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M N Khan

Publications and source records attributed to M N Khan.

At least 55 records · Page 3Linked to original sources
PubMed1989

Internalization and activation of the rat liver insulin receptor kinase in vivo.

The preparation of clearly delineated plasmalemma (PM) and endosomal subcellular fractions from rat liver has allowed us to compare insulin receptor (IR) kinase activity at the cell surface and in hepatic endosomes (ENs) as a function of dose and time after injected insulin. Tyrosine kinase activity in PM and ENs was measured, after solubilization and partial purification by wheat germ agglutinin chromatography (lectin-purified), using poly(Glu:Tyr) as substrate. Following the injection of a subsaturating dose of insulin (1.5 micrograms/100 g body weight), lectin-purified receptor showed peak activation at 30 s in PM and at 2 min in ENs. As observed previously (Khan, M. N., Savoie, S., Bergeron, J. J. M., and Posner, B. I. (1986) J. Biol. Chem. 261, 8462-8472) autophosphorylation activity was also augmented following insulin injection. In a pattern virtually identical to that of exogenous kinase activity, autophosphorylation attained peak activity at 30 s in PM and at 2 min in ENs. The time course of IR autophosphorylation in intact membranes was very similar to that observed for lectin purified receptors and was seen with an injected insulin dose as low as 150 ng/100 g body weight. Phosphatase treatment of the solubilized endosomal receptor abolished its enhanced activity. Hence, insulin treatment led to in vivo receptor phosphorylation which was reflected in the enhancement of both tyrosine kinase and autophosphorylation activities. Significant differences in the phosphorylation activities of PM and ENs were observed. Phosphoamino acid analyses revealed that the activated IR of intact PM was autophosphorylated in vitro, at both serine (55%) and tyrosine (45%) residues; whereas the activated IR of intact ENs was phosphorylated in vitro exclusively on tyrosine autophosphorylation specific activity for the activated IR of ENs was 3- to 4-fold that of the IR of PM. This was observed for the lectin purified IRs as well as for IRs of intact cell fractions. The reduced level of IR autophosphorylation in PM was not due to occlusion of tyrosine acceptor sites by prior in vivo phosphorylation. The rapidity with which activated IR accumulates in ENs as well as the sensitivity of endosomal IR kinase to activation by injected insulin are consistent with the endosomal apparatus serving a physiologically significant site for the regulation of transmembrane signaling.

Animals↗
PubMed1989

Aqueous degradation of N-(hydroxymethyl)phthalimide in the presence of specific and general bases. Kinetic assessment of N-hydroxymethyl derivatives of nitrogen heterocycles as possible prodrugs.

The conversion of N-(hydroxymethyl)phthalimide (NHPH) to phthalimide could not be detected within 300 s at pH 9.0, whereas in 0.18 M NaOH complete conversion of NHPH to phthalimide was observed within 50 s. In the presence of 0.2-0.4 M 1,4-diazabicyclo[2.2.2]octane buffer solutions (pH 9.30-9.54), 40-60% conversion of NHPH to phthalimide occurred within 90-120 s. The initial concentration of NHPH affected the extent of conversion of NHPH to phthalimide.

Alkalies↗
PubMed1989

Effect of retraining on nutrition among tribal women volunteers in Bangladesh.

Training programme on Nutrition was carried out on 42 tribal women nutrition volunteers from Rangamati, Khagrachari and Banderban districts of Chittagong Hill Tracts during July 1987 to June 1988. There were a basic training for 10 days and five retrainings of 6 days on the same group. Assessments on the level of their knowledge before and after each training were conducted by a standard questionnaire. Mean score obtained by the trainees initially was 46.28% which increased to 97.23% in the last round. Although the knowledge gain was found statistically significant in each retraining but the retention of knowledge in pre-test of each retraining was found statistically significant (P less than 0.001) upto second round (i.e. 1st retraining) and thereafter the retention was found insignificant (P greater than 0.05).

Bangladesh↗
PubMed1988

Insulin-like growth factor-binding proteins in hypophysectomized rat liver: characterization and subcellular localization.

