Fusion of lysosomes with late endosomes produces a hybrid organelle of intermediate density and is NSF dependent.

Using a cell-free content mixing assay containing rat liver endosomes and lysosomes in the presence of pig brain cytosol, we demonstrated that after incubation at 37 degrees C, late endosome-lysosome hybrid organelles were formed, which could be isolated by density gradient centrifugation. ImmunoEM showed that the hybrids contained both an endocytosed marker and a lysosomal enzyme. Formation of the hybrid organelles appeared not to require vesicular transport between late endosomes and lysosomes but occurred as a result of direct fusion. Hybrid organelles with similar properties were isolated directly from rat liver homogenates and thus were not an artifact of cell-free incubations. Direct fusion between late endosomes and lysosomes was an N-ethylmaleimide-sensitive factor-dependent event and was inhibited by GDP-dissociation inhibitor, indicating a requirement for a rab protein. We suggest that in cells, delivery of endocytosed ligands to an organelle where proteolytic digestion occurs is mediated by direct fusion of late endosomes with lysosomes. The consequences of this fusion to the maintenance and function of lysosomes are discussed.

Radioimmunoassay for phorbol esters using rabbit antisera against phorbol succinate.

The phorbol nucleus was succinylated and then conjugated to bovine albumin using dicyclohexylcarbodiimide. Rabbits given injections of the conjugate developed antibodies which rose in titer progressively with repeated immunization. By the ninth bleeding, the binding of one antiserum, diluted 1:15,000, was saturated with about 10 nM [3H]phorbol-12,13-dibutyrate [( 3H]-PDBU) and had an average association constant, Ka, of 2.6 X 10(8) M-1. The serological specificity of the antisera was characterized by examining the inhibition of the [3H]PDBU-anti-phorbol succinate immune system by 18 phorbol-related compounds. The specificities of antibodies from two rabbits tested in detail were qualitatively similar. The rank order of inhibitory activity for certain phorbol-related compounds was PDBU [concentration of inhibitor required to give 50% inhibition of PDBU binding (IC50) = 7.6 nM] = phorbol-13-acetate [IC50 = 8.2 nM] greater than phorbol-12,13-dibenzoate greater than 4-beta-phorbol [IC50 = 124 nM] greater than or equal to phorbol-12,13-diacetate greater than or equal to phorbol-12-myristate-13-acetate [IC50 = 184 nM] greater than phorbol-13,20-diacetate greater than phorbol-12-acetate [IC50 = 2300 nM]. The following compounds showed no detectable serological activity: mezerein, 4-0-methylphorbol-12-myristate-13-acetate, ingenol, 4-alpha-phorbol, teleocidin B, and dihydroteleocidin B. These and other results indicated that the 4-beta-phorbol nucleus was required for serological activity, that esterification of the C-13 position with benzoate, acetate, or butyrate enhanced the immunoreactivity of 4-beta-phorbol, and that among the phorbol-related compounds examined there was no direct relationship between serological activity and biological potency as tumor promoters. Using the [3H]PDBU-anti-phorbol succinate immune system, we measured the concentrations of immunoreactive phorbol-related material in crude mixtures such as croton oil and performed pharmacokinetic studies in rats given PDBU s.c.

Thyrotropin-releasing hormone induces redistribution of protein kinase C in GH4C1 rat pituitary cells.

Thyrotropin-releasing hormone (TRH) affects hormone secretion and synthesis in GH4C1 cells, a clonal strain of rat pituitary cells. Recent evidence suggests that the intracellular mediators, inositol 1,4,5-trisphosphate and 1,2-diacylglycerol, which are generated as a result of TRH-induced hydrolysis of the polyphosphatidylinositols, may be responsible for some of the physiological events regulated by TRH. Because diacylglycerol is an activator of protein kinase C, we have examined a role for this enzyme in TRH action. The subcellular distribution of protein kinase C in control and TRH-treated cells was determined by measuring both enzyme activity and 12,13-[3H]phorbol dibutyrate binding in the cytosol and by measuring enzyme activity in the particulate fraction. Acute exposure of GH4C1 cells to TRH resulted in a decrease of cytosolic protein kinase C, and an increase in the level of the enzyme associated with the particulate fraction. The redistribution of protein kinase C induced by TRH was dose- and time-dependent, with maximal effects occurring within the first minute of TRH treatment. Analogs of TRH which do not bind to the TRH receptor did not induce redistribution of protein kinase C, while the active analog, methyl-TRH, did promote redistribution. Treatment of GH4C1 cells with phorbol myristate acetate also resulted in a shift in protein kinase C distribution, although the response was slower than that produced by TRH. TRH-induced redistribution of protein kinase C implies translocation of the enzyme from a soluble to a membrane-associated form. Because protein kinase C requires a lipid environment for activity, association with the membrane fraction of the cell suggests activation of the enzyme; thus, protein kinase C may play a role in some of the actions of TRH on GH4C1 cells.

