PubMed Health⌕ Search

Biomedical subjects

M N Malik

Publications and source records attributed to M N Malik.

At least 19 recordsLinked to original sources
PubMed2005

Regulation of high molecular weight bovine brain neutral protease by phospholipids in vitro.

The activity of the heat stable, glycosylated high molecular weight bovine brain neutral protease (HMW protease) is differentially regulated by phospholipids. While phosphatidylcholine (PC), phosphatidylserine (PS) and phosphatidic acid (PA) had only marginal stimulatory effect (40-75%) on the activity of HMW protease, lysophoshatidylcholine (lysoPC) and lysophosphatidic acid (lysoPA) activated the enzyme by more than two-fold. Both lysoPC and lysoPA exhibited concentration-dependent saturation kinetics for the activation of HMW protease. Surprisingly, phosphoinositides (phosphatidylinositol, PI; phosphatidylinositol 4-phosphate, PIP; and phosphatidylinositol 4,5-bisphosphate, PIP2) modulated the activity of protease differently: activation of the enzyme was higher with PIP (90%) as compared to PI (21%), whereas PIP2 inhibited the enzyme (16%). The inhibition of the protease by PIP2 was concentration-dependent. During receptor-coupled cell activation, phospholipase A2 (PLA2) converts PC and PA to lysoPC and lysoPA, respectively; PI is converted to PIP2 by successive enzymatic phosphorylation by PI 4-kinase and PIP 5-kinase; and phospholipase C (PLC) degrades PIP2 to diacylglycerol and inositol 1,4,5-trisphosphate. Therefore, the data suggest that HMW protease may be coupled to cell signal transduction where PLA2, PI 4-kinase, PIP 5-kinase and PLC are involved.

Animals↗
PubMed1996

Comparison of the hydrophobic grid-membrane filter DNA probe method and the Health Protection Branch standard method for the detection of Listeria monocytogenes in foods.

The standard Health Protection Branch (HPB) method for the detection of L. monocytogenes in foods involves lengthy enrichment, selection and biochemical testing, requiring up to 8 days to complete. A hydrophobic grid-membrane filter (HGMF) method employing a digoxigenin-labelled listeriolysin O probe required 5 days to complete, and included an image-analysis system for electronic data acquisition. A total of 200 food samples encompassing 8 high-risk food groups (soft and semi-soft cheeses, packaged raw vegetables, frozen cooked shrimp, ground poultry, ground pork, ground beef, jellied meats, and pâté) were screened for the presence of L. monocytogenes by the two methods. Overall, 32 (16%) and 30 (15%) of the naturally-contaminated food samples tested positive for L. monocytogenes by the HPB and DNA methods, respectively. The DNA probe method was highly specific in discriminating L. monocytogenes from other Listeria spp. present in 50 of the samples tested. Results showed 94% sensitivity and 100% specificity between the two methods. The HGMF DNA probe method is an efficient and reliable alternative to the HPB standard method for detecting L. monocytogenes in foods.

DNA Probes↗
PubMed1995

Light and electron microscopic immunocytochemical localization of two major proteins in garlic bulb.

Garlic is known as a potent spice and a medicine with broad therapeutic properties ranging from antibacterial to anticancer, antidiabetic, and anticoagulant. Two major proteins of 40 KD and 14 KD constituting approximately 96% of total garlic proteins have been recently purified at our Institute. This immunocytochemical and ultrastructural study revealed that the 40 KD protein was localized in the parenchyma sheath cells (PSC) of garlic bulbs, whereas the 14 KD protein was present in the cortical cells (CC). Immunogold electron microscopy study indicated that the 40 KD protein was specifically localized in the globular granules of the cytoplasmic area of PSC. Each globular granule was amorphous and homogenous with membrane limiting its outermost layer. The yellowish color of PSC in freshly cut slices of garlic bulb suggested that PSC may have sulfur-containing compounds such as allicin, the primary contributor of the pungency and medicinal properties of garlic. Ellman's reagent test quantitatively revealed that there were 17.8 n moles sulfhydryl (SH)/ml of 40 KD garlic protein. Microtubule tubulin in mitotic figures from PHA-stimulated human short-term whole blood cultures reacted strongly with antitubulin antibody but reacted negatively with anti-40 KD garlic protein antibodies and therefore was not related to the 40 KD garlic protein immunocytochemically.