We have characterized binding proteins for insulin-like growth factors (IGFs) in hepatic subcellular fractions and in the washed supernatants of these fractions in normal and hypophysectomized (hypox) rats. In the course of assessing IGF-II-binding sites on rat liver microsomes, we observed that [125I] IGF-II binding to the microsomal membranes of hypox rats was much lower than that in normal rats. Paradoxically, binding increased in hypox animals at low concentrations (0.5-5 ng/ml) of unlabeled IGF-II. After resuspension and centrifugation (washing) of the microsomes, no difference was found in [125I]IGF-II binding to hypox vs. normal microsomes. However, the binding of [125I]IGF-II to the washing supernatant (SN) from hypox rat microsomes was greater than binding to that from normal animals. Binding to SN was inhibited by unlabeled IGF-II in a dose-dependent manner. Scatchard analyses indicated that the affinity constant for binding by hypox SN was higher than that of normal SN and the microsomal fractions of both hypox and normal rats. After further subfractionation of the liver, no binding activity was found in SN from plasmalemma, whereas it was about 20% of input counts per min of [125I]IGF-II in SN from combined Golgi-endosome fractions of hypox rat liver. We next compared IGF-binding moieties in microsomal SN with those in plasma using cross-linking of [125I]IGF-II followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In normal rat plasma, we observed the presence of 42K, 39K, 31K, and 27K binding complexes. In hypox rat plasma only a 42-39K doublet was found. In the SN of normal rat microsomes, the predominant complex migrated at 39K and was distinguishable only after acidification. In the SN of hypox rat microsomes, the 42K complex was predominant, with a minor 34K complex. These studies have identified IGF-binding moieties in hepatic tissues, particularly in hepatic vesicular elements, which interfere in the binding of IGF-II to membrane receptors. Their presence in these receptor-rich elements may influence IGF binding to intracellular receptors and, hence, the biological activity of the peptide.

Animals↗
PubMed1986

Differential kinetics and sensitivity to chloroquine of receptor-mediated insulin and prolactin endocytosis in liver parenchymal cells.

Systemically injected [125I]prolactin or [125I]insulin was accumulated and cleared from rat liver at different rates. Quantitative subcellular fractionation indicated a predominant accumulation of [125I]insulin in liver microsomes while [125I]prolactin was found in both the light-mitochondrial and microsomal fractions. The acidotropic agent chloroquine diminished the rate and extent of loss of each ligand from liver homogenates. In chloroquine treated rats, radiolabeled insulin accumulated in both the light-mitochondrial and the microsomal fractions. Subfraction of microsomes on discontinuous sucrose gradients revealed "early' endosomes in which ligand uptake was maximal at 2-5 min. In contrast, comparable subfraction of the of light mitochondrial fraction revealed "late' endosomes in which ligand uptake was maximal at 10-20 min. Chloroquine-treated rats showed a more marked enhancement of insulin compared to prolactin uptake in the "early' endosomes. It is suggested that "early' endosomes found in the Golgi-intermediate and -heavy fractions floated from parent microsomes may selectively degrade insulin but not prolactin. This could account for the apparently different kinetics of insulin and prolactin uptake into liver parenchyma.

Animals↗
PubMed1986

Epidermal growth factor receptor kinase translocation and activation in vivo.

