Biomedical subjects

J Sredy

Publications and source records attributed to J Sredy.

25 records · Page 2Linked to original sources

PubMed1985

cAMP-dependent phosphorylation of bovine lens alpha-crystallin.

This communication reports that the A1 and B1 chains of bovine lens alpha-crystallin are phosphorylated. The conclusion is based on the following evidence: (i) When soluble preparations from lens cortex are incubated with [gamma-32P]ATP, a cAMP-dependent labeling of a high molecular weight protein is obtained. (ii) After NaDodSO4/PAGE, the label is found in two bands with Mr 22,000 and 20,000, corresponding to the B and A chains of alpha-crystallin, respectively. (iii) Isoelectric focusing indicates that the radioactivity is almost exclusively in bands with pI values of 5.58 and 6.70, corresponding to the A1 and B1 chains, respectively. (iv) Similar results are obtained in experiments of [32P]orthophosphate incorporation in lens organ culture. (v) Analyses of the digested protein indicate the label is exclusively in phosphoserine. (vi) 31P NMR analyses of native, proteolytically digested, and urea-treated alpha-crystallin gives a chemical shift of 4.6 ppm relative to 85% H3PO4 at pH 7.4, suggesting that the phosphate is covalently bound to a serine in the protein. An abundance of approximately one phosphate per four or five monomer units was found. (vii) Similar results were obtained by chemical analyses of independently prepared alpha-crystallin samples. The results are consistent with the view that the A1 and B1 chains arise as result of the phosphorylation of directly synthesized A2 and B2 polypeptides. It is suggested that this metabolically controlled phosphorylation may be associated with the terminal differentiation of the lens epithelial cell and the intracellular organization of the lens fiber cell.

Animals↗

PubMed1985

Phosphorylated intermediates of two Ca++-ATPases in membrane preparations from lens epithelial cells.

By incubating preparations enriched in membranes from lens epithelial cells with [gamma 32P]-ATP and Ca++ at 0 degrees C for 15 seconds followed by SDS-PAGE analysis, it was possible to demonstrate a Ca++-dependent [32P]-phosphate incorporation in two polypeptides with Mr 105,000 and 140,000. Treatment of phosphorylated preparations with 0.06 N hydroxylamine at pH 5.4 and 25 degrees C removed the label from both polypeptides indicating that the phosphate was attached to the proteins by an anhydride linkage characteristic of the phosphorylated intermediates of the ATPases. Membrane preparations from sarcoplasmic reticulum and red blood cell studied under the same conditions showed a Ca++-dependent [32P]-phosphate incorporation into polypeptides with Mr 105,000 and 138,000, respectively, corresponding to the phosphorylated intermediates of the Ca++-ATPases present in these preparations. The results suggest the presence of two Ca++-ATPases in lens epithelial cells which, in terms of Mr, appear to be similar to those present in the sarcoplasmic reticulum and the red blood cell plasma membrane, respectively.

Animals↗

PubMed1984

Phosphorylation of H2O2-treated lens Na+,K+-ATPase.

It has been previously shown that H2O2 inhibits lens 86Rb influx and results in modification of Na+,K+-ATPase with respect to ATP hydrolysis. The effect of H2O2 on ATP hydrolysis was further investigated using [gamma-32P]-ATP to examine the Na+,K+-ATPase phosphorylated intermediate. A Na+-dependent phosphorylated polypeptide with an apparent molecular weight of approximately 100 000 was detected in all lens preparations, irrespective of H2O2 treatment. Similar results were observed for partially purified Na+,K+-ATPase from bovine kidney, porcine brain and canine kidney.

Animals↗

PubMed1984

The phosphorylation of bovine and human lens polypeptides.

The phosphorylation of bovine and human lens polypeptides was examined using an in vitro labeling assay with [gamma-32P]-ATP, followed by SDS-PAGE and radioautography. The major protein phosphorylation in all lens preparations was cAMP-dependent. The effect of cAMP could be depressed by monovalent ions. Phosphorylated polypeptides were detected in capsule-epithelial and outer cortical but not inner cortical-nuclear preparations. Differences in phosphorylated polypeptide distributions and molecular weight patterns were observed between bovine capsule-epithelium and outer cortex fiber cells and bovine and human preparations. Human cataractous lens preparations had the same pattern of phosphorylation as human normal lens preparations but with much less 32P incorporation. Major phosphorylated polypeptides in the 100 000 dalton, 60 000 dalton and 43 000 dalton range were detected in both bovine and human preparations. A 32 000 dalton phosphorylated polypeptide appeared exclusively in the bovine capsule-epithelium and a phosphorylated 23 000 dalton polypeptide was detected only in outer cortical preparations.

