Robert J. Klebe

Localization of the cell attachment region in types I and II collagens

Summary The sites on type I and II collagen which bind to the cell attachment protein (CAP) have been identified using an inhibition assay. Both chains of type I collagen contain binding sites but αI(I) is more active than the α2 chain. The most active peptide obtained from the α1(I) chain corresponds to residues 568 through 835. A comparable region was found to be the only active site on the α1 (II) chain.

A simple method for the quantitation of isozyme patterns.

Isozyme patterns may be analyzed quantitatively, without the use of a densitometer, by performing serial twofold dilutions of a sample to a visual end point. The specific activity (S) of a given dehydrogenase isozyme can be assessed in the presence of other isozymes catalyzing the same reaction, by (1) determining the isozyme titer (T) (defined as mg protein/ml in the last visible band) and (2) applying the formula S=K/T, where K is 1.6×10−3 units/ml in the last visible band. The units/ml (U) in the starting material can be calculated from the equation U=K (2)n−1, where n is the number of the slot producing the last visible band.

Cell attachment to collagen: the ionic requirements.

Abstract This study is concerned with three aspects of the ionic requirements for cell attachment to collagen; namely (a) the divalent, (b) monovalent cation specificities and (c) pH optima for cell interaction with collagen. The divalent cation requirement for cell attachment to collagen can be sufficed by Ca2+, Mg2+ and certain transition group metals; whereas Ba2+, Sr2+, and polyamines are inactive. The pH optimum for cell attachment in this system occurs in the physiological range. The monovalent cation requirement for cell attachment to collagen is satisfied by isotonic NaCl, KCl, LiCl, NH4Cl, sucrose, and glucose. Pronounced inhibition of cell attachment occurs under both hypertonic and hypotonic conditions.

Chemicals which promote cell hybridization

In order to identify new compounds with cell hybridization properties similar to poly(ethylene glycol), the standard method for PEG-induced cell hybridization has been employed as a screening procedure. Of 118 membrane-active agents studied, over 20 compounds were identified which promoted cell hybridization with nearly the same efficiency as PEG. PEG derivatives which retained cell hybridization activity included polymers with branched and charged structures as well as polymers with chemical alterations of the PEG monomer itself. PEG derivatives with hydrophobic moieties were generally inactive. Several chemically modified derivatives of poly(ethylene glycol), which are commercially important in the pharmaceutical and cosmetic industries, were found to be highly efficient cell hybridization agents. The biohazard of such compounds is discussed. A simplified method is presented for the preparation and use of PEG in cell hybridization procedures.

Biochemical selection systems for mammalian cells: the essential amino acids.

The essential amino acid requirement of cultured mammalian cells can be satisfied by 19 amino acid derivatives. This finding (a) confirms the results of animal nutritional studies and (b) identifies 19 essential amino acid derivatives and should permit the isolation of a new class of auxotrophic mutants. Five naturally occurring auxotrophic markers have been detected in this survey; namely, inability to utilize cystathionine and citrulline in Chinese hamster ovary (CHO) cells, inability to metabolize citrulline by HTC+ hepatoma cells, and confirmation of Eagles observation that KB cells can utilize homocystine in place of methionine or cystine and d-cystine in place of l-cystine.

Cell attachment to collagen: Isolation of a cell attachment mutant

Abstract A cell attachment mutant of the Chinese hamster ovary cell line (CHO) was isolated with the aid of an enrichment procedure. The mutant isolated, CHOatt−, was found to be defective in its attachment to collagen in two respects. (1) The mutant requires 100- and 1 000-fold more Mg2+ and Ca2+, respectively, than wild-type cells for 50% cell attachment to collagen; (2) the mutant displays only 5% of the affinity of wild-type cells for a serum cell attachment protein. CHOatt−, which was selected for non-attachment to collagen, is also altered in its ability to attach to synthetic substrata, such as glass and plastic. Collagen adhesive revertants of CHOatt− were isolated. The revertants differed morphologically from wild type CHO cells. The adhesive response to Ca2+, Mg2+, and cell attachment protein in each revertant was either partially or fully restored.

Bioautographic visualization of aminoacylase-1: Assignment of the structural geneACY-1 to chromosome 3 in man

A bioautographic assay was developed for the visualization of aminoacylase-1 (N -acylamino acid aminohydrolase, ACY-1; EC 3.5.1.14) after zone electrophoresis. Bioautography and species differences in electrophoretic mobility of ACY-1 made it possible to investigate the chromosome assignment of the gene for human ACY-1 using human—mouse somatic cell hybrids. Human ACY-1 segregated concordantly with β-galactosidase-A (βGALA;EC 3.2.1.23) but showed discordant segregation with 32 other markers representing 23 linkage groups. The β GALAgene has been previously assigned to chromosome 3. From this evidence and confirming chromosome analyses, ACY-1has been assigned to chromosome 3. A genetic polymorphism in the electrophoretic mobility of ACY was observed in mouse strains, demonstrating that this enzyme can be mapped in genetic crosses of Mus musculus.

Bioautography: A general method for the visualization of isozymes

A new method has been developed for visualization of isozymes which are difficult or impossible to detect with standard histochemical or autoradiographic methods. The principle of this method, bioautography, is the use of a microbial reagent to locate an enzyme after gel electrophoresis. When bioautography was compared to other staining procedures, the bioautographic method yielded identical results to those observed by the histochemical method for lactate dehydrogenase (LDH) or by the autoradiographic method for the adenine phosphoribosyl-transferase (APRT). Using the bioautographic method, stains for enzymes which could not be visualized by any other procedure have been developed: argininosuccinate lyase and branched-chain aminotransferase. By employing appropriately genetically marked bacterial strains, it should be possible to develop new isozyme stains for a large number of unstudied isozymes.

Somatic cell genetic studies of the cystic fibrosis mucociliary inhibitor

A series of cystic fibrosis homozygous fibroblast X mouse cell hybrids were constructed in order to determine the chromosome which carries the gene controlling the cystic fibrosis mucociliary inhibitor (CFMI) phenotype. Several hybrids and their subclones retained the CFMI phenotype for long periods in cell culture. Correlation of the CFMI phenotype with the presence of specific human chromosomes indicated that possible linkage existed between CFMI and human chromosomes 2, 4, 6, 10, and 18. The strongest chance of linkage existed for chromosome 4.

Characterization of naturally occurring auxotrophic mammalian cells.

In a previous study, several cultured cell lines were detected which are naturally occurring auxotrophs. In this investigation, the enzyme deficiencies involved are described. It is demonstrated that the Chinese hamster cell lines CHO(Kl), YH21, RJK-36, and CHW-1102 are deficient in cystathionase and argininosuccinate synthetase. In addition, CHO (Kl) and CHW-1102 were found to lack argininosuccinate lyase. CHW-1102 cells were also found to be unable to proliferate in medium containing branchedchain α-keto acids in place of the corresponding L-amino acids since CHW-1102 cells lack branched-chain aminotransferase.