The promotion of collagen polymerization by lanthanide and calcium ions.

Ca2+ (1-5 mM) and lanthanide (20-250 microM) ions enhance the rate of polymerization of purified calf skin collagen (1.5 mg/ml) at pH 7.0 in the presence of 30mM-Tris/HCl and 0.2 M-NaCl. Both the nucleation phase and the growth phase of polymerization are accelerated. The activation energy of the growth phase, 239.3 +/- 24.3 kJ/mol (57.2 +/- 5.8 kcal/mol), is decreased to 145.6 +/- 9.6 kJ/mol (34.8 +/- 2.3 kcal/mol) by 5 mM-Ca2+ and to 75.3 +/- 4.6 kJ/mol (18.0 +/- 1.1 kcal/mol) by 25 microM-Sm3+. In contrast, the activation energy of the nucleation phase, 191.6 +/- 23.4 kJ/mol (45.8 +/- 5.6 kcal/mol), is only slightly decreased by Ca2+ or Sm3+. Collagen fibrils formed in the presence of Sm3+ are thinner than control fibrils, and more thermoresistant.     

Age-related changes in chemical composition and physical properties of mucus glycoproteins from rat small intestine.

Mucus glycoproteins from newborn and adult rat small intestine were radiolabelled in vivo with Na2 35SO4 and isolated from mucosal homogenates by using Sepharose 4B column chromatography followed by CsCl-density-gradient centrifugation. Non-covalently bound proteins, lipids and nucleic acids were not detected in the purified glycoproteins. Amino acid, carbohydrate and sulphate compositions were similar to chemical compositions reported for other intestinal mucus glycoproteins, as were sedimentation properties. There were, however, important differences in the chemical and physical characteristics of the mucus glycoproteins from newborn and adult animals. The buoyant density in CsCl was higher for the glycoproteins from newborn rats (1.55 g/ml versus 1.47 g/ml). On sodium dodecyl sulphate/polyacrylamide/agarose-gel electrophoresis, the glycoprotein from newborn rats had a greater mobility than the adult-rat sample. Although both preparations had similar general amino acid compositions, variations were observed for individual amino acids. The total protein content was greater in the glycoprotein from newborn animals (27%, w/w, versus 18%, w/w). The molar ratio of carbohydrate to protein was less in the newborn, primarily owing to a decreased fucose and N-acetylgalactosamine content. Comparison of the molar ratio of fucose and sialic acid to galactose for both glycoproteins demonstrated a reciprocal relationship similar to that described by Dische [(1963) Ann. N.Y. Acad. Sci. 106, 259-270]. The sulphate content was greater in the glycoprotein from newborn rats (5.5%, w/w, versus 0.9%, w/w). Both had similar sedimentation coefficients in a dissociative solvent. These results suggest an age-related difference in the types of mucus glycoproteins synthesized by small intestine.     

Mechanism for the ''anti-lipolytic'' action of vasopressin in the starved rat.

The marked decrease in blood non-esterified fatty acids and ketone bodies after vasopressin infusion into starved rats [Rofe & Williamson (1983) Biochem. J. 212, 231-239] was investigated. Vasopressin did not inhibit lipolysis in isolated rat adipocytes. The metabolic effects in vivo were still present after pretreatment of rats with indomethacin, indicating that the effect is not secondary to the release of prostaglandins. Vasopressin significantly decreased blood flow through the retroperitoneal, epididymal and mesenteric fat depots, by 80%, 76% and 46% respectively. The specific haemodynamic effect of vasopressin on adipose tissue is considered to be the primary cause of the major metabolic changes seen in the starved rat.     

Changes with starvation in the rat of the lipoprotein lipase activity and hydrolysis of triacylglycerols from triacylglycerol-rich lipoproteins in adipose tissue preparations.

