98
ALBERTO MONROY AND RACHELE MAGGIO
and Barnes, 1953; Tyler et al., 1956), a quantitative determination of
Na+ in the homogenate (which can be done very quickly and easily by
flame photometry) has been suggested as a convenient method of
estimating the amount of contaminating sea-water present (Aiello and
Maggio, 1961). In this way, at least, homogenates found to be too largely
contaminated can be discarded, thus avoiding one serious pitfall.
Washing the eggs with isotonic Ca-free solutions or with solutions
containing chelating agents may be quite useful, although one must
make sure that such washing does not act deleteriously per se on the
eggs. The addition of chelating agents to the solutions used for homogenization may help to remove excess of divalent ions. This process has
been found to be quite advantageous for the preparation of mitochondria,
while on the other hand it seems that microsomes, at least in the case of
liver, are dissolved in the presence of EDTA| (Palade and Siekevitz,
1956). However, recent observations indicate that, in the case of the
sea urchin egg, EDTA preserves the structure and function of microsomes (Allison and Clark, 1960). This may be a further indication of how
risky it is to apply a method developed on one particular material to
another without carrying out appropriate checks.
Before the introduction of EDTA to preserve mitochondria during
cell fractionation, it had been a practice in our Laboratory to prepare
the sucrose solutions for homogenization in citrate buffer. This in fact
had been found to be an essential prerequisite for the preparation of
apparently intact pigment granules from sea urchin eggs and embryos
(Monroy and De Nicola, 1952). Since then we have found it advantageous
to use the citrate to buffer the sucrose even when EDTA is added.
Another relevant question is that of the molarity and composition of
the solutions used for homogenization. For the preparation of the
mitochondria a 0-44 M solution of sucrose (in citrate and EDTA) has
commonly been used as we have found that at this molarity (actually
between 0-5 and 0-3 M) swelling of the mitochondria is minimal (Monroy,
1957a). A preliminary electron microscopic study of ultra-thin sections
of mitochondrial pellets prepared by this technique shows that they are
quite well preserved. Lowering the sucrose concentration to 0*25 M
results in mitochondrial damage. Also, the addition of polyvinylpirrolidone has proved to be deleterious to the mitochondria (Vittorelli,
t The following abbreviations have been used : EDTA : ethylendiaminetetracetic acid ;
ATP: adenosine triphosphate ; ADP: adenosinediphosphate ; AMP: adenosinemonophosphate; ATPase: adenosine triphosphatase; DNA: deoxyribonucleic acid; RNA:
ribonucleic acid; DNAse: deoxyribonuclease; RNAse: ribonuclease; TCA: trichloroacetic acid; DNP: dinitrophenol; UMP: uridylic acid; CMP: cytidylic acid; dCMP:
5'-deoxycytidylic acid; dUMP: 5'-deoxyuridylic acid; dTMP: 5-thymidylic acid;
MedCMP: 5-methyl-deoxycytidylic acid; CH 2 OHdCMP: 5-hydroxymethyldeoxyuridylic acid.
ALBERTO MONROY AND RACHELE MAGGIO
and Barnes, 1953; Tyler et al., 1956), a quantitative determination of
Na+ in the homogenate (which can be done very quickly and easily by
flame photometry) has been suggested as a convenient method of
estimating the amount of contaminating sea-water present (Aiello and
Maggio, 1961). In this way, at least, homogenates found to be too largely
contaminated can be discarded, thus avoiding one serious pitfall.
Washing the eggs with isotonic Ca-free solutions or with solutions
containing chelating agents may be quite useful, although one must
make sure that such washing does not act deleteriously per se on the
eggs. The addition of chelating agents to the solutions used for homogenization may help to remove excess of divalent ions. This process has
been found to be quite advantageous for the preparation of mitochondria,
while on the other hand it seems that microsomes, at least in the case of
liver, are dissolved in the presence of EDTA| (Palade and Siekevitz,
1956). However, recent observations indicate that, in the case of the
sea urchin egg, EDTA preserves the structure and function of microsomes (Allison and Clark, 1960). This may be a further indication of how
risky it is to apply a method developed on one particular material to
another without carrying out appropriate checks.
Before the introduction of EDTA to preserve mitochondria during
cell fractionation, it had been a practice in our Laboratory to prepare
the sucrose solutions for homogenization in citrate buffer. This in fact
had been found to be an essential prerequisite for the preparation of
apparently intact pigment granules from sea urchin eggs and embryos
(Monroy and De Nicola, 1952). Since then we have found it advantageous
to use the citrate to buffer the sucrose even when EDTA is added.
Another relevant question is that of the molarity and composition of
the solutions used for homogenization. For the preparation of the
mitochondria a 0-44 M solution of sucrose (in citrate and EDTA) has
commonly been used as we have found that at this molarity (actually
between 0-5 and 0-3 M) swelling of the mitochondria is minimal (Monroy,
1957a). A preliminary electron microscopic study of ultra-thin sections
of mitochondrial pellets prepared by this technique shows that they are
quite well preserved. Lowering the sucrose concentration to 0*25 M
results in mitochondrial damage. Also, the addition of polyvinylpirrolidone has proved to be deleterious to the mitochondria (Vittorelli,
t The following abbreviations have been used : EDTA : ethylendiaminetetracetic acid ;
ATP: adenosine triphosphate ; ADP: adenosinediphosphate ; AMP: adenosinemonophosphate; ATPase: adenosine triphosphatase; DNA: deoxyribonucleic acid; RNA:
ribonucleic acid; DNAse: deoxyribonuclease; RNAse: ribonuclease; TCA: trichloroacetic acid; DNP: dinitrophenol; UMP: uridylic acid; CMP: cytidylic acid; dCMP:
5'-deoxycytidylic acid; dUMP: 5'-deoxyuridylic acid; dTMP: 5-thymidylic acid;
MedCMP: 5-methyl-deoxycytidylic acid; CH 2 OHdCMP: 5-hydroxymethyldeoxyuridylic acid.