Isolation and characterization of human glucose-6-phosphate isomerase isoforms containing two different size subunits

Previously undetected isoforms of human glucose-6-phosphate isomerase (GPI) have been isolated utilizing substrate-induced elution of the enzyme from spherical cross-linked phosphocellulose as an affinity ligand and subjected to a series of physical and chemical studies. The two major isoforms (1, 48%, pI 9.13; 2, 36%, pI 9.00) are homodimers of subunits of 63.2 kDa (Type-A) and are charge isomers, probably representing deamidation of specific Asn-Gly sequences as in other species. Isoform 3 (13%, pI 8.84) is a heterodimer composed of the Type-A subunit and a previously unreported larger subunit of 69.8 kDa (Type-B). Isoform 4 (3%, pI 8.62) is a BB-homodimer. Structural differences in the two types of subunits are also apparent from CNBr fragmentation patterns. Carbohydrate analyses show that, even though potential N- and O-linked glycosylation sites exist, the isoforms are not due to glycosylation. Recently recognized sequence similarities between GPI and the neurotropic lymphokine, neuroleukin (NLK) suggest that GPI and NLK are either derived from the same gene or represent modifications of the same protein. The possibility of NLK-GPI dimers exists, but the new isoforms identified in this study do not appear to represent hybrids of GPI subunits with mature NLK.     

Lipid conjugates of antiretroviral agents. II. Disodium palmityl phosphonoformate: anti-HIV activity, physical properties, and interaction with plasma proteins

Disodium palmityl phosphonoformate, a novel lipid phosphoester of the anti HIV agent phosphonoformate (foscarnet), inhibits HIV replication in H9 cells and syncytia formation in MOLT-3 cells as effectively as foscarnet itself, as shown by dose-response data from assays for expression of p17 and p24 viral antigens and syncytia formation. Protein binding studies indicate that in serum, the derivative exists bound to albumin and the lipoproteins, and would therefore be likely to exhibit improved serum lifetime in vivo.     

Large scale purification and immunolocalization of bovine uroplakins I, II, and III. Molecular markers of urothelial differentiation

The differentiation of mammalian urothelium culminates in the formation of asymmetrical unit membrane (AUM). Using gradient centrifugation and detergent wash, we purified milligram quantities of AUMs which, interestingly, contained three major proteins (15, 27, and 47 kDa) that appeared to be identical to the three immunoaffinity purified, putatively AUM-associated proteins that we described earlier (Yu, J., Manabe, M., Wu, X.-R., Xu, C., Surya, B., and Sun, T.-T. (1990) J. Cell Biol., 111, 1207-1216). Peptide mapping and immunoblotting established that these three proteins were distinct molecules. Using monospecific antibodies to these three proteins, we showed that they were all restricted to the superficial urothelial cells and were AUM-associated. The 27- and 15-kDa proteins were detected exclusively on the luminal side of mature, apical AUMs. In contrast, epitopes of the 47-kDa protein were detected on both sides of apical AUMs suggesting a transmembranous configuration. These results (i) provide the strongest evidence thus far that AUM contains three major proteins (the 27-kDa uroplakin I, 15-kDa uroplakin II, and 47-kDa uroplakin III) which form an extremely insoluble complex, (ii) suggest that uroplakin II, like uroplakin I (Yu, J., Manabe, M., Wu, X.-R., Xu, C., Surya, B., and Sun, T.-T. (1990) J. Cell. Biol. 111, 1207-1216), translocates from one side of the membrane to another during AUM maturation, (iii) indicate that uroplakin III may play a different structural role than uroplakins I and II in AUM formation, and (iv) establish the three uroplakins as markers for an advanced stage of urothelial differentiation.     