Lipoprotein lipase activity was higher in fat-pad pieces than in isolated adipocytes from the same fed rats, whereas hydrolysis of triacylglycerols from triacylglycerol-rich lipoproteins was similar in the two preparations when incubated either in basal conditions or in the presence of heparin. In both preparations there was a similar release of lipoprotein lipase activity into the medium during basal incubation, enhanced by the presence of heparin. In fat-pad pieces, but not in isolated adipocytes, incubation with heparin produced a decrease in the lipoprotein lipase activity measured in the tissue preparation. In fat-pad pieces from 24 h-starved rats, lipoprotein lipase activity was the same as in isolated adipocytes from the same animals and incubation with heparin did not affect the appearance of lipoprotein lipase in the medium or the utilization of triacylglycerols from triacylglycerol-rich lipoproteins. These results support the following conclusions. (1) The effectiveness of lipoprotein lipase in adipose tissue preparations in vitro depends more on its availability to the substrate than on its total activity. (2) Heparin acts on adipose tissue preparations from fed animals both by enhancing the release of pre-existing extracellular enzyme (which is absent in isolated adipocytes) and by enhancing the transfer outside the cells of the intracellular (and mainly undetectable) enzyme that is activated in the secretion process. (3) In adipose tissue from starved animals there is not only a decrease in the active extracellular form of lipoprotein lipase activity but also a reduction in the intracellular (and mainly undetectable) pool of the enzyme.     

The cartilage-resorbing protein catabolin is made by synovial fibroblasts and its production is increased by phorbol myristate acetate.

Pig synovial fibroblasts were shown to produce a protein that caused live cartilage to resorb its proteoglycan matrix in vitro. Fibroblasts were obtained either from synovial tissue digest or by allowing them to grow out of explants. The population derived from the digests was homogeneous and free of macrophage-like cells after two passages, but was still producing the cartilage-resorbing protein after seven passages. The active protein was found to have Mr 20,000 on gell filtration, and pI 4.8 on isoelectric focussing in polyacrylamide gel. It was indistinguishable from a protein with the same activity from pig mononuclear leucocytes, which has been called catabolin. Production of the protein was increased if the synovial fibroblasts were cultured with the tumour promoter phorbol 12-myristate 13-acetate. Fibroblasts from other sources (joint capsule and peritoneum) also apparently made the protein. The possibility that catabolin is the same as interleukin-1 is discussed: if they are, then the results suggest that fibroblasts can make an interleukin-1-life protein.     

The stimulation of glycogenolysis and gluconeogenesis in isolated hepatocytes by opioid peptides.

The opioid agonists [leucine]enkephalin, [D-Ala2,D-Leu5]enkephalin and dynorphin-(1-13)-peptide, but not morphine, stimulated the conversion of [2-14C]pyruvate into glucose and glycogenolysis when added directly to isolated hepatocytes. Naloxone produced a small but significant inhibition of both the basal and stimulated rate of incorporation of label into glucose but had no effect on the total glucose output by the cells. The effects of the opioid peptides were mediated by a cyclic AMP-independent mechanism.     

Slow passive diffusion of NAD+ between intact isolated plant mitochondria and suspending medium.

Isolated potato (Solanum tuberosum) tuber mitochondria purified by isopycnic centrifugation in density gradients of Percoll were found to be highly intact, to be devoid of extramitochondrial contaminations and to retain a high rate of O2 consumption. When suspended in a medium that avoided rupture of the outer membrane, intact purified mitochondria progressively lost their NAD+ content by passive diffusion. This led to a slow decrease of oxoglutarate-dependent O2 consumption by isolated mitochondria. Addition of NAD+ to the medium restored the initial State-3 rate of oxoglutarate oxidation. The rate of NAD+ accumulation in the matrix space was concentration-dependent, exhibited Michaelis-Menten kinetics and was strongly inhibited by the analogue N-4-azido-2-nitrophenyl-4-aminobutyryl-NAD+.     

Glycosylation of proteins from sugar nucleotides by whole cells. Effect of ammonium chloride treatment on mouse thymocytes.