Antibodies, Monoclonal↗
PubMed1994

Immunocytochemical localization of the high molecular weight protease in brain.

Polyclonal antiserum against a high molecular weight glycosylated protease, purified from calf brain cytosol, was raised in rabbit and purified by immunoaffinity. The antibody specifically immunoreacted with the M(r) = 165,000 and 155,000 polypeptides of the protease. Immunocytochemical localization data revealed that the protease is localized in the pyramidal neurons, granular and glial cells of the hippocampus. Microscopic analysis of the pyramidal neurons indicates that the protease is present in the cytoplasm and extends to the dendrite and axon. The nuclei of these neurons remain unstained.

Animals↗
PubMed1991

High molecular weight glycosylated protease in calf brain. Purification and partial characterization.

A high molecular weight protease has been purified to homogeneity from calf brain cytosol. The purification procedure involves ammonium sulfate fractionation of the cytosol followed by chromatography on DEAE-Sephacel, hydroxylapatite, concanavalin A-Sepharose 4B and Sephacryl S-300. The molecular weight of the native protease was estimated to be Mr = 465,000 by high pressure liquid chromatography. It is composed of a closely moving doublet of Mr = 165,000 and 155,000, as determined on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It degrades [methyl-14C] alpha-casein with a broad pH optimum of 6.8-8.5. [methyl-14C]bovine serum albumin and 125I-bovine serum albumin are hydrolyzed to the same extent as [methyl-14C]alpha-casein, whereas [methyl-14C]methemoglobin is hydrolyzed to half the extent of [methyl-14C] alpha-casein. Divalent cations, nucleotides, and known protease inhibitors (phenylmethylsulfonyl fluoride, p-chloromercuribenzoate, iodoacetic acid, N-ethylmaleimide, leupeptin, antipain, pepstatin, and hemin) have no effect on the activity of the protease. The protease is glycosylated and appears to aggregate readily. Aggregation may be reversed by treating the protease with certain organic solvents. The protease seems to maintain full activity after heat treatment. Electron microscopic data reveals a spherical structure of 20-nm diameter.

Amino Acids↗
PubMed1987

Third form of calcium-activated neutral proteinase from calf brain: purification, partial characterization and comparison of properties with other forms.

A third form (CANP3) of calcium-activated neutral proteinase (CANP) has been purified, 3900-fold, to near homogeneity from calf brain cortex. The purification procedure is based on the one recently developed for the purification of CANP1 and CANP2. The molecular weight of CANP3, as judged on SDS-polyacrylamide gel electrophoresis was Mr 78,000. A protein with an apparent Mr 17,000 co-purified with the proteinase. At neutral pH (7.2), it was maximally active at 260 microM CaCl2. In the presence of CaCl2, CANP1 and CANP3 were autolyzed very rapidly, whereas the autolysis of CANP2 was slow and gradual. The autolyzed CANP1 and CANP3 responded differently to CaCl2; CANP1 lost activity completely, whereas CANP3 was fully active at 0.5 microM CaCl2. Despite the opposite behavior of these proteinases in the presence of Ca2+, no significant differences in the peptide maps of the three proteinases were observed. Neurofilaments, neurotubules and myelin basic protein (MBP) were degraded by each of the proteinases. Monoclonal antibodies raised against CANP2 reacted almost equally with CANP1 and CANP3. As with CANP1 and CANP2, leupeptin and sulfhydryl-modifying compounds, NEM and iodoacetic acid, inhibited the activity of CANP3.

Animals↗
PubMed1987

Micromolar Ca2+ requiring protease from human platelets: purification, partial characterization and effect on the cytoskeletal proteins.

