On the monoglyceride content of hog pancreas

B I O C H I M I C A E T B I O P H Y S 1 C A ACTA

373

Short Communications On the

monoglyceridec o n t e n t

of

hog pancreas

JONES, KOCK, HEATH AND ~{UNSON1 reported in 1949 that they had found ~-monopalmitin in hog-pancreas tissue in a remarkably high concentration, i.e. 1-1.2 ~, of the fresh weight of the organ. The authors considered the possibility that the e-monoglyceride could have been formed through autolysis catalyzed by the potent lipolytic enzymes present in the pancreatic gland but found the same yield of monoglyceride if the pancreas was ground and extracted immediately after the slaughter. The monoglycerides of the pancreas therefore seemed to be a singnificant and meaningful constituent of the living pancreas. The results of the above authors, however, do not exclude the possibility that the ~-monoglyceride could be formed during the grinding or extraction of the organ and it appeared to us that the level of free f a t t y acid in the extracted lipids might better indicate the origin of the monoglycerides. Hog pancreas was obtained as early as possible in the slaughter house and treated as follows: (A) immediately frozen in dry ice, the frozen pieces ground in a Waring blendor with chloroform-methanol (2:1), the volume of which was made up to a final volume 2o times that of the weight of the gland ; (B) raw pancreas was ground in a Waring blendor, left for 15 rain at room temperature and then chloroform-methanol added; (C) part of the gland was ground and extracted by ethanol as described bv JONES et al. The chloroform-methanol extracts from (A) and (B) were left overnight at room temperature, filtered and equilibrated with 0. 4 vol. of o.5 o/.,osolutions of acid phosphate. After equilibration the phases were allowed to separate and the chloroform phase removed, dried over Na2SO ~ and taken to a small volume. The total lipids were then separated into a neutral fat plus free fatty acid fraction and a phospholipid fraction on silicic acid columns. The lipids extracted in (C) were freed of phospholipids on a silicic acid column and the neutral fat plus fatty acid fraction analysed as the other fractions. The free f a t t y acid content was obtained by titration. ~-Monoglycerides were determined by HIO~ oxidation and iodometric titration. Finally an analysis of mono-, di- and tri-glyceride was made after separation on silicic acid columns. Approx. 4 mg of the neutral fat plus free f a t t y acid fraction were put on i-g columns of silicie acid (Unisil ; Clarkson Chemical Company, Williamsport, Pa.); tri-, di-and mono-glycerides were eluted with 3 ° m l of respectively 5 and 20 O//odiethyl ether in light petroleum and diethvl, ether. Aliquots from the eluates were used for glyceride glycerol determinations. The purity of the fractions obtained was checked by silicic acid thin-layer chromatography. The results are given in Table I. The monoglyceride and free f a t t y acid content of the frozen gland is very low (A). Homogenizing the gland at room temperature for 15 min leads to an appreciable increase in monoglyceride and free f a t t y acid content and a decrease in triglycerides (B). Extraction of the homogenized gland by ethanol Biochim. Biophys..qcta, 5 t (1961) 373--374

374

SHORT COMMUNICATIONS

according to the procedure of JONES et al. (C) results in even more pronounced increase in monoglyceride and decreased triglyceride content. This is probably an effect of a somewhat longer incubation period of the homogenized gland. In case (B) and (C) the monoglyceride content corresponds to approx. 1 % of gland wet wt. The difference in total glyceride in the different samples must be due to unequal distribution of mesenteric adipose tissue contaminating the pancreas. The free fatty acid content in TABLE I LIPID

COMPOSITION

OF HOG-PANCREATIC

GLAND

For m e t h o d of t r e a t m e n t of s a m p l e s A, B a n d C, see text.

Sample

N e u t r a l fat + free f a t t y acids (mg/g wet wt. pancreas) Monoglyceride (/~moles/loo t, inoles glycerides) Diglyceride (/,moles/Ioo /,moles glycerides) Triglyceride (lsinoles/loo /~moles glycerides) Free f a t t y acids (/~moles/mo Hmoles glycerides)

A

B

C

35-5 2 14 84 5

94.5 31 18 51 79

27.5 69 16 15 84

sample (C) is much too low compared to the fatty acid deficit of the glycerides, a possible explanation for this discrepancy being incomplete extraction of free fatty acids with the system of JONES et al. Determination of the monoglyceride content by H I Q oxidation gave figures very close to those obtained by glyceride glycerol deternlinations after silicic acid chromatography indicating that only ~-monoglycerides were present in the extracted lipids. The results clearly show that monoglyceride and free fatty acid content of the pancreatic gland is very low and that the high content of monoglyceride reported by J O N E S et al. must be due to lipolysis in vitro.

DeISartment of Physiological Chemistry, University of Lund, Lund (Sweden)

B. BORGSTR65~

z M. E. JONES, F. C. KOCK, A. E. HEATH AND P. L. MUNSON, J. Biol. Chem., 181 (1940) 755.

R eceived March Ist, 1961 Biochim. Biophys. Acla, 5 t (1961) 373-374

The inhibition of M 9 (ll)-enolase by other activating metal ions Enolase can be activated by several divalent metal ions I. However, the maximal velocities obtained with the various ions differ, the Mg(II)-enzyme having the highest activity. If anotber activating ion, e.g. Mn ~+, is added to Mg(II)-enolase, the activity is decreased. The kinetics of this inhibition were studied by WOLD AND BALLOU2 who concluded that their data, unexpectedly, indicated an uncompetitive inhibition. Since this behavior is not consistent with the well-supported picture l,a that enolase activation involves the binding of the activating ion to one specific site on the enzyme molecule, a reinvestigation of this problem seemed desirable. Biochim. Biophys..4cta, 51 (1961) 374-376