Animals↗

PubMed1984

Identification of two of the major phosphorylated polypeptides of the bovine lens utilizing a lens cAMP-dependent protein kinase system.

Two of the major in vitro phosphorylated polypeptides of the bovine lens have been identified. Analysis by means of two-dimensional gel electrophoresis (IEF) has demonstrated that the lens phosphorylated 57,000 and 43,000 dalton polypeptides correspond in mobility to purified phosphorylated bovine lens vimentin and chicken gizzard actin, respectively. Purified actin and vimentin were phosphorylated by a partially purified cAMP-dependent protein kinase isolated from the outer cortex water soluble fraction. All detectable bovine lens vimentin isoelectric variants were phosphorylated. In both the lens fiber cell and chicken gizzard actin preparations, the phosphorylated actin isoelectric variants did not correspond in mobility to the major actin isoelectric variant, but were more acidic. Phosphorylation in all preparations occurred at serine residues.

Actins↗

PubMedDate not supplied

Prevention of urinary albumin excretion in 6 month streptozocin-diabetic rats with the aldose reductase inhibitor tolrestat.

Recent clinical data strongly suggest that elevated urinary albumin excretion (UAE) identifies diabetic subjects at risk of developing nephropathy. Elevated UAE is attributed to increased transglomerular pressure, which is associated with poor metabolic control in rats. Because excess glucose in diabetes is metabolized via the polyol pathway, we were interested in whether the diabetes-induced elevation in UAE in rats could be prevented by inhibiting aldose reductase (AR), the first enzyme in the polyol pathway, with the AR inhibitor tolrestat. In fact, in rats made diabetic with streptozocin (35 mg/kg IV), treatment for 6 months with tolrestat (25 mg/kg/day in the diet) prevented both sorbitol accumulation in the kidney and the increase in UAE. Sorbitol accumulation and the increased UAE were not associated with statistically significant mesangial expansion, and the thickening of glomerular basement membranes was not affected by tolrestat treatment. The authors conclude that the 4.7-fold elevation in UAE in chronically diabetic rats is linked to the increased flux of glucose through the polyol pathway since it was prevented by inhibiting aldose reductase with tolrestat.

Albuminuria↗

PubMedDate not supplied

Polyol pathway activity in nervous tissues of diabetic and galactose-fed rats: effect of dietary galactose withdrawal or tolrestat intervention therapy.

Enhanced polyol pathway activity resulting in an accumulation of sorbitol and a depletion of myoinositol in nervous tissues has been proposed to be important in development of diabetic neuropathies. This investigation demonstrated that in two models of diabetic complications, streptozocin (STZ)-induced diabetic rats and galactose-fed rats, 5 weeks of disease led to an accumulation of sorbitol or galactitol, respectively, in three cranial nerves (the optic (II), trigeminal (V), and vagus (X) nerves), as well as the sciatic nerve, cerebral cortex, and retina. In both models, the cranial nerves and cerebral cortex contained lower levels of accumulated polyol than the sciatic nerve. In addition, myoinositol depletion was observed in the sciatic nerve only. In a second galactose-fed rat study, returning 5-week galactose-fed rats to a normal diet for 6 weeks led to complete elimination of galactitol from the optic nerve, sciatic nerve, and retina and normalization of the sciatic nerve myoinositol concentration. Similarly, continuing the galactose diet for 6 more weeks (ie, a total of 11 weeks) as well as administration of the aldose reductase inhibitor (ARI) tolrestat (20 and 40 mg/kg/day), caused the sciatic nerve to contain a normal myoinositol concentration and a galactitol concentration that was 95% below the level observed in galactose-fed controls. In the optic nerve and retina, tolrestat was less effective, resulting in 69-78% lower galactitol levels. In conclusion, these findings indicate that sorbitol and galactitol accumulate in cranial nerves, brain, and retina without a concomitant decrease in myoinositol. Either withdrawal of the galactose diet or intervention with tolrestat normalized polyol levels in the sciatic nerve.(ABSTRACT TRUNCATED AT 250 WORDS)

Aldehyde Reductase↗