yet be one of the most specific markers of microvascular complications in diabetes than any other serum protein.

Aged↗

PubMed2000

Alkaline protease from Spilosoma obliqua: potential applications in bio-formulations.

Some properties of the purified alkaline protease from larvae of the insect Spilosoma obliqua (Lepidoptera) and its potential application as an additive in various bio-formulations are reported. The novel feature of the present study is the use of insect protease. The protease was found to be compatible with some of the commercial detergents tested, and was also effective in cleaving various protein substrates tested, albeit to different extents, implying broader substrate specificity and effectiveness of the protease against a wide variety of stains. This property of the protease can also be exploited by using it as an active component in enzymic debriders in view of its ability to digest various protein substrates. The insect protease appears to be potentially useful as an additive in detergent, stain remover and other bio-formulations.

Animals↗

PubMed1999

Bioaffinity based immobilization of enzymes.

Procedures that utilize the affinities of biomolecules and ligands for the immobilization of enzymes are gaining increasing acceptance in the construction of sensitive enzyme-based analytical devices as well as for other applications. The strong affinity of polyclonal/monoclonal antibodies for specific enzymes and those of lectins for glycoenzymes bearing appropriate oligosaccharides have been generally employed for the purpose. Potential of affinity pairs like cellulose-cellulose binding domain bearing enzymes and immobilized metal ionsurface histidine bearing enzymes has also been recognised. The bioaffinity based immobilization procedures usually yield preparations exhibiting high catalytic activity and improved stability against denaturation. Bioaffinity based immobilizations are usually reversible facilitating the reuse of support matrix, orient the enzymes favourably and offer the possibility of enzyme immobilization directly from partially pure enzyme preparations or even cell lysates. Enzyme lacking innate ability to bind to various affinity supports can be made to bind to them by chemically or genetically linking the enzymes with appropriate polypeptides/domains like the cellulose binding domain, protein A, histidine-rich peptides, single chain antibodies, etc.

Antibodies↗

PubMed1999

Immunoaffinity layering of enzymes. Stabilization and use in flow injection analysis of glucose and hydrogen peroxide.

A general procedure for the high yield immobilization of enzymes with the help of specific anti-enzyme antibodies is described. Polyclonal antibodies were raised against Aspergillus niger glucose oxidase and horseradish peroxidase in rabbits and the gamma globulin (IgG) fraction from the immune sera isolated by ammonium sulphate fractionation followed by ion-exchange chromatography. Immobilization of glucose oxidase and horseradish peroxidase was achieved by initially binding the enzymes to a Sepharose matrix coupled with IgG isolated from anti-(glucose oxidase) and anti-(horseradish peroxidase) sera, respectively. This was followed by alternate incubation with the IgG and the enzyme to assemble layers of enzyme and antibody on the support. The immunoaffinity-layered preparations obtained thus were highly active and, after six binding cycles, the amount of enzyme immobilized could be raised about 25 times over that bound initially. It was also possible to assemble layers of glucose oxidase using unfractionated antiserum in place of the IgG. The bioaffinity-layered preparations of glucose oxidase and horseradish peroxidase exhibited good enzyme activities and improved resistance to heat-induced inactivation. The sensitivity of a flow injection analysis system for measuring glucose and hydrogen peroxide could be remarkably improved using immunoaffinity-layered glucose oxidase and horseradish peroxidase. For the detection of glucose, a Clark-type oxygen electrode, constructed as a small flow-through cell integrated with a cartridge bearing immunoaffinity-layered glucose oxidase was employed. The hydrogen peroxide concentration was analysed spectrophotometrically using a flow-through cell and the layered horseradish peroxidase packed into a cartridge. The immunoaffinity-layered enzymes could be conveniently solubilized at acid pH and fresh enzyme loaded onto the support. Immunoaffinity-layered glucose oxidase was successfully used for the on-line monitoring of the glucose concentration during the cultivation of Streptomyces cerevisiae.

Animals↗

PubMed1998

Role of naturally occurring autoantibodies in senescence of normal and ATP depleted goat erythrocytes.

