Roland Henning

pH gradient across the lysosomal membrane generated by selective cation permeability and donnan equilibrium

The pH within isolated Triton WR 1339-filled rat liver lysosomes was determined by measuring the distribution of [14C]methylamine between the intra- and extralysosomal space. The intralysosomal pH was found to be approximately one pH unit lower than that of the surrounding medium. Increasing the extralysosomal cation concentration lowered the pH gradient by a cation exchange indicating the presence of a Donnan equilibrium. The lysosomal membrane was found to be significantly more permeable to protons than to other cations. The relative mobility of cations through the lysosomal membrane is H+ greater than Cs+ greater than Rb+ greater than K greater than Na+ greater than Li+ greater than Mg2+, Ca2+. The presented data suggest that the acidity within isolated Triton WR 1339-filled lysosomes is maintained by: (1) a Donnan equilibrium resulting from the intralysosomal accumulation of nondiffusible anions and (2) a selective permeability of the lysosomal membrane to cations.

Membrane lipids of rat liver lysosomes prepared by freeflow electrophoresis

Abstract Rat liver lysosomes were isolated by free-flow electrophoresis and were examined morphologically and enzymatically for purity. Their membrane fraction was prepared by osmotic shock and analyzed for cholesterol, phospholipids and fatty acids. The results were compared with the membrane fraction of Triton WR 1339-filled lysosomes and with mitochondria. The cholesterol content (0.269 M cholesterol per M lipid phosphorus), the sphingomyelin concentration (7.9% of total lipid phosphorus) and the degree of unsaturation of fatty acids (38–45%) were found to be intermediate between those of membranes of Triton WR 1339-filled lysosomes (“plasma membrane-like”) and mitochondria (“endoplasmic reticulum-like”). The similarity of these results with corresponding data for the Golgi apparatus support the present view concerning the formation of primary lysosomes via the Golgi apparatus. The drastic changes in the lipid composition found after overloading with Triton WR 1339 confirm that the plasma membrane participates in the formation of the secondary lysosomal membrane. The data presented here underline the significance of the analysis of membrane lipids in evaluating correlations between morphologically different but functionally closely related membrane types.

Nature and localization of acidic groups on lysosomal membranes

Abstract pH-dependent reversible binding of lysosomal enzymes to membranes of Triton WR 1339-filled rat liver lysosomes indicates the presence of acidic groups on lysosomal membranes. Increasing the ionic strength as well as addition of nonlysosomal proteins inhibit binding of enzymes at pH 4. Digestion of lysosomal membranes with proteases and phospholipase C has no effect on the reversibility of enzyme-membrane binding. Only neuraminidase treatment of lysosomal membranes significantly reduces the enzyme membrane binding at pH 4 indicating that sialic acid is the main anionic group at this pH. Electron micrographs of colloidal iron-stained lysosomes show a preferential localization of sialic acid on the inside of the lysosomal membrane. Neuraminidase-treated lysosomal membranes show no or considerably reduced iron staining. pH-dependent binding curves of lysosomal enzymes to a strong acid cation-exchanger (phosphocellulose) show a similar shape as those obtained with lysosomal membranes. The intralysosomal accumulation of nondiffusible anions might produce a Donnan equilibrium serving as an energy independent means to maintain an intra-lysosomal acid milieu.

SV40-transformed cells express SV40 T antigen-related antigens on the cell surface

Abstract SV40 tumor antigen (T-Ag)-related antigens were detected serologically on the surface (surface T) of living SV40-transformed human and mouse monolayer cells by an 125 I-protein A binding assay. In immunofluorescence analysis, these cells were negative for surface T. However, on mKSA, a SV40-transformed mouse cell line grown in suspension or on SV40-transformed human and mouse monolayer cells put into suspension, surface T could be visualized by immunofluorescence microscopy. The antisera used in these experiments were raised in rabbits with purified, SDS-denatured SV40 T-Ag or came from hamsters bearing SV40 tumors. Both types of antisera had in common high titers against SV40 T-Ag (⩾1:1000). All these antisera were negative on normal cells or on polyoma virus-transformed cells. The specificity of both antisera for SV40 T-Ag-related binding sites on the surface of SV40-transformed cells were demonstrated by an 125 I-IgG blocking assay in which preincubation of the cells with rabbit anti-T-Ag serum inhibited the binding of hamster SV40 tumor serum to the cell surface by about 85%. These results demonstrate the expression of T-Ag-related antigens on the surface of living cells and, therefore, support the hypothesis that SV40 T-Ag-related antigens participate in the formation of the SV40-specific tumor transplantation antigen (TSTA).

Isolation of rat liver lysosomes by loading with colloidal gold

Abstract Rat liver lysosomes were isolated by density-gradient centrifugation after loading with colloidal gold with good yield (0.74 mg protein/g rat liver) and purity. Contamination by mitochondria, endoplasmic reticulum, plasma membranes and peroxisomes is low, as judged by the determination of marker enzymes. Purity and yield, as well as the enrichment factors of various lysosomal enzymes (acid phosphatase, 17 × ; β-galactosidase, 15 × ; β-glucosidase, 17 × ; β - N -acetyl-glucosaminidase, 47 × ; β-glucuronidase, 9 × ; arylsulfatase A, 32 ×) compare favorably with those obtained by other methods to which the new procedure is proposed as a simple alternative.