The rat liver epidermal growth factor (EGF) receptor was assessed for EGF-dependent autophosphorylation as well as phosphorylation of a defined exogenous substrate in purified plasmalemma and Golgiendosome fractions isolated from rat liver homogenates. While EGF-dependent kinase activity was readily detected in plasmalemma the corresponding activity in Golgi-endosome fractions required detergent. Consequent to the systemic injection of EGF in vivo, the majority (approximately 60%) of receptor as evaluated by 125I-EGF binding was rapidly lost (T 1/2 approximately 8 min) from the plasmalemma and correspondingly accumulated in the Golgi-endosome fraction in a dose-dependent manner. Electron microscope radioautography of 125I-EGF uptake into Golgi-endosome fractions identified internalization into lipoprotein-filled vesicles of heterogenous size and shape but not into stacked saccules of the Golgi apparatus. Evaluation of receptor kinase activity in plasmalemma fractions isolated at various times after EGF injection in vivo showed more rapid loss of EGF-dependent autophosphorylation activity (T 1/2 approximately 10 s) than of receptor content (T 1/2 approximately 8 min). In contrast to the EGF receptor kinase of the plasmalemma fraction, kinase activity accumulating in endosomes was activated, i.e. maximally stimulated, in the absence of EGF or Triton X-100 in vitro. Furthermore, following the peak time of accumulation of EGF receptor kinase in endosomes (5-15 min) EGF-dependent autophosphorylation activity and EGF receptor content were lost more slowly (T 1/2 approximately 27 and 87 min for the loss of autophosphorylation activity and receptor content, respectively). The rapidity of translocation of activated EGF receptor into endosomes (30 s) and the dose response to low levels (1 microgram) of EGF injected are consistent with a physiological role for internalized EGF receptor kinase activity.

Animals↗
PubMed1986

Characterization of rat liver endosomal fractions. In vivo activation of insulin-stimulable receptor kinase in these structures.

A protocol employing discontinuous sucrose gradient centrifugation was developed to prepare light mitochondrial (L) and Golgi fraction endosomes from simultaneously prepared parent L and microsomal fractions. As judged by the concentration of labeled hormone postinjection, L intermediate and heavy endosome subfractions were 40- to 175-fold purified and Golgi intermediate and heavy endosome subfractions were 30- to 45-fold purified. On electron microscopy, L endosomal fractions contained a predominance of lipoprotein-filled vesicles and were less heterogeneous than corresponding Golgi endosomal fractions. All endosomal fractions were enriched in receptors for insulin and prolactin but binding sites for the former were more broadly distributed in other subfractions than those for the latter. On Percoll gradient centrifugation, L endosomal fractions yielded one peak (rho 1.057) corresponding to the heavier of two peaks seen in Golgi endosomal fractions. The protein composition of high density L and Golgi endosomes, as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was similar. The bulk of marker enzymes assayed did not migrate with the endosomal components. Combined acid phosphatase cytochemistry and electron microscope radioautography established that about 80% of the L endosomes contained no acid phosphatase. By affinity labeling and immunological titration with insulin receptor antibody, insulin receptors were identical in L and Golgi endosomes. Insulin-stimulable receptor kinase was demonstrable in both L and Golgi endosome fractions. Following in vivo insulin administration, the insulin receptor kinase in both L and Golgi endosomes was significantly activated. This activated state was not inhibited by a large excess of antiserum to insulin and thus not due to insulin contaminating the partially purified receptor preparation. These observations are compatible with the maintenance and/or initiation of hormone-dependent phosphorylations intracellularly.

Animals↗
PubMed1986

Differential and analytical subfractionation of rat liver components internalizing insulin and prolactin.

Receptor-mediated endocytosis of 125I-insulin and 125I-prolactin into liver parenchymal cells has been studied by quantitative subcellular fractionation. Differential centrifugation yielded three particulate fractions, N (nuclear), ML (large granule), and P (microsomes), and a final supernatant (S). Quantitative differences in the extent and rates of accumulation of 125I-insulin and 125I-prolactin into the fractions were observed. The acidotropic agent chloroquine and the microtubule disrupting agent colchicine were administered separately to rats. The agents increased significantly the T 1/2 of hormone clearance from the liver and augmented the accumulation of both ligands in the low-speed ML fraction. However, differences in the rates of accumulation of insulin and prolactin into all cell fractions were still maintained. Analytical centrifugation of each of the particulate fractions was carried out in order to determine if different endocytic components were specific to insulin or prolactin internalization. This was not the case. An "early" endosomal component of density 1.11 was identified in microsomes. A "late" endosome of density 1.10 was identified in the large granule (ML) fraction. Both endosomal components appeared to accumulate insulin and prolactin but at different rates. Marker enzyme analysis identified the presumed plasma membrane component in microsomes (density approximately 1.155). This component showed a significant difference in the rate of loss of 125I-insulin (T 1/2 approximately 4.1 min) as compared to that of 125I-prolactin (T 1/2 approximately 12.7 min). A further difference in the handling of the ligands was observed in early endosomes.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1985

Terminal glycosylation in rat hepatic Golgi fractions: heterogeneous locations for sialic acid and galactose acceptors and their transferases.

