Helmuth Hilz

Permeation of dibutyryl cAMP into HeLa cells and its conversion to monobutyryl cAMP

Summary 3H-Dibutyryl cAMP (DBcAMP) when exposed to HeLa cultures proved rather resistant to extracellular degradation. It was taken up by the cells and led to an accumulation of monobutyryl cAMP (MBcAMP), which — in contrast to DBcAMP — showed a high affinity to Gilmans (1) cAMP-binding protein. cAMP and DBcAMP were not accumulated in the cells to a comparable degree under these conditions. Other than DBcAMP, cAMP was rapidly degraded extracellulary to various metabolites including adenosine which was taken up by the cells and converted to purine nucleotides. Only at high extracellular concentrations cAMP could permeate the cells and lead to a transient rise in intracellular cAMP levels. These levels, however, never reached the steadily increasing concentrations of protein kinase-binding substances (MBcAMP + cAMP) when similar concentrations of DBcAMP were added to cultures. These results indicate that the sustained hormone-like actions of DBcAMP come about mainly by a high resistance to extracellular and intracellular phosphodiesterase as well as by the enzymic conversion to MBcAMP accumulating in the cells.

Specific inhibition of poly adpribose polymerase by thymidine and nicotinamide in HeLa cells

The enzyme forming poly ADPR seems to be related to DNA in a yet unknown manner. The exclusive localisation in the nucleus [l-3] , the even distribution over the chromatin [4, 51, the firm association with nuclear proteins (6, 71, its inhibitory action on “endogenous” DNA polymerase activity [ 81, and the correlation of poly ADPR polymerase activity with the DNA content of various malignant and nonmalignant tissues [9] point to a close connection with the nuclear DNA. In spite of these findings, however, the true function of the polymer is still obscure, and additional information is needed. We wish to report on a highly specific inhibition of the poly ADPR-forming enzyme from HeLa cells by thymidine and by nicotinamide.

A new method using ‘proteinase K’ to prevent mRNA degradation during isolation from HeLa cells

Abstract Diethyl pyrocarbonate (DEP) degrades RNA in a time-dependent reaction at neutral pH with the production of acid-soluble nucleotides. In spite of its inactivating effect on ribonuclease, diethyl pyrocarbonate present during isolation induces considerably degradation of pulse-labeled mRNA of HeLa cells. This can be explained by the above-mentioned reaction. A new method was developed using ‘proteinase K’ to inactivate endogenous ribonuclease leading to high specific activities of mRNA extracted from pulse-labeled HeLa cells.

Stoichiometry of cAMP binding and limited proteolysis of protein kinase regulatory subunits R I and R II

Abstract Protein kinase regulatory subunits type I (rabbit skeletal muscle) and type II (bovine heart) were isolated by a rapid two step procedure which involved affinity chromatography on an 8-thio cAMP matrix. The R proteins were analyzed for cAMP binding capacity using three different methods for the separation of bound from free cAMP, and various methods for protein determination. Regulatory subunits type I as well as type II were both found to contain two high affinity cAMP binding sites per R monomer corresponding to a formula for the native R proteins of R 2 ·cAMP 4 . - Kinetic analyses of limited proteolysis by various proteases revealed striking differences between R I and R II with respect to loss of cAMP binding capacity, ability to inhibit the catalytic subunit C, and susceptibility to further degradation. Some of the products had lost about one half of the cAMP binding capacity supporting the presence of two binding sites in R while other degradation products showed no change in high affinity binding sites. By contrast, the ability to inhibit the catalytic subunit C was lost in all products of limited proteolysis except one.

The age-dependent decrease in creatine kinase and aldolase activities in human striated muscle is not caused by an accumulation of faulty proteins

In human striated muscle obtained in surgery, an age-dependent decrease in aldolase and creatine kinase specific activities and an increase in DNA content per wet weight was found. In the group of the elderly (64-84 years), the enzymes decreased by 40-60% when compared with a group between 24 and 47 years old, while DNA content rose by a factor of 1.53 indicating loss of tissue water. Titration of aldolase and creatine kinase molecules by specific antibodies against aldolase A and creatine kinase MM isozymes, respectively, revealed very little accumulation of aldolase cross-reacting materials in the old age group (1.13 fold), and no accumulation of inactive creatine kinase molecules. Similar conclusions can be drawn from thermostability analyses of these two enzymes. The data do not support the view that accumulation of modified proteins due to random errors or to post-translational alternations is a general or causative phenomenon of aging in human muscle tissue.

Molecular heterogeneity of free PSA in sera of patients with benign and malignant prostate tumors.