A calcium-activated neutral protease (CANP) has been purified 2,800 fold, to near homogeneity, from human platelets. The purification procedure involved ammonium sulfate fractionation of the platelet cytosol followed by chromatography on Sephacryl S-200, DEAE-Sephacel, Agarose-Hexylamine, Agarose-Octylamine and alpha-casein-Sepharose 4B affinity gel. The protease consisted of two polypeptides of Mr = 74,000 and 28,000 as judged on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It hydrolyzed [methyl-14C] alpha-casein at a significant rate of 37 degrees C which was, therefore, used as an exogenous substrate. Microtubules and intermediate filament proteins were also susceptible to hydrolysis by the purified protease. It attained maximum activity at 0.06 uM CaCl2 and displayed two pH maxima: one at 5.5 and the other at 6.5. The protease was fully active in the presence of MnCl2 and was about 75% active with BaCl2 and SrCl2. Among the actinomycete protease inhibitors, leupeptin, antipain and pepstatin, the order of inhibition was: leupeptin greater than antipain greater than pepstatin. The protease was also inhibited by sulfhydryl modifying agents.

Blood Platelets↗
PubMed1986

Purification and degradation of purified neurofilament proteins by the brain calcium-activated neutral proteases.

The effect of the three forms (CANP1, CANP2 & CANP3) of calf brain calcium activated neutral protease (CANP) on the hydrolysis of purified neurofilament triplet proteins was investigated. It was observed that: each of the purified neurofilament proteins, was hydrolyzed slowly by CANP2 whereas the hydrolysis of 150 KDa and 68 kDa proteins by CANP1 & CANP3 was rapid; when assembled neurofilaments were used as a substrate, again differences in the rate and extent of degradation of the triplet proteins by the three proteases were observed. For example, little cleavage of the 68 kDa protein by CANP2 and CANP3 was noted whereas 210 kDa and 150 kDa proteins remained largely intact. CANP1 degraded the 150 kDa and 68 kDa proteins more rapidly than 210 kDa protein, where only a slight effect was noted. These data provide further proof of the existence of three different forms of CANP in the brain, and indications of the resistance of 210 kDa protein to proteolysis which may be compatible with its proposed special role in crossbridge formation.

Animals↗
PubMed1984

Folic acid therapy in the fragile X syndrome.

Two brothers with fra(X) positive X-linked mental retardation (XLMR) were treated with folic acid. Initially a double blind cross-over design was employed followed by a long-term high dose trial. A decrease in the frequency of fra(X) positive cells was observed when low folic acid culture medium was used but not when an FUdR induction system was employed. Selected behavioral characteristics improved in both while receiving folic acid. Decreased hyperactivity, greater attention span, increased motor coordination, increased quantity and quality of speech were noted. Improvement in Leiter mental age and regression after cessation of treatment was seen in one subject but not in the other. Further controlled trials with larger numbers of subjects using high doses of folic acid over longer periods of time are needed to assess the possible benefits of this experimental form of treatment.

Adolescent↗
PubMed1984

Purification and partial characterization of two forms of Ca2+-activated neutral protease from calf brain synaptosomes and spinal cord.

Two forms ( CANP1 and CANP2 ) of a calcium activated neutral protease (CANP) have been purified to near homogeneity from calf brain synaptosomes and spinal cord. The procedure involves ammonium sulfate fractionation of the brain synaptosome or spinal cord cytosol followed by chromatography on DEAE-Sephacel, Hydroxylapatite and alpha-casein-CH-Sepharose 4B affinity gel. The molecular mass of each of the proteases is 78,000 as judged on SDS-PAGE. A protein with apparent molecular mass of 17,000 copurifies with each of the proteases. CANP1 was maximally active at 600 microM while CANP2 exhibited maximum activity at about 2 microM Ca2+. Both of the proteases were inhibited by sulfhydryl modifying agents and leupeptin.

Animals↗
PubMed1983

Purification and characterization of two forms of Ca2+-activated neutral protease from calf brain.

