RESPIRATION AND OXIDATIVE PHOSPHORYLATION 101 



centrifugations to attain the required final cell concentration of 5 X 

 10 8 /ml. The spermatozoa were suspended in the above modified 

 Ringer, in seminal fluid, or in mixtures of both, buffered at pH 7.4 by 

 0.02M glycylglycine. Motility was high in the presence of glucose or 

 seminal fluid. 



Spisula spermatozoa were collected from the punctured gonads of 

 ripe animals. The sperm suspension was passed through cheesecloth, 

 washed once, centrifuged at 3000 rpm, and finally resuspended in sea 

 water buffered by 0.02M glycylglycine at pH 7.5. Final cell concen- 

 tration was 10 to 30 X 10 8 cells/ml. Motility was high in the samples 

 studied. These studies on clam sperm were performed at the Marine 

 Biological Laboratory (Woods Hole) and the Johnson Foundation 

 (University of Pennsylvania). 



Methods 



Absorption spectra at room temperature were obtained with the 

 wavelength-scanning, recording spectrophotometer described by 

 Yang and Legallais (1954). Spectra at the temperature of liquid ni- 

 trogen, run in the presence of glycerol (Keilin and Hartree, 1949), 

 were recorded by the procedure of Estabrook (1958). At liquid nitro- 

 gen temperature, difference spectra, without addition of glycerol, 

 were also obtained on "stabilized steady states" according to the 

 method of Chance and Spencer (1959). Oxygen consumption was 

 determined on aliquots before and after spectroscopic measurements. 

 The recording platinum and silver electrodes were dipped in a ro- 

 tating cuvette (0.5 to 2 ml) and polarized at —600 mv (Davies and 

 Brink, 1942; Williams, 1961). Motility was estimated by microscopic 

 examination of pairs of drops pipetted from treated and reference 

 samples. In later stages of the work an apparatus modified from that 

 of Bosselaar and Spronk (1952) and of Rikmenspoel (1957) was used 

 which gave relative motility ratings under controlled conditions and 

 at the same time registered pH changes and oxygen concentration 

 (Gonse, 1960). 



Calculations 



The concentrations of respiratory enzymes may be obtained from 

 optical density changes at selected pairs of wavelength on difference 

 spectra at 25 °C. One applies the molecular extinction coefficients 



