Merwin Moskowitz

Differences in the rates of protein degradation in untransformed and transformed cell lines

Abstract The rate of protein degradation in 3T3, 3T3/41, SV40 virus-transformed 3T3, BHK21, Rous sarcoma virus-transformed BHK21 and MRC-5 cells was assayed in monolayer and suspension culture by pre-labeling the cells with radioactive leucine and measuring the loss of radioactivity from the cells. The rate of protein degradation of 3T3 and MRC-5 cells in suspension culture, under which condition they do not grow, was greater than that of other cell lines which are capable of growing in suspension. In all cell lines it was greater in suspension culture than in monolayer culture. There was an inverse correlation between the rate of protein degradation and the growth rate of the cells. The addition of insulin to 3T3 cells in suspension culture causes the reduction of the rate of protein degradation, increase of cellular protein and stimulation of DNA synthesis.

Studies on the turnover of plasma membranes in cultured mammalian cells.: II. Rates of synthesis and degradation of plasma membrane proteins and carbohydrates

The rate of turnover of membrane proteins and membrane-bound carbohydrates in exponentially growing and in confluent contact-inhibited cultures of strain MK-2 cells was investigated. Cells were labelled with [14-C]leucine and [3-H]glucosamine, incubated in isotope-free medium and, at various times thereafter, the cells were harvested and membranes isolated from them. The rate of decay of the protein and carbohydrate components was determined from specific activity dilution of the labeled components in the isolated membranes. Although the rate of membrane synthesis is different in exponential and contact-inhibited cells, the rate of degradation (turnover) of membrane proteins and carbohydrates was found to be the same (25% per generation (42 h) or 0.6%/h).

Studies on the turnover of plasma membranes in cultured mammalian cells.: II. Demonstration of heterogeneous rates of turnover for plasma membrane proteins and glycoproteins

The relative rate of turnover of individual membrane proteins and glycoproteins in exponentially growing and contact-inhibited MK2 cells was investigated. Plasma membranes were isolated from cells that had been sequentially labelled with 14-C and 3-H isotopes of leucine and glucosamine. The membranes were then solubilized in sodium dodecylsulfate and their polypeptides separated by acrylamide gel electrophoresis. The 3-H/14-C ratios of the individual polypeptides reflected their relative rates of turnover. The proteins and glycoproteins of the exponentially growing cells exhibited markedly heterogeneous rates of turnover. In contrast, polypeptides in membranes of contact-inhibited cells exhibited a lesser degree of heterogeneity of turnover. In both exponential and contacted cell membranes a glycoprotein with a high apparent molecular weight exhibited the fastest rate of turnover.

The effects of low molecular weight materials derived from animal tissues on the growth of animal cells in vitro

Abstract A low molecular weight fraction was isolated by gel filtration from commercially prepared tissue hydrolysates. These low molecular weight materials could replace the serum supplement for the growth of some established cell lines in vitro , and they enabled the cultured cells to grow at a rate comparable to that observed with the serum supplement. It would appear that the active material is a mixture of products, possibly peptide in nature, with molecular weight between 700 to 1600. A high molecular weight fraction separated from these hydrolysates did not stimulate the growth of the cells to the same extent and, under some conditions, caused the cells to clump.

Sensitivity of Cultured Mammalian Cells to Streptomycin and Dihydrostreptomycin

Cultured mammalian cells killed by streptomycin were essentially unaffected by an identical concentration of dihydrostreptomycin.

Arrest of cell growth in the G1 phase of the cell cycle by serine deprivation.

Abstract BHK21 cells cultured in minimal essential medium (Eagle) supplemented with 10% dialyzed fetal calf serum did not grow as they did in whole serum containing medium. Logarithmic growth was, however, initiated after a lag period, the length of which was dependent upon the cell density: medium volume ratio. The quiescent cells conditioned the medium during this lag period, and growth stimulation was apparently due to the release of serine into the medium. Cells cultured in 10% dialyzed serum plus the low molecular weight fraction of serum (serum dialysate), grew with kinetics similar to cells cultured in serum containing medium. When serum dialysate was chromatographed on Bio-gel P-2 the growth promoting activity eluted with the amino acids. Each of the non-essential amino acids was tested for its ability to stimulate the growth of cells in 10% dialyzed serum. Serine was capable of stimulating cell growth to the same extent as 10% serum dialysate and its concentration optimum was similar to its concentration in 10% serum dialysate. The remaining non-essential amino acids were either slightly stimulatory or had no effect on cell growth. Shifting a logarithmically growing population of cells to serine-free medium resulted in the accumulation of 95% of the cells in the G1 phase of the cell cycle within 24 h. Escape from the G1 block could occur if serine was added to the medium or if the cells were allowed to condition the medium. Entry of cells into S phase after the addition of 0.05 μmoles/ml of serine followed a 4–6 h lag and 80% of the cells were synthesizing DNA 12 h after shift-up.

Growth promoting activity for mammalian cells in fractions of tissue extracts

Abstract Growth promoting and growth inhibiting factors were separated from an extract of porcine pancreas by dialysis. The growth promoting fraction in the dialysate initially had a slight toxic effect on the cells, but upon recovery they grew to a greater extent than in serum supplemented medium. The addition of human serum albumin to dialysate supplemented medium protected the cells from initial toxic effects and growth in this medium was as much as three times greater than in serum supplemented medium.

Studies on the effect of streptomycin on mammalian cells in culture: I. Isolation of resistant strains☆

Abstract Cells resistant to the action of relatively large quantities of streptomycin have been isolated from a streptomycin sensitive strain of mammalian cells. Sensitive and resistant clone cultures were also separated from the parent strain by cloning techniques in the absence of streptomycin. Streptomycin resistance and sensitivity is maintained after repeated subculturing in the absence of streptomycin. Some of the factors affecting the resistance of cells to streptomycin have been studied and it was found that the nutritional and physical environment of the growing culture affected the behavior of the cells in streptomycin. Streptomycin sensitive cells appear to release more 260 mμ absorbing material than streptomycin resistant cells.

Separation of Growth Promoting Activity from Horse Serum by Dialysis.

Summary A method for separation of a fraction from horse serum which does not react in tests for protein and which is capable of substituting for whole serum in tissue culture medium has been described. When this fraction was used as a supplement in synthetic medium, cell growth was obtained which compared favorably with controls grown in serum supplemented medium.

Polyacrylamide gel electrophoresis of glycoproteins on single concentration and gradient gels

Abstract A comparison of the distribution patterns of glycoproteins released into the medium by normal and oncogenic virus-transformed mammalian cells on 5% and 2–12% linear gradient sodium dodecylsulfate-polyacrylamide gels was made. Marked differences were observed in the distribution patterns obtained with the two types of gels. The gradient gels separate the glycoproteins into three discrete groups, while the 5% gels show only numerous small peaks. It appears that gradient gels may be of unique value in the separation of glycoproteins and may permit the identification of groups of compounds not distinguishable on single porosity gels.