Simian virus 40 T-antigen phosphorylation is variable

Simian virus 40 (SV40)-transformed tumor cells contain at least two SV40 coded proteins: Little t(MW 2 1 000) and large T-antigen (MW 94 000). Large T-antigen (T-Ag) is located in the cell nucleus [I] and seems to be involved in the synthesis of viral DNA and RNA in permissive cells, as well as of host cell DNA and RNA in transformed cells. Furthermore, this protein is apparently responsible for the initiation and the mainten~ce of cell transformation (review in [2J). Besides the amino acid sequence of T-Ag which can be derived from the known nucleotide sequence of SV40 DNA [3,4], only a few biochemical properties of T-Ag are known: (i) In 1977 P. Tegtmeyer reported that T-Ag is a phosphoprotein [5]; (ii) T-Ag has a nonspeci~c binding affinity to double-stranded (ds) and single’stranded (ss) DNA [6-S] and a specific binding affinity to the region of the origin of replication of SV40 DNA [7,9,10]. This report describes that the phosphorylation of T-Ag is variable and it gives a preliminary characterization of an apparently positive correlation between the phosphorylation and the binding afIinity of T-Ag to calf thymus ds-DNA.

Enhanced protein phosphorylation in SV40-transformed and -infected cells

We have studied the phosphorylation of cellular phosphoproteins and, in more detail, of SV40 T antigen and the cellular protein p53 in SV40 tsA-transformed cells. As detected by radiolabeling cold-sensitive tsA1499- or heat-sensitive tsA58-transformed rat fibroblasts with [32P]orthophosphate or by in vitro labeling extracts with [gamma-32P]ATP the hyperphosphorylation of certain cellular phosphoproteins including p53 and also of free SV40 large T antigen and T antigen complexed with p53 is strictly correlated with the expression of the transformed phenotype. This hyperphosphorylation can be observed as early as 30 min after shifting to the temperature where the cells expressed the transformed phenotype and, furthermore, it is dependent on protein synthesis. To evaluate the influence of a functional T antigen and to exclude properties of individual transformants we 32P labeled in vitro cellular proteins from rat F111, mouse NIH 3T3, and monkey TC-7 cells infected with tsA58 or tsA1499. In tsA58-infected cells we found a heat-sensitive enhancement of protein phosphorylation just as in tsA58 transformants. In tsA1499-infected monkey cells we observed a heat-sensitive and in abortively infected rat or mouse cells a cold-sensitive hyperphosphorylation of proteins. Thus in tsA-transformants and in various tsA-infected cells we found a strong correlation among the transformed phenotype, functions of T antigen, and the phosphorylation of various cellular proteins and in particular T antigen and p53.

Regions of SV40 large T antigen necessary for oligomerization and complex formation with the cellular oncoprotein p53

The simian virus 40 (SV40) T antigen is composed of 708 amino acids and forms monomers and various oligomers and, in small amounts, heterologous complexes with the cellular oncoprotein p53 (T‐p53). Using SV40 mutants coding for T antigen fragments which are either deleted in the N‐terminal half or truncated by various lengths at the C‐terminal end, we found that a region between amino acids 114 and 152 and a C‐terminal region up to amino acid 669 are essential for the formation of high Mr oligomers of T antigen. Furthermore, only the C‐terminal end up to amino acid 669 is essential for T‐p53 complex formation but not the N‐terminus up to amino acid 152.

Oligomerization of simian virus 40 tumor antigen may be involved in viral DNA replication

Biological implications of the oligomerization of simian virus 40 (SV40) large T antigen for viral DNA replication were studied by using two temperature-sensitive SV40 A-gene mutants, tsA 58 and tsA 1499. Both mutants are defective at elevated temperature for viral DNA replication whereas tsA 58 is like most other tsA mutants additionally heat sensitive for cell transformation. We found that in contrast to tsA 58 encoded T antigen, tsA 1499 T antigen is thermostable in the ability to bind specifically to the origin of replication of SV40 DNA. Detailed structural analysis of tsA 1499 T antigen by sucrose density gradient centrifugation revealed that it is strictly temperature sensitive for the formation of homologous oligomers but, as we reported previously (M. Montenarh, M. Kohler, and R. Henning, 1984, J. Virol, 49, 658-664), not for the association with the cellular phosphoprotein p53. These observations are compatible with the idea that, in addition to the specific origin-binding ability as well as other functional features, the oligomerization of T antigen may be essential for viral DNA replication.

Cell surface binding affinity of Simian virus 40 T-antigen

Abstract Simian virus 40-transformed cells are characterized by a virus-induced tumor transplantation antigen (SV40 TSTA) defined in vivo by the rejection of tumorigenic SV40-transformed cells in SV40-immunized mice and in vitro by SV40 tumor cell-specific cytotoxic T cells. Several experimental findings support the notion that SV40-infected and -transformed cells express SV40 large tumor antigen (TAg) or closely related antigens on the cell surface (surface T). In this report, evidence is presented for a cell surface binding affinity of SV40 TAg solubilized and extracted by disruption of SV40-transformed and SV40-infected cells in growth medium. Incubation of various transformed and nontransformed living monolayer cells in situ with these extracts led to a significant uptake of TAg to the cell surface (called “externally bound TAg”) up to two to five times higher amounts in comparison to native surface T on SV40-transformed cells. This was demonstrated by the highly sensitive 125 I-protein A assay using rabbit antisera directed against purified SV40 TAg. Serological analysis of TAg in cellular extracts and of externally bound TAg revealed no apparent differences suggesting the cell surface binding affinity as a new property of SV40 TAg. We interpret our results as an indication that this property enables purified TAg to initiate the cellular immune response necessary for the SV40-tumor rejection in mice.