The possible role of autoantibodies in the senescence of goat erythrocytes has been investigated. For this purpose goat gammaglobulin was purified to homogeneity and antigoat gammaglobulin was raised in rabbits. Using the peroxidase labelled antigoat gammaglobulin it was possible to detect the presence of auto anti band-3 antibodies in goat sera. The goat erythrocytes were aged in vitro by ATP-depletion, which resulted in appearance of a 52,000 mol. wt. polypeptide. The in vitro aged goat erythrocyte membrane bound substantial amounts of auto anti band-3 antibodies as visualized by immunoblots.

Adenosine Triphosphate↗

PubMed1997

Bioaffinity layering: a novel strategy for the immobilization of large quantities of glycoenzymes.

A simple strategy for increasing considerably the quantities of glycoenzymes immobilized on insoluble supports is described. The strategy that we call bioaffinity layering makes use of the multivalent nature of concanavalin A (Con A) and the multiple oligosaccharide chains of most glycoenzymes to build alternating lectin and glycoenzyme layers on a Sepharose matrix with precoupled Con A. Using this procedure, it was possible to increase the amounts of several glycoenzymes immobilized on Sepharose and 19.0 mg glucose oxidase could be associated with one ml Sepharose matrix after seven Con A/glucose oxidase incubation cycles. Bioaffinity layered preparations of glycoenzymes exhibited high activities as indicated by very high effectiveness factor (eta) values and those of glucose oxidase and invertase exhibited a layer-by-layer increase in thermostability. The sensitivity of a flow-through glucose monitoring cartridge integrated into a flow injection analysis (FIA) system was enhanced significantly by increasing the amount of immobilized glucose oxidase via bioaffinity layering. A cartridge bearing six layers of glucose oxidase on Sepharose support was used effectively and repeatedly for analysis of medium glucose concentration during a fed-batch cultivation of the yeast Saccharomyces cerevisiae.

Biosensing Techniques↗

PubMed1996

Phenylhydrazine causes sulfhydryl oxidation and protein aggregation in hemoglobin-free human erythrocyte membranes.

Phenylhydrazine induces a remarkable decrease in total free sulfhydryls as well as spectrin and most other polypeptides in hemoglobin-free human erythrocyte ghosts. A parallel increase in coomassie blue stainable material that was retained on the gel tops was also observed. EDTA but not DM protected the membrane from phenylhydrazine-induced alterations, while antioxidants, catalase and SOD were ineffective. The aggregated material isolated from phenylhydrazine-treated membranes could be partially dissociated in presence of beta-mercaptoethanol to yield most of the membrane polypeptides.

Antioxidants↗

PubMed1996

Immobilization and stabilization of horseradish peroxidase isoforms.

Purified anionic and cationic isoforms of horseradish peroxidase (HRP) immobilized by coupling the amino acid side-chain amino groups and/or carbohydrate moieties to Sepharose have been studied for their resistance to denaturation. The isoforms were treated with periodate followed by ethylenediamine to generate additional amino groups in the glycosyl residues. The immobilized preparations were: Preparation I (Sp-aHRP, Sp-cHRP), in which HRP was covalently immobilized via side-chain amino groups exclusively; Preparation II (Sp-NHaHRP, Sp-NHcHRP), in which periodate and ethylenediamine-treated HRP was covalently immobilized via side-chain amino groups and amino groups incorporated into glycosyl residues. HRP isoforms in preparation II lacked about 33-55% carbohydrate. Both strategies of immobilization induced significant stabilization against denaturation. Inclusion of 2 mM calcium enhanced isoenzyme stability significantly.

Calcium↗

PubMed1995

Unlike its human counterpart, band 3 anion exchange protein from goat erythrocyte membrane shows a lack of reactivity against various -SH oxidants and protease treatments.

Studies involving a number of -SH oxidants and proteases were made to analyse the organization of band 3 in goat erythrocyte membrane. -SH oxidizing agents such as diamide, Cu2+.o-phenanthroline and phenylene dimaleimide, known to cause cross-linking of human erythrocyte band 3, failed to show any cross-linking in the case of goat band 3 protein. When resolved to their individual components using -SH reducing agent beta-mercaptoethanol, high molecular weight protein adducts formed as a result of diamide treatment did not show any band 3 on two-dimensional electrophoresis. Also no proteolysis of band 3 was detected when intact goat erythrocytes were exposed to pronase, though marked proteolysis was noticed in the case of human band 3 proteins under similar conditions. These studies involving -SH oxidant and protease treatments suggest a different organization for goat erythrocyte band 3 protein as compared to that of human in erythrocyte membrane.