Two forms (CANP1 and CANP2) of a calcium-activated neutral protease (CANP) have been purified, 1,950- and 1,250-fold, respectively, to near homogeneity from calf brain. The purification procedure involves ammonium sulfate fractionation of the brain cytosol followed by chromatography on DEAE-Sephacel, hydroxylapatite, and alpha-casein-CH-Sepharose 4B affinity gel. A protein with apparent Mr = 17,000 copurifies with each of the proteases. This protein was separated by chromatography on a reactive red-120 agarose. Preliminary experiments indicate that, in the absence of this protein, the activity of each of the proteases was reduced. These observations raise the possibility that the 17,000-Da protein may regulate the activity of these proteases. Each of the proteases have similar apparent Mr = 78,000 as judged on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Except for casein, hemocyanin, and hemoglobin, no other exogenous proteins tested were significantly hydrolyzed by either of the proteases. [ methyl-14C ]alpha-Casein or methemoglobin was routinely used as a substrate for both of the enzymes. The endogenous proteins, neurotubules (microtubule-associated proteins and tubulin), neurofilament triplet proteins and desmin from smooth muscle were extensively hydrolyzed by both of the proteases. A marked difference was found in their requirement for CaCl2. CANP1 was maximally active at 700 microM while CANP2 exhibited highest activity at 2 microM CaCl2. Both displayed maximum activity at pH 7.5, although the overall pH profiles were slightly different. Among the actinomycete protease inhibitors, antipain, leupeptin, and pepstatin, leupeptin was highly effective in inhibiting the activities of both enzymes. Both of the proteases were also inhibited by sulfhydryl modifying agents. Metal ions other than CaCl2 were poor activators of the activity of either protease.

Aluminum↗
PubMed1983

Purification and characterization of myosin from calf brain.

Actomyosin complex was extracted from the brain cortex in a medium consisting of low salt, ATP, and EDTA, in the presence of protease inhibitors, followed by ammonium sulfate fractionation. Myosin was then purified from the actomyosin. Myosin obtained according to the procedure used was significantly contaminated with actin high (greater than 200,000 dalton) and low molecular weight proteins. Therefore, an alternative method based on affinity chromatography (Blue Dextran/Sepharose) and gel filtration (Sepharose 4B) was developed to purify myosin. This procedure yielded myosin that was greater than 95% pure as judged by electron microscopy and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The subunit composition of purified brain myosin was monitored by sodium dodecyl sulfate-polyacrylamide gel also containing a urea gradient. A closely migrating triplet in the heavy chain and three light chains, LC1, LC2, and LC3, of Mr 21,000, 19,000, and 17,000, respectively, were observed. These findings raise the possibility of the existence of myosin isoenzymes in the brain. Brain myosin formed bipolar thick filaments in 0.075 M KCl and MgCl2. At low ionic strength, the Mg2+-ATPase activity of myosin was stimulated 3- to 3.5-fold in the presence of skeletal muscle f-actin. Brain myosin also hydrolyzed other nucleotides; the rate of hydrolysis was ITP greater than ATP approximately equal to CTP greater than GTP approximately equal to UTP. The substrate (ATP) saturation curve in the presence of 10 mM CaCl2 and 0.6 M KCl was complex and consisted of plateau regions. The Arrhenius plot of the Ca-ATPase data was linear, whereas with ITPase, it was biphasic with a break occurring around 20 degrees C.

Actins↗
PubMed1978

Steady-state kinetics of smooth muscle myosin.

The kinetic properties of purified smooth muscle myosin, free of actin, have been examined. Analysis of the steady-state kinetic data revealed an intermediary plateau region on the substrate saturation curves. In addition, these data, when analyzed by Hill and Lineweaver and Burk plots, indicate both positive and negative cooperativity, suggesting at least four substrate binding sites. The plateau region was abolished when the kinetic measurements were made at pH 5.5 and 9.0. Both positive and negative cooperative effects were absent at pH 9.0 and hyperbolic kinetics was observed. In contrast, at pH 5.5, although the plateau region was abolished, the enzyme exhibited positive cooperativity of substrate binding. When either heated or urea treated enzyme was used for kinetic measurements: (i) the plateau region shifted toward higher substrate concentration range; (ii) the cooperativity of binding sites was lost at low substrate concentrations but was instead seen at higher concentrations; and (iii) the Vmax was doubled. These data have been interpreted as due to ligand-induced conformational changes in the enzyme according to J. Teipel and D. E. Koshland, Jr. (1969).

Adenosine Triphosphatases↗