When thymocytes are treated with iso-osmotic NH4Cl, the sugar incorporation into endogenous acceptors from labelled sugar nucleotides is largely increased compared with that in control thymocytes. This effect was obtained with labelled GDP-mannose, UDP-galactose and CMP-N-acetylneuraminic acid. The stimulation observed with NH4Cl-treated thymocytes does not involve the glycosylation of exogenous acceptors, and it was proved that the NH4Cl treatment (1) does not stimulate glycosyltransferase activities themselves, (2) does not lead to the release of soluble glycosyltransferases as the result of an extensive lysis of the thymocytes and (3) does not cause the emergence of glycosyltransferases at the cell surface. In fact, electron-microscopy observations showed that, although marked changes had occurred in the cytoplasm, the plasma membrane is sufficiently maintained to allow the cell to keep roughly its original shape and to retain the intracellular vesicles. We thus demonstrate that this stimulation is due to an enhancement of the entry of sugar nucleotides into the cell. As demonstrated by the inclusion of Trypan Blue within the cells, and the non-stimulation of glycosylation of exogenous large-molecular-mass acceptors, the effect of NH4Cl seems to be limited to the penetration of small-molecular-sized compounds through the plasma membrane. Thus NH4Cl treatment allows the labelled sugar nucleotides to penetrate the cell and to behave as the cellular pool to be utilized for glycosylation by intracellular vesicles.     

The effect of counterions on melittin aggregation.

Melittin, a surface-active polypeptide from bee venom, has an overall hydrophobic N-terminus, with basic residues clustered at the C-terminus. In aqueous solution melittin exists as a mixture of monomer and tetramer, the monomer adopting a predominantly random-coil configuration, whereas the tetramer is rich in alpha-helix. The tendency of melittin to aggregate is dependent on the counter-anions present in solution, the effect being most marked with phosphate, decreasing in the order HPO4(2-) greater than SO4(2-) greater than ClO4- greater than Cl-.     

Subcellular location of an abundant substrate (p36) for tyrosine-specific protein kinases.

A 36,000-dalton cellular protein (p36) has been identified previously as an abundant substrate for phosphorylation by tyrosine-specific protein kinases. Since several of the responsible kinases are associated with the plasma membrane, we explored the subcellular distribution of p36. Biochemical fractionations located p36 on the plasma membrane of both normal and retrovirus-transformed cells. Approximately half of the p36 was bound to the membrane with the affinity of a peripheral membrane protein; the remainder was even more tightly bound. The distribution of p36 among subcellular fractions and its affinity for the plasma membrane were not affected by tyrosine phosphorylation. We determined that p36 is synthesized in the soluble compartment of the cell and then moves rapidly to the membranous compartment. Immunofluorescence microscopy with antibodies directed against p36 revealed two distinct distributions of the antigen: (i) a sharply demarcated crenelated pattern within or immediately beneath the plasma membrane, which we presume to be a correlary of the distribution of p36 in biochemical fractionations; and (ii) diffuse staining in a cytoplasmic location that could not be attributed to a specific feature of cytoarchitecture and could not be easily reconciled with the results of biochemical fractionations. Efforts to detect the secretion of p36 were unsuccessful. No evidence was obtained for exposure of p36 on the cell surface, and no changes in localization were observed as a consequence of neoplastic transformation. During the course of this study, we had the opportunity to pursue a previous report that p36 is a component of the enzyme malate dehydrogenase (Rubsamen et al., Proc. Natl. Acad. Sci. U.S.A. 79:228-232, 1982). We were unable to substantiate this claim. We conclude that at least a substantial fraction of p36 is located on the cytoplasmic aspect of the plasma membrane, where it could be well situated to serve as a substrate for several identified tyrosine-specific kinases. But the function of p36 and its role, if any, in neoplastic transformation of cells by retroviruses possessing tyrosine-specific kinases remain enigmatic.