Animals↗

PubMed1995

Studies on the differential morphological alterations in human and goat erythrocytes against ATP depletion and Ca(2+)-induced stresses.

Erythrocyte membranes obtained from goat exhibited Ca2+/calmodulin-insensitive (Ca(2+)-Mg2+)-ATPase activity and the activity levels were significantly lower as compared to those obtained from human erythrocyte membranes. Although Ca2+/ionophore A23187 -treatment enhanced the intracellular Ca2+ levels in both the freshly collected human and goat erythrocytes, negligible loss of ATP was noticed in goat erythrocytes under such conditions. Also, fluoride, a known metabolic inhibitor, caused a lesser ATP depletion in goat erythrocytes compared to that of human. Goat erythrocytes showed remarkable rigidity to shape change and unlike human erythrocytes, did not undergo echinocytic formation as a result of metabolic depletion and Ca2+/ionophore A23187-treatments. These studies suggest the participation of (Ca(2+)-Mg2+)-ATPase -independent mechanism(s) for Ca(2+)-extrusion.

Adenosine Triphosphate↗

PubMed1994

A crosslinked tetrameric alpha 2M that binds but incompletely entraps trypsin.

A crosslinked preparation of alpha-2-macroglobulin was obtained by treatment of the purified human plasma inhibitor with glutaraldehyde at low temperature. The preparation migrated as a 780 KDa polypeptide in SDS-PAGE and with mobility comparable with that of trypsinized native alpha-2-macroglobulin under nondenaturing conditions. Trypsinization of the glutaraldehyde treated alpha-2-macroglobulin further increased its electrophoretic mobility in non-denaturing gels and resulted in the association of the proteinase with the crosslinked inhibitor. Trypsin associated with crosslinked alpha-2-macroglobulin, unlike that associated with native inhibitor was incompletely protected from soybean trypsin inhibition.

Electrophoresis, Polyacrylamide Gel↗

PubMed1993

Entrapment of nonproteolytic enzymes in alpha 2-macroglobulin using immobilized trypsin.

Entrapment in human alpha 2-macroglobulin (alpha 2M) of non-proteolytic enzymes was achieved with the help of trypsin covalently attached to Sepharose matrix. While it was also possible to achieve entrapment by the exposure of the alpha 2M: enzyme mixtures to soluble trypsin, use of the immobilized proteinase resulted in improved entrapment yields and also prevented the coentrapment of trypsin. Both soluble and immobilized trypsin transformed alpha 2M to the electrophoretically fast form but the immobilized trypsin required relatively longer incubation to bring about the transformation. Horseradish peroxidase was entrapped in higher yield in alpha 2M compared to the relatively high-molecular-weight invertase. alpha 2M-entrapped peroxidase and invertase appeared highly accessible to their respective substrates, as evident from their relatively unaltered Km values. alpha 2M-associated invertase, in spite of its large dimensions, failed to crossreact with the rabbit anti-invertase antiserum, indicating its physical entrapment rather than any other form of association.

Animals↗

PubMed1993

Immobilization and stabilization of invertase using specific polyclonal antibodies.

Antisera raised in rabbits to baker's-yeast invertase significantly activated the enzyme in vitro. The antisera contained precipitating antibodies, a significant fraction of which appeared to be directed against the glycosyl residues of the enzyme. Invertase could be immobilized as insoluble enzyme antibody adducts or by binding to a Sepharose matrix precoupled with the gamma-globulin fraction derived from the antisera. The immobilized invertase preparations exhibited high enzyme activity and had markedly enhanced thermal stability, which could be further improved by cross-linking with glutaraldehyde.

Animals↗

PubMed1993

Ca(2+)-induced alterations in the activity of membrane (Ca(2+)-Mg2+)-ATPase of human and rat erythrocytes.

Effect of intraerythrocyte Ca2+ elevation on human and rat erythrocyte membrane (Ca(2+)-Mg2+)-ATPase along with that of incubation of the erythrocyte ghosts in their own hemolysates enriched with Ca2+ has been studied. While the membrane (Ca(2+)-Mg2+)-ATPase levels of Ca(2+)-loaded human erythrocytes showed an initial increase and subsequent decline, membranes incubated in their own hemolysate showed a consistent decrease in the enzyme activity. Calmodulin sensitivity was retained by the preparations in contrast to the earlier observations made with washed erythrocyte membranes. Similar changes in (Ca(2+)-Mg2+)-ATPase activity but of greater magnitude were observed in response to Ca2+ in the calpain-rich rat erythrocytes. Considerable crosslinking and proteolysis was observed in case of human and rat erythrocytes exposed to high Ca2+ concentrations. The Ca(2+)-activable transglutaminase, however, did not play any role in the activation of the (Ca(2+)-Mg2+)-ATPase.