Ionomycin inhibits thyrotropin-releasing hormone-induced translocation of protein kinase C in GH4C1 pituitary cells.

Thyrotropin-releasing hormone (TRH) induces rapid and transient conversion of protein kinase C (Ca2+/phospholipid-dependent enzyme) from a soluble to a particulate-bound form in GH4C1 rat pituitary cells. Ionomycin (200 nM), a calcium ionophore, had no effect by itself on the subcellular distribution of protein kinase C. However, pretreatment of the cells with 200 nM ionomycin inhibited by greater than 50% the ability of TRH to cause translocation of protein kinase C from the cytosol to the particulate cell fraction. Inhibition by ionomycin required that the cells be incubated with the ionophore for at least 10 s before TRH addition. Ionomycin pretreatment did not alter the kinetics of TRH-induced protein kinase C redistribution. Incubation of the cells with 43 mM potassium prior to TRH addition almost completely reversed the inhibition induced by ionomycin. We propose that the mechanism by which ionomycin attenuates TRH action on protein kinase C may involve the capacity of the ionophore to empty the intracellular calcium reservoir which normally releases calcium into the cytosol in response to TRH. Our result provides evidence that the rise in intracellular calcium, which accompanies diacylglycerol formation following TRH action on polyphosphatidylinositide hydrolysis, may be required to achieve maximal conversion of protein kinase C to its presumed active, membrane-bound form in these cells.

Identification of the active-site residue of gamma-cystathionase labeled by the suicide inactivator beta, beta, beta-trifluoroalanine.

Inactivation of gamma-cystathionase by beta, beta, beta-trifluoroalanine, a suicide inactivator of the enzyme, results in covalent labeling of an amino group of the protein [Silverman, R. B., & Abeles, R. H. (1977) Biochemistry 16, 5515-5520]. We have established that this modified amino function is the epsilon-NH2 group of a lysine residue. A heptapeptide which includes this modified lysine residue was isolated, and its sequence was found to be Cys-Ser-Ala-Thr-Lys-Tyr-Met. The amino acid sequence was the same as that determined for peptides containing the active-site lysine residue which forms a Schiff base with pyridoxal phosphate. Therefore the epsilon-NH2 group of the active-site lysine which binds pyridoxal phosphate is capable of interacting with the beta carbon of trifluoroalanine, and presumably the beta carbon of normal substrates. We therefore propose that this lysine residue may function as a proton-transfer agent in the reactions catalyzed by gamma-cystathionase.

The molecular basis of beta thalassaemia in Punjabi and Maharashtran Indians includes a multilocus aetiology involving triplicated alpha-globin loci.

We have analysed 201 beta-thalassaemia (beta-thal) genes from natives of the Punjab (156) and Maharashtra states of India and found the causative mutation in 200 of them. The most common beta-globin gene mutations differed significantly between these two groups and between these groups and Indian immigrants in the U.S.A. and the U.K. In the Punjabi Indians the IVS-1, nt 1 (G-T) mutation accounted for nearly one-quarter of beta-thal genes, whereas it was 5% or less in the other groups. Likewise, the cap + 1 mutation was much more prevalent in the Punjabis, whereas the nonsense codon 15 allele had a higher frequency in the Maharashtrans of the Bombay region. The common IVS-1, nt5 allele had a frequency of 60% of beta-thal genes in the Maharastrans, 35% in North American immigrants, and only 23% in the Punjabis. Two-thirds of all beta-thal genes in Punjab were found in the merchant caste (Khatri-Arora), whereas the menial caste (Shudra) was highly represented among those with beta-thal genes in Maharashtra. Two novel beta-globin alleles were each found once; a frameshift codon 55 (+A) in Maharashtrans and a frameshift codons 47-48 (+ATCT) in Punjabis. Of three Punjabi patients with beta-thal intermedia in whom only a single severe beta-globin gene mutation was found, two had six alpha-globin genes (homozygosity for a triplicated alpha-globin locus) instead of the normal alpha-globin gene number of four. Thus, these two individuals had a multilocus aetiology of beta-thal and their parents have the unusual recurrence risk of 1 in 8 for conceiving a third with beta-thal intermedia. Since 15% of 126 alpha-globin clusters studies in Punjabis contained either single (10%) or triplicated (5%) alpha-globin genes, the alpha-globin gene number is a frequent modifier of the phenotype of beta-thal in this ethnic group.