SummaryThe kinetics of the release of glycopeptides by the action of rennin on bovine whole milk have been studied. The initial rate of release of glycopeptides was proportional to the concentration of rennin. Using the integrated form of the Michaelis–Menten equation, values have been obtained forKmandV. These values varied between milk samples from individual cows, and for a single milk sample the value forKmincreased with increasing rennin concentration. It is suggested that theKmvalue is partly dependent on the carbohydrate composition of individual κ-casein molecules.

SummaryA method based on salt fractionation, iso-electric precipitation and gel filtration chromatography is described for the purification of the enzyme rennin (E.C. 3.4.4.3).

acids and possibly a sterol ester. Similar classes of neutral lipids were found in the sporophore of A. bi8porua (Hughes, 1962). The acetone eluate from the column contains the acylated form of the carbohydrates found in the aqueous fraction. The composition of this fraction will be described elsewhere.Phospholipids were found only in the chloroformmethanol eluate. T.l.c. in chloroform-methanolwater (65:25:4, by vol.) and spraying with specific reagents showed principally phosphatidylinositol and phosphatidylethanolamine. Phosphatidylcholine was absent. These identifications were confirmed by acid hydrolysis and paper chromatography, which demonstrated glycerol, inositol, ethanolamine and traces of serine, but no choline. Mild alkaline hydrolysis and paper chromatography in propan-2-ol-aq. ammonia (sp.gr. 0.88)-water (7:1: 2, by vol.) showed glycerylphosphorylinositol, glycerylphosphorylethanolamine and glycerylphosphorylserine.This work is the early part of a study of the composition of A. bi8poruw, in the fruiting and vegetative stages, which may provide some insight into the metabolic differences underlying the initiation of sporophore development. It may also help to elucidate the role of lipids in stimulating spore germination (L6sel, 1967).

The kinetics of the primary phase of rennin action on casein micelles and on total casein prepared by ultracentrifugation have been studied. It was observed that the values of K m and V varied for the different casein samples. Addition of whey proteins to the reaction mixture or variation of the concentrations of Na + and K+ had no effect on K m and V, but there was a consistent increase in the values of both these parameters when /c-casein was added. It was concluded that these changes were due to an increase in the accessibility of the /c-casein resulting from the rearrangement of the casein micelles. These results are in agreement with our earlier conclusion that the K m for rennin action on casein is dependent on the carbohydrate composition of the molecule.In a previous paper (Castle & Wheelock, 1972) we showed that there was a wide variation between cows in the values of K m and F obtained for the primary phase of rennin action on whole milk. It was also observed that for an individual milk sample the value of K m but not of V varied with rennin concentration. It was suggested that this variation was partly due to differences between milks in the amount and composition of the /c-casein and possibly also in the composition of the non-casein fraction of the milk.The present experiments were designed to study the kinetics of rennin action on casein in order to obtain further information on the factors which contribute to the differences observed for K m and V between milks of different animals. MATERIALS AND METHODSMilk samples were obtained from individual cows which were known to be free from bacterial infections of the udder.Casein micelles and total casein (casein micelles + soluble casein) were prepared by an adaptation of the method of Bohren & Wenner (1961). The micelles were sedimented by centrifuging skim-milk in a Superspeed 40 (Measuring and Scientific Equipment Ltd, Crawley, Sussex) preparative ultracentrifuge at 100000 g for 1 h at 20 °C. The total casein was prepared in exactly the same way except that 2-0 ml of 3-0 M-CaCl 2 /100 ml milk was added prior to centrifugation in order that the soluble casein would form a sediment with the micelles. After the addition of the CaCl 2 , the pH of the milk was adjusted to its original value with 1-0 M-KOH.