Animals↗

PubMed1992

Inactivation and reactivation of horseradish peroxidase immobilized by various procedures.

Horseradish peroxidase (HRP) immobilized by coupling the amino acid side chain amino groups or carbohydrate spikes to the matrix has been studied for its resistance to heat, urea-induced inactivation and ability to regain activity after denaturation in order to understand the influence of the nature of immobilization procedure on these processes. The various immobilized preparations were obtained and their properties studied: Sp-HRP was obtained by direct coupling of HRP to cyanogen bromide-activated Sepharose, Sp-NHHRP by coupling periodate oxidized and diamine-treated enzyme to the cyanogen bromide activated Sepharose, SpNH-COHRP by coupling periodate-treated enzyme to amino-Sepharose and SpCon A-HRP by binding of the enzyme on Con A-Sepharose. All the immobilized preparations exhibited higher stability against heat-induced inactivation as compared to the native HRP. Sp-NHHRP was most stable followed by Sp-HRP, SpNH-COHRP and SpCon A-HRP. Sp-NHHRP was also superior in its ability to regain enzyme activity after thermal denaturation, although Sp-HRP regained maximum activity after urea denaturation. Inclusion of Ca2+ was essential for the reactivation of all preparations subsequent to denaturation by urea.

Enzymes, Immobilized↗

PubMed1991

Concanavalin A: a useful ligand for glycoenzyme immobilization--a review.

Concanavalin A is finding increasing applications as a useful ligand in glycoenzyme immobilization. An attempt therefore, has been made to summarize the work available in the area. Glycoenzymes that are recalcitrant to immobilization procedures involving covalent coupling to solid supports can be immobilized in high yields by binding to matrices precoupled with concanavalin A. In addition, glycoenzymes associated with concanavalin A matrices usually exhibit high retention of activity and enhanced stability against various forms of inactivation. Binding of the glycoenzymes on the concanavalin A supports, being noncovalent, can be reversed by incubating the preparation with a high concentration of sugars/glycosides or at acidic pH. The association can be, however, rendered covalent by crosslinking the preparations with bifunctional reagents like glutaraldehyde. Crosslinking may be accompanied by further increase in stability, albeit at the expense of the loss of some enzyme activity. Several laboratory-size reactors containing concanavalin A matrix-bound glycoenzyme have been successfully operated for reasonably long durations with only small losses in catalytic activity. Insoluble glycoenzyme preparation can also be obtained by precipitating them from solution as concanavalin A complexes. Such complexes have small particle dimensions but can be successfully used in column reactors after a subsequent immobilization step. Insoluble concanavalin A-flocculates containing various microorganisms and glycoenzymes that successfully carry out multistep transformations have also been obtained by several investigators.

Concanavalin A↗

PubMed1991

Goat erythrocyte calmodulin is not abnormal.

Calmodulin was purified from goat erythrocyte hemolysate using heat treatment and Sephadex G-100 gel filtration chromatography. The molecular weight and Stokes, radius of the purified calmodulin was determined. The goat erythrocyte calmodulin stimulated (Ca(2+)-Mg2+)-ATPase but not (Mg2+)-ATPase and (Na(+)-K(+)-Mg2+)-ATPase. The (Ca(2+)-Mg2+)-ATPase of the erythrocyte membrane derived from human, rat, rabbit and pig were significantly stimulated.

Animals↗

PubMed1988

Lack of some Ca2+-mediated processes in goat erythrocytes.

Ca2+ does not promote crosslinking of proteins nor stimulate proteolysis in goat and sheep erythrocyte membranes. Neither crosslinking nor proteolysis was observed even when the goat erythrocytes were loaded with calcium with the help of calcium ionophore A23187. Membrane-free human erythrocyte hemolysate, however, induced Ca2+-dependent crosslinking in goat erythrocyte membranes.

Animals↗