Selective-Differential Medium for Isolation and Differentiation of Pectinatus from Other Brewery Microorganisms

An agar medium, LL-agar (lactate-lead acetate) was designed to selectively differentiate members of the genus Pectinatus (S. Y. Lee, M. S. Mabee, and N. O. Jangaard, Int. J. Syst. Bacteriol. 28:582-594, 1978; S. Y. Lee, M. S. Mabee, N. O. Jangaard, and E. K. Horiuchi, J. Inst. Brew. 86:28-30, 1980) from other brewery microorganisms. Selectivity was achieved by the use of sodium lactate as the sole source of carbon and phenylethyl alcohol as an inhibitor for aerobic gram-negative bacteria and yeast. Differentiation was established by the introduction of lead acetate into the medium, which reacted with the H₂S liberated by Pectinatus and resulted in a blackening of the Pectinatus colonies while the other brewery organisms, when present, remained white. In combination with the Lee tube (J. E. Ogg, S. Y. Lee, and B. J. Ogg, Can. J. Microbiol. 25:987-990, 1979) and this medium, isolation of Pectinatus organisms from beer samples was accomplished with convenience and simplicity.     

Preparation of two dinor-PGI2 metabolites from 6-keto-PGF1α by mycobacterium rhodochrous

The transformation of 6-keto-PGF_1α to two prostacyclin metabolites, 2,3-dinor-6-keto-PGF_1α (I) and 2,3-dinor-6,15-diketo-13,14-dihydro-PGF_1α (II) by $\text{Mycobacterium rhodochrous}$ UC-6176 is described. The finding that the bacterium oxidized 6-keto-PGF_1α to the 6,15-diketo metabolite II shows that it contains 15-hydroxy prostaglandin dehydrogenase and Δ¹³ reductase enzyme systems.     

Ectoprotein kinase activity of the isolated rat adipocyte.

Intact adipocytes exhibit ectoprotein kinase activity as reflected by their ability to catalyze the transfer of the terminal phosphate of (γ-³²P) ATP to histone added to a cell suspension. This activity is substrate, time and cell number dependent. Lineweaver-Burk plots gave Km and Vmax values for ATP of 5 × 10^?5 M and 7.14 pmoles/min/1.5 × 10⁵ cells. Cyclic AMP but not cyclic GMP in μM concentrations stimulates ectoprotein kinase activity. The controlled tryptic digestion of intact cells results in reduction of ectoprotein kinase activity. This activity is not due to leakage of intracellular protein kinases during the preparative procedure nor to penetration of histone into the cells. Additional phosphoproteins not accessible to endogenous protein kinase activity are also localized on the external surface of the intact fat cell.     

The amino acid sequence of chick skin collagen α1-CB7: The presence of a previously unrecognized triplet

Because alignment of the amino acid sequences of chick skin collagen α2-CB3 (1) with the relevant portion of chick skin collagen α1-CB7 (2) suggested that a Gly-X-Y triplet may have been missed in the latter, the peptide TM-2, produced by tryptic digestion of maleylated α1-CB7, was reinvestigated. Cleavage by trypsin at the unblocked lysine at position 18, and isolation of the resulting COOH-terminal peptide, showed this to be a 15-residue peptide containing a previously unrecognized Gly-Pro-Hyp triplet. Sequencing of the peptide showed this to occupy positions 4 through 6, or 56 through 58 of α1-CB7. The latter thus has 271 instead of 268 residues, and the α1[I] chain 1055 instead of 1052.     

Influence of clay minerals on sorption of bacteriolytic enzymes

Myxobacteria presumably produce extracellular bacteriolytic enzymes when they are growing in soil. In order to study their ecological significance, adsorption experiments were performed with lytic enzymes produced byMyxococcus virescens in casitone media. Different soils as well as montmorillonite and kaolinite can rapidly adsorb the bacteriolytic but not the proteolytic enzymes. About 1 gm of montmorillonite per liter of cell-free culture solution is enough for the adsorption of 97% of the bacteriolytic enzymes. The adsorption per unit weight is about 100 times greater on montmorillonite than on kaolinite. About 40% of the adsorbed enzymes can be eluted with solutions of high pH or high ionic strength. The only desorbed bacteriolytic enzyme is the alanyl-∈-N-lysine endopeptidase.