A myosin I isoform in the nucleus.

A nuclear isoform of myosin I beta that contains a unique 16-amino acid amino-terminal extension has been identified. An affinity-purified antibody to the 16-amino acid peptide demonstrated nuclear staining. Confocal and electron microscopy revealed that nuclear myosin I beta colocalized with RNA polymerase II in an alpha-amanitin- and actinomycin D-sensitive manner. The antibody coimmunoprecipitated RNA polymerase II and blocked in vitro RNA synthesis. This isoform of myosin I beta appears to be in a complex with RNA polymerase II and may affect transcription.

Microheterogeneity of rat serum alkaline phosphatase in fasting state: characterization of two duodenal alkaline phosphatase glycoforms.

Using concanavalin A-Sepharose affinity chromatography (Con A) we found that the serum of normal fasted adult rats contains two alkaline phosphatase (APase) glycoforms, one weakly bound (II) and the other strongly (III) bound to the column. Both serum APase glycoforms had an apparent molecular mass of 163 kD on Sepharose CL-6B and 118 kD on SDS-PAGE under nondenaturing conditions. We consider the molecular forms as dimeric, since monomers of 60.3 and 58.5 kD for the Con A weakly and strongly bound glycoform, respectively, were obtained. However, these two dimeric glycoforms were different in their pH optimum, affinity to p-nitrophenyl phosphate as substrate, the degree of L-phenylalanine inhibition and relative thermostability. Judging by the relative thermostability and by L-phenylalanine inhibition, it seems that both serum APase glycoforms in fasted rats are mainly of duodenal mucosal cell origin. The Con A weakly bound (II) glycoform could be derived from the cytosol, and the Con A strongly bound (III) one from both the cytosolic and membranous fractions of duodenal mucosal cells. However, in addition to the heat-stable component, the Con A strongly bound serum APase glycoform also contains a minor heat-labile and L-phenylalanine-resistant component which could be of nonspecific tissue origin since such a fraction was not discovered by us in rat duodenal mucosal cells.

Selective inhibition of duodenal and jejunal villous cell alkaline phosphatase by the duodenal ulcerogen cysteamine.

We have found that cysteamine-HCl, a potent duodenal ulcerogen, after a single subcutaneous injection (30 mg/100 g body weight), inhibited villous cell duodenal and jejunal alkaline phosphatase (APase) under in vivo and under in vitro conditions. The duodenal and jejunal crypt-cell APase was not susceptible to cysteamine inhibition. Ileal APase from both the villous and the crypt cells was unaffected by cysteamine. Tissue-nonspecific APase from the kidney and liver was not affected by cysteamine either. The differences in tissue and cellular accumulation of cysteamine, the submolecular differences in APase molecules, and its anatomical localization in mucosal cells along the small intestine could explain the different degrees of susceptibility to cysteamine inhibition. The extent of duodenal APase inhibition by cysteamine was highly pH-dependent and varied from 5% to 85% within a pH range of 7.5-10.5. A shift in pH optimum from 9.6 to 9.3 was found in the presence of cysteamine. The inhibition of duodenal villous cell APase was greatly dependent on cysteamine concentration (Ki = 2.65 mM). At a fixed concentration of cysteamine it was not influenced by substrate concentration. Cysteamine did not change the Km value for duodenal APase but did decrease its Vmax to 46% and 15% of the controls when added in the assay or injected subcutaneously, respectively, indicating that the inhibition was of the linear, 'noncompetitive' type. Somehow cysteamine increased the requirement in the activation energy for substrate hydrolysis as well. The data indicate that macromolecular transformations could take place in the mucosal cells of duodenum after cysteamine administration.

Effect of duodenal ulcerogens mepirizole and propionitrile on small intestinal and liver alkaline phosphatase activity in rats.

Duodenal ulcer could be induced in rats by a single subcutaneous injection of the anti-inflammatory agent mepirizole (M) or by the alkyl chemical propionitrile (P). Contrary to M, P provokes HCl hypersecretion as well. We used these animal models of duodenal ulcer to study the preulcerogenic molecular changes in the mucosal cells and their causal relationship to HCl hypersecretion. It was found that M and P induced the dose- and time-dependent decrease of duodenal alkaline phosphatase (DAP) activity. The decrease was detected at 4 h, with a nadir at 12 h after the injection of both drugs. The decrease of alkaline phosphatase activity was organ-specific after P administration and it was even regional-specific along the small intestine after M administration. At the level of mucosal cells of duodenum the effect of the ulcerogens was enzyme-selective. Both ulcerogens decreased protein and alkaline phosphatase activities; however, they had no effect on lysosomal acid phosphatase. Contrary to cysteamine (C), the effect of M and P on DAP depletion could not be reproduced under in vitro conditions. An interference of P and a slight additive effect of C on DAP depletion after simultaneous subcutaneous administration with M to the rats was found. The results indicate that the DAP depletion after in vivo administration of the three duodenal ulcerogens could be provoked by at least two different mechanisms. The decrease of DAP activity seems to be a general property of the duodenal ulcerogens independent of their effects on gastric acid secretion or on the suppression of alkaline secretion in the duodenum. As a late molecular event most probably elicited by the early morphological changes, the decrease of DAP activity could rather be related to ulcer healing than to its pathogenesis.