Objective: To analyze free prostate-specific antigen (f-PSA) in sera from patients with prostate cancer (PCa) and benign prostatic hyperplasia (BPH), and to detect possible differences in subtypes as potential diagnostic parameters. Materials and Methods: PSA was purified from sera by an immunoaffinity procedure developed on the basis of oriented antibody immobilization, and subjected to size exclusion chromatography (SEC), Western blotting, and N-terminal amino acid sequencing. Results: The novel procedure allowed the purification of PSA with high yield from sera containing PSA <10 ng/ml. SEC under nonreducing conditions as well as Western blots demonstrated the presence of several molecular forms of f-PSA. Three of the smaller polypeptides exhibited the N-terminal sequence of PSA while one represented the C-terminal fragment Lys146–Pro237. Shortening of some polypeptides by the N-terminal amino acid Ile1 suggestive of aminopeptidase action was also observed. No propeptide sequence could be detected, and none of the bands from patient sera reacted with antibodies raised against propeptide antigens. BPH sera expressed higher proportions of smaller PSA fragments per unit p33, and contained significant amounts of fragments <14,000 which appeared to be very low or absent from most PCa sera. Conclusions: f-PSA as obtained from BPH and PCa sera represents a heterogeneous fraction. The major component (p33) is not in the nicked form and does not contain proPSA. Diagnostic potential could arise from the quantitative differences of the smaller PSA derivatives seen between PCa and BPH sera.

Rapid isolation of undegraded polysomal RNA without phenol

Recently we have described a method for the isolation of mRNA from polysomes using proteinase K to suppress ribonuclease action and to degrade ribosomal proteins [ 1 ] . In these experiments, the proteinase itself was finally removed. by phenol treatment. We present now a simpler way’of isolating mRNA avoiding the use of phenol, and leading to nearly complete yield of mRNA even in ‘small samples.

Opposite effects of cyclic AMP and its dibutyryl derivative on glycogen levels in HeLa cells.

Abstract In HeLa S3 cells, N 6 ,O 2′ -dibutyryl-3′,5′-cyclic-AMP (DBcAMP) leads to a decrease in glycogen content within two hours. This effect is reinforced by theophylline and counteracted by insulin and imidazole. In contrast to DBcAMP, 3′,5′-cyclic AMP (CAMP) itself acts like insulin by increasing the glycogen content. It is synergistic with imidazole and insulin, and counteracts the theophylline effect partially. When added together, the dibutyryl derivative completely abolishes the anabolic action of cAMP on glycogen content.

Isolation and partial characterization of the ADP-ribosylated nuclear proteins from Ehrlich ascites tumor cells

Abstract The (ADP-ribose)n protein conjugates formed by incubation of Ehrlich ascites tumor cell nuclei with 1 mM (3H)NAD were isolated by chromatography on boronate cellulose columns with a yield of >85%. Possible contamination by glycoproteins was excluded by rechromatography after specific release of the (ADP-ribose)n residues from their acceptors. Dodecyl sulfate gel electrophoresis revealed numerous protein bands which coincided with the (3H)ADP-ribose bands obtained by fluorography of the gels. 40% of the acceptor proteins were identified as the nucleosomal core histones. Most of these histones, however, appeared in the non-histone fraction because of extensive modification by poly(ADP-ribose). Drastic changes in properties were also seen in the true non-histone proteins which comprised 60% of the total conjugated protein. Besides several prominent acceptor proteins (Mr = 12,000; 31,000; 125,000) numerous proteins were detected indicating a considerable heterogeneity of non-histone acceptors.

A new cAMP affinity matrix for the rapid purification of protein kinase regulatory subunits.

Affinity chromatography has been described as a useful step in the purification of protein kinase subunits (cf. [l-S]). Coupling of the CAMP derivatives to Sepharose was usually performed with ~NBr-activated matrices. The stability of the linkages formed appears reasonably good when ligation involved more than one bond, as in the coupling of proteins. If, however, the ligand is bound via a single linkage, stability of the conjugate is drastically reduced as in the case of Sepharose~oup~ed ~-substituted cAMP derivatives [6]. Such matrices, then, may lead to significant losses of CAMP binding proteins when present in low concentrations. An additional disadvantage of most cAMP affinity matrices resides in the hydrophobic spacer region, which tends to bind unrelated proteins leading to contamination of the final product. This paper describes the coupling of an 8substituted thio-CAMP derivative to epoxy-activated Sepharose via a hydrophilic spacer. The resulting CAMP affinity matrix was rather stable and could be used successfully to isolate protein kinase I and II regulatory subunits in pure form and high yield, R II prepared in this way exhibited two binding sites for CAMP corresponding to an R*scAMP, formula.