Endogenous acceptors for N-acetylglucosamine (GlcNAc), galactose (Gal) or sialic acid (NeuAc) transfer were labeled to high activities when purified hepatic Golgi fractions were incubated with the corresponding radiolabeled nucleotide sugar in the absence of detergent. The in vitro conditions which were optimal for the endogenous glycosylation of GlcNAc and Gal acceptors (Mn2+, ATP) also promoted fusion within a subset of Golgi membranes. Electron microscope radioautography revealed that the majority of NeuAc acceptors were associated with unfused Golgi membranes, whereas the majority of Gal acceptors were localized to fused membranes. GlcNAc acceptors were approximately equally distributed between fused and unfused membranes. Under conditions in which Golgi membrane fusion was absent (-Mn2+), only NeuAc transfer was active. The majority of endogenous NeuAc acceptors were consequently assigned to the more trans regions of the hepatic Golgi apparatus as concluded from a combination of radioautography (NeuAc transfer) and acid NADPase cytochemistry (reactive medial and trans Golgi saccules). The distribution of NeuAc and Gal transferases was assessed after Percoll gradient centrifugation of disrupted Golgi fractions. The median density of NeuAc transferase was lower than that of Gal transferase. The studies are indicative of distinct Golgi components harboring the majority of acceptors and enzymes for terminal glycosylation.

Acetylglucosamine↗
PubMed1985

Prolactin uptake into liver endocytic components. Reduced sensitivity to chloroquine.

Subfractionation of hepatic Golgi fractions on Percoll gradients revealed two populations of lactogen-receptor enriched components with one pool of rho = 1.040-1.050 and a high-density component of rho = 1.053-1.064. 125I-labelled prolactin uptake into Percoll gradient subfractions demonstrated rapid accumulation into low-density elements and slower accumulation in high-density structures. Electron microscope radioautography demonstrated that silver grains were largely associated with lipoprotein-filled structures. Radiolabel was highly concentrated in these components especially the very pure higher-density component where enrichment over the homogenate was 184-fold at the peak time after injection (10 min). Administered chloroquine accumulated in the higher-density component but not in low-density elements, suggesting that the former are at an acid intraluminal pH. In contrast to the marked effect of chloroquine on insulin uptake into endocytic structures found in Golgi fractions (Posner, B.I., Patel, B.A., Khan, M.N. and Bergeron, J.J.M. (1982) J. Biol. Chem. 257, 5789-5799), little effect of this pH disrupting agent was observed on prolactin uptake. The difference between the effect of chloroquine on 125I-labelled prolactin and 125I-labelled insulin uptake may reflect the greater stability of prolactin-receptor complexes in a low-pH environment.

Animals↗
PubMed1985

NH2-terminal specificity and axonal localization of adrenocorticotropin binding sites in rat median eminence.

Adrenocorticotropin binding sites in the rat median eminence have been localized in vivo. These binding sites occur in the basalar zone, which is rich in axonal endings. Using competitive binding and quantitative light-microscope radioautography, we found that the median-eminence binding site, in contradistinction to the adrenal receptor, binds specifically the residue 4-10 region of the adrenocorticotropin molecule. Using quantitative electron-microscope radioautography and median-eminence deafferentation, we localized the binding sites to axon terminals in this region. In time-delayed uptake studies using light-microscope radioautography, we failed to observe concentration of radiolabel in neurons of the medial basal hypothalamus after the direct injection of radioiodinated adrenocorticotropin(1-24) into the median eminence.

Adrenal Glands↗